identifier
stringlengths 11
32
| pdf_url
stringlengths 17
4.62k
⌀ | lang
stringclasses 120
values | error
stringclasses 1
value | title
stringlengths 2
500
⌀ | source_name
stringlengths 1
435
⌀ | publication_year
float64 1.9k
2.02k
| license
stringclasses 3
values | word_count
int64 0
1.64M
| text
stringlengths 1
9.75M
|
|---|---|---|---|---|---|---|---|---|---|
https://openalex.org/W2011359630
|
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0083742&type=printable
|
English
| null |
Adherence to Hypothermia Guidelines: A French Multicenter Study of Fullterm Neonates
|
PloS one
| 2,013
|
cc-by
| 6,761
|
Received July 23, 2013; Accepted November 7, 2013; Published December 31, 2013 Received July 23, 2013; Accepted November 7, 2013; Published December 31, 2013 Copyright: 2013 Chevallier et al. This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. vallier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: These authors have no support or funding to report. Funding: These authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: TDebillon@chu-grenoble.fr " Membership of the Mouse Genome Sequencing Consortium is provided in the Acknowledgments. Over the last fifteen years, several randomized trials on the
neuroprotective effect of hypothermia (HT) have been published
[12–18]. The results are summarized in four meta-analyzes, which
show a reduction of 25% in the combined risk of death and major
neuro-developmental disability at 18 months (RR = 0.76 [0.65 to
0.89]) [18–21]. Currently, HT is the standard treatment for HIE,
and in 2009, the French Society of Neonatology (SFN) published
guidelines on both the indications for HT and how it should be
performed [22]. The role of HT on the medium and long term
prognosis of these children remains unclear. Databases set up in
England and the USA (Toby Cooling Register and Vermont
Oxford Register) could help provide answers to this question
[9,23,24]. In France, in May 2010, the SFN set up a database with
similar objectives. It is intended to register all HIE cases admitted
to neonatal intensive care units (NICU), whether treated or not
with HT, and to ensure their subsequent follow-up. Marie Chevallier1, Anne Ego2, Christine Cans3, Thierry Debillon1*", on behalf of the French Society
of Neonatology 1 Neonatology and Pediatric Intensive Care Unit, Grenoble University Hospital, Grenoble, France, 2 Clinical Research Center (CICO3), Grenoble University Hospital
Grenoble, France, 3 THEMAS (Techniques pour l’e´valuation et la mode´lisation des actions de sante´), Joseph Fourier University-Grenoble1, Grenoble, France Abstract Aim: The objective of this study was to describe the French practice of hypothermia treatment (HT) in full-term newborns
with hypoxic-ischemic encephalopathy (HIE) and to analyze the deviations from the guidelines of the French Society of
Neonatology. Materials and Methods: From May 2010 to March 2012 we recorded all cases of HIE treated by HT in a French national
database. The population was divided into three groups, "optimal HT" (OHT), ‘‘late HT’’ (LHT) and ‘‘non-indicated’’ HT (NIHT),
according to the guidelines. Results: Of the 311 newborns registered in the database and having HT, 65% were classified in the OHT group, 22% and
13% in the LHT and NIHT groups respectively. The severity of asphyxia and HIE were comparable between newborns with
OHT and LHT, apart from EEG. HT was initiated at a mean time of 12 hours of life in the LHT group. An acute obstetrical
event was more likely to be identified among newborns with LHT (46%), compared to OHT (34%) and NIHT (22%). There was
a gradation in the rate of complications from the NIHT group (29%) to the LHT (38%) group and the OHT group (52%). Despite an insignificant difference in the rates of death or abnormal neurological examination at discharge, nearly 60% of
newborns in the OHT group had an MRI showing abnormalities, compared to 44% and 49% in the LHT and NIHT groups
respectively. Conclusion: The conduct of the HT for HIE newborns is not consistent with French guidelines for 35% of newborns, 22%
being explained by an excessive delay in the start of HT, 13% by the lack of adherence to the clinical indications. This first
report illustrates the difficulties in implementing guidelines for HT and should argue for an optimization of perinatal care for
HIE. Citation: Chevallier M, Ego A, Cans C, Debillon T, on behalf of the French Society of Neonatology (2013) Adherence to Hypothermia Guidelines: A French
Multicenter Study of Fullterm Neonates. PLoS ONE 8(12): e83742. doi:10.1371/journal.pone.0083742 Materials and Methods Since May 2010, the full-term neonate HIE database records
newborns of gestational age (GA) $ 36 weeks, weighing $ 1800g
and presenting with HIE (mild, moderate or severe), regardless of
the treatment (HT or standard care). All French Level III NICU
(including overseas territories) have secure access and reporting of
cases is declarative, without control of completeness. During the
study period, 33 level III NICUs among 57 nationwide included
newborns in the database, with a mean number of infants reported
per center of 9.2 (+ / –8.2). As of March 22, 2012, the database
contained 465 cases registered in 23 months. Not all French Level
III NICU participated, so the number of newborns included is not
exhaustive. Adverse events associated with HT such as thrombocy-
topenia and bleeding disorders, hemorrhage, inadvertent
overcooling, overheating, hypomagnesemia, and skin
lesions. –
Visceral complications linked to hypoxia-ischemia (HI)
such as liver cytolysis or failure, shock or isolated
hypotension, hypertension, renal failure, pulmonary
hypertension, glucose intolerance and a paralytic ileus. Selection of the study population and variables studied Our study included all newborns reported in the database
between May 2010 and March 2012 except newborns with
missing data concerning at least one of the criteria for the
indication of HT (n = 40) and those with HIE but not treated by
HT (n = 114) (Figure 1). According to our definition, a lack of
clinical indications was observed for 58 newborns (50.9%) among
114. Among the remaining 56 untreated cases, the main reasons
reported by neonatologists were the lack of electrophysiological
anomalies or clinical indications for HT (n = 16), the presence of
contraindications (n = 15), late admission to the NICU (n = 13) and
the restriction of neonatal care for critically ill neonates (n = 11). Hypothermia for Neonates To our knowledge, few studies have been published on the
evaluation of the practice of HT in the various countries where
this treatment is recommended. This is necessary before any
assessment of the long term impact of this new treatment can be
made. The aim of this study was to describe the practice in France
for full-term newborns with HIE treated by HT, and to analyze
deviations from the SFN guidelines and the reasons for these. Short-term outcomes (death, clinical examination results
according to the Amiel-Tison scoring [25] and brain
imaging data at discharge). For the neurological exami-
nation, 3 degrees of neurological abnormalities were
retained (normal or mild, moderate, and severe). For
cerebral imaging, 4 types of abnormalities were distin-
guished i) basal ganglia and/or cortical or subcortical
lesions, ii) white matter lesions apart from hemorrhagic
petechial lesions, iii) isolated hemorrhagic petechial
lesions, iii) brainstem or cerebellar lesions. Definition of optimal and sub-optimal HT According to the SFN guidelines, the clinical, paraclinical, and
organizational criteria justifying the initiation of HT are the
following: Ethics statement Data collection was approved by the French authority entitled
‘‘Commission Nationale de l’Informatique et des Liberte´s’’ (National Data
Protection Authority, Ref: AT/FLR/DI103637, Authorization
Nu1426721, 2010–266). This data collection was initiated in 2010
to study the newborns with HIE and to assess the implementation
of the hypothermia treatment in France. No written consent from
the parents was required by this French authority. According to
their guidelines for an observational study, we advised clinicians to
provide a parent information leaflet about data collection. If
parents express a disagreement with this survey, none data
collection on their child was performed but we did not collect this
information from each centers. The main finality of this data base,
specified
by
the
approving
of
the
Commission
Nationale
de
l’Informatique et des Liberte´s, is to use the data for statistical and
epidemiological analysis on order to ameliorate the management
of newborns with HIE. For that, we use anonymous data. 1. GA $ 36 weeks and birth weight $ 1800 g; 2. Clinical or paraclinical symptoms of asphyxia during
delivery: Apgar score ,5 at 5 minutes and/or a pH
,7.00, and/or base deficit . -13 mmol/L, and/or lactate
levels . 11 mmol /L in cord blood or in the first hour of
life; and/or need for assisted ventilation (endotracheal or
face mask) at 10 minutes of life; 3. Abnormal neurological examination according to Sarnat’s
classification (Stage II or III); 4. Abnormal electrophysiology in standard EEG or ampli-
tude-integrated EEG (low voltage, periodic and/or
paroxysmal trace, convulsion); 5. HT started in the first six hours of life. Due to missing data (46% of the data base population), criterion
4 concerning EEG was not taken into account. HT was qualified
as i) optimal (OHT) if all other criteria were met, ii) late HT
(LHT), if criteria 1 to 3 were met, but hypothermia was initiated
more than 6 hours after birth, iii) non indicated HT (NIHT), if
one or more of the 3 first clinical criteria was missing (either
gestational age/birthweight, or asphyxia, or neurological exami-
nation), whatever the time to initiate hypothermia. Introduction The incidence of hypoxic ischemic encephalopathy (HIE) in
newborns is currently imprecise with numbers ranging from 1 to 8
per 1000 live births worldwide [1,2]. The method of identifying
cases, the definition of HIE and the source study population have
an impact on the reported incidence [3,4]. In France, apart from a
study by Pierrat et al in the Nord Pas de Calais region, where an
incidence of 0.86 per 1000 was found, little precise epidemiological
data on HIE is available [5]. However the prognosis is severe and
mortality can reach 20 to 40% of cases. The rate of adverse
outcomes (death, cerebral palsy, severe cognitive deficit) reaches
30 to 50% in cases with Sarnat stage II HIE and 100% for stage
III [6–8]. Some studies report a 5–6 increased risk of epilepsy
[10,11]. PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e83742 1 Hypothermia for Neonates Statistical Analysis Several hypotheses were retained to deal with missing data. As
"need for assisted ventilation at 10 minutes of life" (criteria 2) was
not mentioned as such in the database, newborns requiring
intubation and/or non-invasive ventilation with an Apgar score
,10 at 10 min of life were considered to present this criterion. When data concerning the time to initiation of HT was missing
(n = 16), the time between birth and admission to the NICU was
used. Considering that this choice may underestimate this delay, a
sensitivity
analysis
was
performed:
the
alternative
solution
consisting in adding the mean time from admission to initiation
of HT observed among babies with complete data was considered. The distribution of OHT, NIHT and LHT, and the factors
associated with the practice of HT were reassessed according to
this scenario. The variables recorded in the database and analyzed were: –
Delivery characteristics (place, date, inborn or outborn,
time to admission), –
Clinical and paraclinical characteristics of the neonate
(term, weight, sex, care in the delivery room, clinical stage
of HIE severity, temperature on admission, umbical cord
acid-base balance (or within the first hour of life), EEG
performed before initiation of HT, –
Conditions of HT (time, material used), December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org 2 Hypothermia for Neonates Figure 1. Flow chart of the population study. doi:10.1371/journal.pone.0083742.g001 Figure 1. Flow chart of the population study. doi:10.1371/journal.pone.0083742.g001 As all French level III centers did not participate, main
characteristics of participating and non-participating NICUs were
compared. (13%) in the NIHT group (Figure 1). Table 2 shows the criteria
involved in this classification. By definition, the clinical context in
the group of newborns with NIHT is less severe than in the two
other groups. The mean GA of babies in the NIHT group was one
week lower (38.6w versus 39.3w), criteria suggesting asphyxia are
lacking in more than one half of these babies, and HIE is
considered as mild for 5 of them (12%). It should be noted that HT
was initiated before 6 hours for only 71% of them. The groups of
OHT and LHT are more comparable. About two thirds of
neonates in these two subgroups presented severe HIE. EEG was
more often performed in the group of OHT (55 versus 27%), and
abnormal (97 versus 80%). Nearly 12 hours were needed before
starting HT among newborns with LHT. Statistical Analysis Newborns were classed into three groups, OHT, LHT and
NIHT, as defined above. We compared the organizational factors
at birth, the newborns clinical and paraclinical characteristics, and
the conditions in which HT was performed. The Chi2 test (or
Fisher exact test if insufficient numbers) and Student’s t test or an
analysis of variance were used to analyze the qualitative and
quantitative variables respectively. The threshold of type 1 risk was
set at 5%. Statistical analysis was performed using STATA
software (Stata / IC 10.0 for Windows). Table 3 shows different factors (organization of care, temper-
ature at admission, and obstetrical circumstances) associated with
the different groups of HT. Inborn birth tended to be more
common in the OHT group. The size of the obstetric unit and the
frequency of birth outside office hours (18h-8H) were similar
between the three groups. The late initiation of HT in the LHT
group was the result of a long delay between admission and
treatment (nearly 8 hours compared to less than 1 hour in the
OHT group, p,10-3). The mean temperature at admission to
NICU was lower in the group of OHT compared with the two
others (34.9 versus 35.74 and 35.3). An acute obstetrical event was
more likely to be identified among newborns with LHT (46%)
compared to OHT (34%) and NIHT (22%). Nevertheless, apart
from uterine rupture, no other circumstance of delivery differed
significantly between the three groups. None of HIE in the OHT
and LHT groups were post-mature births, while 17% of HIE were
concerned. Results Among the 68 NICU in France, 33 (49%) participated. These
NICUs were more likely to be teaching Hospitals (75.8% versus
38.2%, p,1022), but did not differ for the size of the maternity
unit, the size of the neonatal unit or the type of intensive care
(pediatric and neonatal intensive care, or exclusively neonatal
intensive care) (data not shown). For the whole study population, the main characteristics are
presented in Table 1. The breakdown by severity of HIE into
grade of I, II, III was 2%, 64% and 34%, respectively. Information
regarding the intrapartum context associated with HIE was
missing in 35% (n = 108) of cases. When details were recorded, the
main complications were funicular pathologies (prolapse, circular
loop) for 25% of cases, and dystocia during delivery for 19% of
cases. A maternal infection was reported in 6% of cases. A life
threatening event (at between 15 and 120 min of life) was reported
for seven newborns. There was no difference in the method used to achieve HT (ice
packs, switching off the incubator) and in the duration of HT
between the groups (Table 4). There was a gradation in the rates Our present study population is composed of 311 neonates, 202
(65%) in the OHT group, 68 (22%) in the LHT group, and 41 December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org 3 Hypothermia for Neonates Table 1. General characteristics of the whole study population. Category
Subcategory
N
n (%) or mean +/– [SD]
Gestational age (weeks)
311
39.2 [1.5]
Birthweight (g)
311
3170 [547]
Male
311
163 (52.4)
Level of care in birth place
Home
311
4 (1.3)
Level I
311
74 (23.8)
Level II
311
125 (40.2)
Level III
311
106 (34.1)
No identified obstetrical event
311
108 (34.7)
Apgar ,5 at 5min
301
181 (60.1)
Severity of HIE
Mild
311
5 (1.6)
Moderate
311
198 (63.7)
Severe
311
108 (34.7)
EEG performed before hypothermia
301
168 (55.8)
Time to initiate hypothermia (hours)
308
5h41 [4h33]
Technique used to induce hypothermia
CriticoolH
311
129 (41.5)
TecothermH
311
90 (28.9)
Artisanal*
311
51 (16.4)
Me´dithermH
311
24 (7.7)
BlanketrollH
311
16 (5.1)
CoolcapH
311
1 (0.3)
Abnormal MRI
284
155 (54.6)
In-hospital mortality
310
63 (20.3)
Abnormal neurological examination among survivors
238
69 (29.0)
*: Switch off incubator or ice packs. Table 2. Criteria indicating HT according to HT subgroups. Table 2. g
doi:10.1371/journal.pone.0083742.t002 Results Criteria indicating HT according to HT subgroups. OHT (n = 202)
LHT (n = 208)
NIHT (n = 41)
Category
Sub category
n (%) or mean +/– [SD]
n (%) or mean +/– [SD]
n (%) or mean +/– [SD]
p
Gestational age (weeks)
39.3 [1.5]
39.3 [1.5]
38.6 [1.9]
*
Birthweight (g)
3201 [575]
3144 [475]
3073 [508]
ns
Asphyxia criteria
Apgar,5 at 5 min
129 (65.2)
43 (60.1)
9 (25.0)
***
pH cord ,7
98 (63.6)
24 (53.3)
7 (28.0)
**
Lactate in cord .11mmol/L
89 (64.5)
24 (53.3)
4 (15.4)
***
Base deficit in cord ,–16
15 (46.9)
5 (62.5)
2 (40.0)
ns
Ventilation
146 (72.3)
54 (79.4)
16 (39)
***
Intubation
172 (85.1)
53 (77.9)
24 (58.5)
***
Chest Compression
85 (42.1)
23 (33.8)
11 (26.8)
ns
Adrenalin
58 (28.7)
12 (17.6)
11 (26.8)
ns
Severity of HIE
Mild
0
0
5 (12.2)
***
Moderate
131 (64.9)
44 (64.7)
23 (56.1)
Ns
Severe
71 (35.1)
24 (35.32)
13 (31.7)
ns
EEG performed before hypothermia
111 (54.9)
18 (26.5)
14 (34.2)
***
Abnormal EEG
84 (96.5)
39 (79.6)
23 (88.5)
**
Age at start of hypothermia (h)
3h35 [1.36]
11h54 [3.21]
5h41 [1.10]
***
Hypothermia started before 6h
202 (100)
0
27 (71.1)
***
OHT: optimal hypothermia treatment, LHT: late hypothermia treatment, NIHT: non indicated hypothermia treatment, *** p,1023, **1023#p,1022, *1022#p,0.05,
ns : not significant. doi:10.1371/journal.pone.0083742.t002 December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org 4 Hypothermia for Neonates Table 3. Organization of care and birth circumstances according to HT subgroups. Discussion This study shows that for 35% of newborns with HIE and
treated with HT the conduct of the treatment is not consistent with
the guidelines published by the French Society of Neonatology. The main reason for non-compliance (62%) is a delay in the start
of treatment, beyond the 6 hours recommended. The second
reason is the lack of adherence to the clinical indications for HT
(38%). Apart from late transfer to the NICU, the main
characteristics of newborns in the SOHT group are reduced
severity of HIE and less frequent need for resuscitation in the
delivery room. Finally, the complications generally associated with
HT are significantly less frequent in the groups LHT and NIHT. In our study, HT was started at or over 6 hours of life for 22%,
and at or over 8 hours of life for 18% of all neonates. This rate is
higher than that in two recent studies evaluating the practice of
HT in the UK and in Belgium and The Netherlands, estimated at
about 5%, and at 1% of newborns between 8 and 12 hours of life
[9,26].However, the study design, inclusion criteria and/or the
characteristics of participating centers (centers with a high level of
awareness to HT due to their participation in large randomized
studies), might explain the disparities with our study which reflects
the daily practice of French NICUs. This study shows that for 35% of newborns with HIE and
treated with HT the conduct of the treatment is not consistent with
the guidelines published by the French Society of Neonatology. The main reason for non-compliance (62%) is a delay in the start
of treatment, beyond the 6 hours recommended. The second
reason is the lack of adherence to the clinical indications for HT
(38%). Apart from late transfer to the NICU, the main
characteristics of newborns in the SOHT group are reduced
severity of HIE and less frequent need for resuscitation in the
delivery room. Finally, the complications generally associated with
HT are significantly less frequent in the groups LHT and NIHT. The rates of death or abnormal neurological examination at
discharge were similar between the three groups. In contrast,
nearly 60% of newborns in the OHT group had an MRI showing
abnormalities compared to 44% and 49% in the LHT and NIHT
groups respectively. No difference in the type of abnormalities was
found. Results OHT (n = 202)
LHT (n = 208)
NIHT (n = 41)
Category
Sub category
n (%) or mean
+/– [SD]
n (%) or mean
+/– [SD]
n (%) or mean
+/– [SD]
p
Newborn characteristics
inborn
62 (30.7)
15 (22.1)
8 (19.5)
ns
male
102 (50.5)
37 (54.4)
24 (58.5)
ns
Temperature at admission to NICU (uC)
34.9 [1.4]
35.4 [1.4]
35.3 [1.4]
ns
Size of maternity
Less than1000 births/year
23 (11.4)
3 (4.4)
4 (9.8)
ns
1000 – 2000
0
0
1 (2.4)
ns
2000 – 3000
79 (39.1)
26 (38.2)
17 (41.5)
ns
3000 – 4000
76 (37.6)
28 (41.2)
14 (34.1)
ns
Over 4000
24 (11.8)
11 (16.2)
5 (12.2)
ns
Delay
Age at start of hypothermia (h)
3h35 [1.36]
11h54 [3.21]
5h41 [1.1]
***
Time between birth and admission (h)
2h54 [2.28]
3h36 [2.2]
3h17 [2.08]
***
Time between admission and start of HT
0h44 [2.43]
7h56 [5.28]
3h09 [3.5]
***
Obstetrical Circumstances
Funicular cause
34 (16.8)
11 (16.2)
6 (14.6)
ns
Dystocia
25 (12.4)
7 (10.3)
6 (14.6)
ns
Retro-placental hematoma
24 (11.9)
7 (10.3)
3 (7.3)
ns
Uterine rupture
22 (10.9)
2 (2.9)
1 (2.4)
*
Infection
8 (4.0)
2 (2.9)
3 (7.3)
ns
Velamentous insertion of the cord
7 (3.5)
1 (1.5)
2 (4.9)
ns
Feto-maternal hemorrhage
5 (2.5)
1 (1.5)
2 (4.9)
ns
Post-maturity
0
0
7 (17.1)
***
No causal circumstances found or reported
68 (33.7)
31 (45.6)
9 (22)
*
OHT: optimal hypothermia treatment, LHT: late hypothermia treatment, NIHT: non indicated hypothermia treatment, *** p,1023, **1023#p,1022, *1022#p,0.05,
ns : not significant,
doi:10.1371/journal.pone.0083742.t003 OHT: optimal hypothermia treatment, LHT: late hypothermia treatment, NIHT: non indicated hypothermia treatment, *** p,1023, **1023#p,1022, *1022#p,0.05,
ns : not significant,
doi:10.1371/journal.pone.0083742.t003 of adverse events and complications from the NIHT group to the
LHT group and the OHT group, although this increase did not
reach significance for adverse events. About one in five infants
suffered adverse events during HT. One half of newborns in the
OHT group presented at least one complication, the most
frequent being liver failure or cytolysis (28.7%), renal insufficiency
(24.8%), shock (24.3%), and pulmonary hypertension (11.4%). Three in ten newborns with NIHT suffered complication(s). No
significant difference was observed between the types of compli-
cations linked to HI in each subgroup. Results of adverse events and complications from the NIHT group to the
LHT group and the OHT group, although this increase did not
reach significance for adverse events. About one in five infants
suffered adverse events during HT. One half of newborns in the
OHT group presented at least one complication, the most
frequent being liver failure or cytolysis (28.7%), renal insufficiency
(24.8%), shock (24.3%), and pulmonary hypertension (11.4%). Three in ten newborns with NIHT suffered complication(s). No
significant difference was observed between the types of compli-
cations linked to HI in each subgroup. December 2013 | Volume 8 | Issue 12 | e83742 Discussion In our study, HT was started at or over 6 hours of life for 22%,
and at or over 8 hours of life for 18% of all neonates. This rate is
higher than that in two recent studies evaluating the practice of
HT in the UK and in Belgium and The Netherlands, estimated at
about 5%, and at 1% of newborns between 8 and 12 hours of life
[9,26].However, the study design, inclusion criteria and/or the
characteristics of participating centers (centers with a high level of
awareness to HT due to their participation in large randomized
studies), might explain the disparities with our study which reflects
the daily practice of French NICUs. Finally, we performed a sensitivity analysis consisting in adding
the mean time between admission and initiation of HT observed
among babies with complete data, when time to initiate HT was
unknown. Among the 16 cases concerned, 4 were initially
classified as NIHT, 1 as LHT and 11 as OHT. Under the
hypothesis tested as part of the sensitivity analysis, the first 5
children remained in the same group. In contrast, time to initiate
hypothermia became greater than 6 hours for 4 of the remaining
11 children, and these newborns were reclassified in the LHT
group. The corresponding rates of OHT and LHT were
respectively 64 (n = 198) and 23% (n = 72). This new distribution
did not affect our previous findings about the factors associated
with the different HT subgroups. Regarding adverse events associated with HT and complica-
tions linked to HI, our findings are similar to previous studies
[9,26]. It is difficult to distinguish those associated with the natural
history of HIE and those particularly related to HT. In our
opinion, the most severe side effects attributable to HT are
cysteatonecrose and persistent pulmonary hypertension. In our December 2013 | Volume 8 | Issue 12 | e83742 December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org 5 Hypothermia for Neonates Table 4. Characteristics of HT, adverse events, complications and short-term outcomes according to the different HT subgroups. Discussion OHT
(n = 202)
LHT (n = 208)
NIHT (n = 41)
Category
Sub category
n (%)
n (%)
n (%)
p
Procedural for HT
Hypothermia continued for 72h
180 (29.1)
58 (85.3)
36 (87.8)
ns
No special hypothermia equipment +
32 (15.8)
12 (17.6)
7 (17.1)
ns
Adverse events associated with HT
At least one adverse event
58 (29)
16 (23.5)
7 (17.1)
ns
Thrombopenia
16 (8)
6 (8.8)
4 (9.8)
ns
Hypomagnesemia
9 (4.5)
5 (7.4)
0
ns
Excessive cooling
12 (6)
5 (7.4)
0
ns
Cutaneous lesions
2 (1.0)
0
0
ns
Hemorrhage
10 (5.0)
3 (4.4)
0
ns
Complications linked to HI
At least one complication
105 (52.0)
36 (38.2)
12 (29.3)
**
Liver failure/cytolysis
58 (28.7)
13 (19.1)
6 (14.6)
ns
Shock
49 (24.3)
17 (25.0)
5 (12.2)
ns
Hypertension
15 (7.4)
2 (2.9)
1 (2.4)
ns
Renal insufficiency
50 (24.8)
10 (14.7)
5 (12.2)
ns
Pulmonary hypertension
23 (11.4)
7 (10.7)
3 (10.3)
ns
Hypotension
7 (3.5)
2 (2.9)
1 (2.4)
ns
Glucose intolerance
4 (2.0)
1 (1.5)
3 (7.3)
ns
Ileus
1 (0.5)
0
0
ns
Abnormal RMI
All lesions
110 (59.5)
27 (43.5)
18 (46.6)
ns
Basal Ganglia and/or cortical or subcortical
80 (43.2)
19 (30.6)
16 (43.2)
ns
White matter lesions apart from hemorrhagic petechial
43 (23.2)
12 (19.4)
9 (24.3)
ns
Isolated hemorrhagic petechial lesions
18 (9.7)
4 (6.4)
0
ns
Brainstem or cerebellar lesion
11 (5.9)
3 (4.8)
1 (2.7)
ns
In-hospital mortality
All causes
45 (22.3)
10 (14.9)
8 (19.5)
ns
Neurological cause without limitation of care
6 (3.0)
0
1 (2.4)
ns
Neurological cause with limitation of care
36 (17.8)
9 (13.4)
7 (17.1)
ns
Other cause
3 (1.5)
1 (1.5)
0
ns
Neurological examination for survivors
Normal at discharge from NICU
104 (68.9)
42 (75)
23 (74.2)
ns
Moderate at discharge from NICU
43 (28.5)
13 (23.2)
7 (22.6)
ns
Severe at discharge from NICU
4 (2.6)
1 (1.8)
1 (3.2)
ns
OHT: optimal hypothermia treatment, LHT: late hypothermia treatment, NIHT: non indicated hypothermia treatment, +Switch off incubator or ice packs, *** p,1023,
**1023#p,1022, *1022#p,0.05, ns : not significant. doi:10.1371/journal.pone.0083742.t004 and several studies have tested this solution. This strategy
sometimes obtains the target temperature of 33.5 u C, either by
passive or active HT [28]. December 2013 | Volume 8 | Issue 12 | e83742 References 10. Bergamasco B, Benna P, Ferrero P, Gavinelli R (1984) Neonatal hypoxia and
epileptic risk: a clinical prospective study. Epilepsia 25: 131–136. 1. Thornberg E, Thiringer K, Odeback A, Milsom I (1995) Birth asphyxia:
incidence, clinical course and outcome in a Swedish population. Acta Paediatr
84: 927–932. 11. Glass HC, Hong KJ, Rogers EE, Jeremy RJ, Bonifacio SL, et al. (2011) Risk
factors for epilepsy in children with neonatal encephalopathy. Pediatr Res 70:
535–540. 2. Kurinczuk J J, White-Koning M, Badawi N (2010) Epidemiology of neonatal
encephalopathy and hypoxic-ischaemic encephalopathy. Early Hum Dev 86:
329–338. 12. Lin Z-L, Yu H-M, Lin J, Chen S-Q , Liang Z-Q et al. (2006) Mild hypothermia
via selective head cooling as neuroprotective therapy in term neonates with
perinatal asphyxia: an experience from a single neonatal intensive care unit. J
Perinatol 26: 180–184. 3. Graham EM, Ruis KA, Hartman AL, Fox HE (2008) A systematic review of the
role of intrapartum hypoxia-ischemia in the causation of neonatal encephalop-
athy. Am J Obstet Gynecol 199: 587–595. 4. Yates HL, McCullough S, Harrison C, Gill AB (2012) Hypoxic ischaemic
encephalopathy: accuracy of the reported incidence. Arch Dis Child Fetal
Neonatal Ed 97: F77–78. 13. Eicher DJ, Wagner CL, Katikaneni LP, Hulsey TC, Bass WT et al. (2005)
Moderate hypothermia in neonatal encephalopathy: efficacy outcomes. Pediatr
Neurol 32: 11–17. 5. Pierrat V (2005) Prevalence, causes, and outcome at 2 years of age of newborn
encephalopathy: population based study. Arch Dis Child Fetal Neonatal Ed 90:
F257–261. 14. Inder TE, Hunt RW, Morley CJ, Coleman L, Stewart M, et al. (2004)
Randomized trial of systemic hypothermia selectively protects the cortex on
MRI in term hypoxic-ischemic encephalopathy. J Pediatr 145: 835–837. 6. Sarnat HB, Sarnat MS (1976) Neonatal encephalopathy following fetal distress. A clinical and electroencephalographic study. Arch Neurol 33: 696–705. 15. Gluckman PD, Wyatt JS, Azzopardi D, Ballard R, Edwards AD, et al. (2005)
Selective head cooling with mild systemic hypothermia after neonatal
encephalopathy: multicentre randomised trial. Lancet 365: 663–670. 7. Pin TW, Eldridge B, Galea MP (2009) A review of developmental outcomes of
term infants with post-asphyxia neonatal encephalopathy. Eur J Paediatr Neurol
13: 224–234. 16. Simbruner G, Mittal RA, Rohlmann F, Muche R (2010) Systemic hypothermia
after neonatal encephalopathy: outcomes of neo.nEURO.network RCT. Pediatrics 126: e771–778. 8. Azzopardi DV, Strohm B, Edwards AD, Dyet L, Halliday HL, et al. (2009)
Moderate hypothermia to treat perinatal asphyxial encephalopathy. Discussion There is however a risk of excessively
lowering HT to below 33.5 u C, which is limited by means of the
development of mobile devices. The drawback of this solution is to
compromise the interpretation of the first EEG [29], while in most
randomized trials about cooling, an electrophysiological exami-
nation before hypothermia is recommended so as confirm the
indication for HT, increasing thus avoiding the risk of over-
treatment. sample, these adverse events were rare, respectively 2 and 5 cases. Concerning the NIHT, the adverse events were comparable to
those of other groups. Among the 5 cases of mild HIE treated with
HT, no adverse events were observed and only one newborn
presented several complications. Most treatment guidelines recommend a delay of less than
6 hours between birth and the start of HT treatment. The
effectiveness of late treatment, initiated between 6 and 24 hours is
currently under study, but in the absence of known results, it
cannot be recommended at present [27]. The ways to reduce the
delay before starting HT remain unclear, particularly in the
context of an unpredictable acute perinatal pathology. Reducing
the time of transfer by ambulance to the NICU is probably
unfeasible in France. Nevertheless, doctors in charge of the
ambulance services should be reminded of the urgency to be given
to calls for neonatal neurological distress, to ensure rapid transfer
of the newborn to a level III NICU. The initiation of HT during
transport prior to arriving at the NICU is proposed by some teams Failure to recognize the indications for HT is the second reason
for non-adherence to the guidelines. In our study, there were 41
newborns (13.2%) for whom no clinical or biological signs of
perinatal asphyxia were recorded. Of these, the GA at birth was
,36 weeks for four, four others did not show all the neurological
signs of HIE, and for two newborns two or more of these three
criteria were missing. Some authors suggest that the indication for
HT should be extended to moderately premature infants (34–36 December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org 6 Hypothermia for Neonates be used for this study while it is crucial for determining the severity
of HIE and thus in classing the newborns. be used for this study while it is crucial for determining the severity
of HIE and thus in classing the newborns. Discussion weeks GA), or to infants with less severe HIE [30]. A feasibility
study of brain HT for preterms between 32 and 36 weeks is
ongoing [31]. Currently, the level of evidence does not justify an
extension of the indication. We observed that 46% of newborns
had no electrophysiological examination prior to HT. Difficulties
in performing and obtaining an interpretation of a standard EEG,
especially during the night may partly explain this result. Greater
dissemination of amplitude integrated EEG, which is simpler to
implement and interpret, should make it easier to obtain an
electrophysiological assessment before treatment [32]. Further-
more, the sensitivity and specificity in assessing the prognosis is
between 70 and 100% depending on the study [33,34]. Conclusions This is the first multicenter French study to look at the practice
of HT nationally. Although the results should be treated with
caution since our database is only declarative, it reveals non-
compliance with the guidelines in 35% of cases. These correspond
to excess treatments. Our results attest to the difficulty of
implementing the guidelines in clinical practice. This initial study
should prompt a better organization of perinatal care, in particular
to reduce the transportation time of newborns with HIE, as well as
the diffusion of mobile equipment allowing the early initiation of
HT in safe conditions. We also suggest that more systematic use of
amplitude EEG examinations in neonatal units could improve the
rate of electrophysiological assessment before HT treatment. Finally this audit of daily practice might prompt the widespread
use of this recent neuro-protective strategy, and improve the
awareness of HT among neonatologists. p
g
y
For 35% of our population, no perinatal circumstances that
could explain the intrapartum asphyxia are mentioned in the
database. This has already been observed in another French study
for 19% of newborns with stage II or III encephalopathy [5]. In
the randomized trial of Shankaran et al, a complication during
delivery was only noted in 68 cases among the 102 newborns
treated with HT [35]. This raises the question of the anoxic-
ischemic character of encephalopathy for some cases in our series. Moreover, the designation ‘‘HIE’’ is debated in the literature,
some authors preferring to use the term ‘‘neonatal encephalop-
athy’’ in view of the difficulty in proving an anoxic-ischemic cause
of neonatal neurological distress. Author Contributions Conceived and designed the experiments: AE TD. Performed the
experiments: MC. Analyzed the data: MC AE CC. Contributed
reagents/materials/analysis tools: MC AE TD. Wrote the paper: MC
AE TD. Conceived and designed the experiments: AE TD. Performed the
experiments: MC. Analyzed the data: MC AE CC. Contributed
reagents/materials/analysis tools: MC AE TD. Wrote the paper: MC
AE TD. Acknowledgments We thank Dr Alison Foote (Grenoble Clinical Research Centre) for
translating the manuscript. We thank all the participating neonatologists,
for their contribution to the data base : G. Krim (Amiens), S. Le Bouedec
(Angers), G Thiriez (Besanc¸on), J. Sizun (Brest), B Guillois (Caen),
?M Deiber (Chambery), V Zupan (Clamart), B Bœuf (Clermont Ferrand),
L Desfrere (Colombes), F Decobert (Cre´teil), C Chantegret (Dijon),
?M Moktari (Le Kremlin Biceˆtre), H Bruel (Le Havre), C Morisot (Lens),
?A Bedu (Limoges), O Claris (Lyon), L Colettto and JC Picaud (Lyon),
?V Millet (Marseille), I Rayet (St Etienne), G Cambonie (Montpellier),
?P Daoud (Montreuil), C Flamant (Nantes), JB Mariette (Nimes), PH
Jarreau (Paris), S Soudee (Paris), V Meau-Petit (Paris), JF Magny (Paris),
?A Beuchee (Rennes), S Marret (Rouen), P Bolot (St Denis), RP Dupuy (St
Brieuc), P Kuhn and D Astruc (Strasbourg), MO Marcoux (Toulouse),
?E Saliba (Tours), F Lapeyre (Valenciennes). A major limitation of our study is the declarative nature of the
database. It is likely that the population of newborns treated for
HIE is selected and not completely representative, but the aspects
influencing the selection and thus how they influenced our results
are undocumented. Therefore the SFN database does not
necessarily reflect the general practice of HT in France, as the
registration of all cases is still not achieved. To maximize the
number of reported cases, we contacted all the centers in
December 2011. For the 26 most recently reported cases, most
(n = 20) were classified in the OHT group suggesting some
improvement in practice over time. Our management of missing
data may have generated a classification bias. Concerning the time
to initiate HT, we have checked through the sensitivity analysis to
ensure that our strategy, which potentially underestimated this
delay, was not likely to change our findings. As electrophysiology
results were not available for 46% of cases, this criterion could not Hypothermia for Neonates 18. Jacobs S, Hunt R, Tarnow-Mordi W, Inder T, Davis P (2007) Cooling for
newborns with hypoxic ischaemic encephalopathy. Cochrane Database Syst Rev
CD003311. 28. O’Reilly D, Labrecque M, O’Melia M, Bacic J, Hansen A, et al. (2013) Passive
cooling during transport of asphyxiated term newborns. J Perinatol 33: 435–440. 29. Azzopardi, D. on behalf of the TOBY study group (2013) Predictive value of the
amplitude integrated EEG in infants with hypoxic ischaemic encephalopathy:
data from a randomised trial of therapeutic hypothermia. Arch Dis Child Fetal
Neonatal Ed Jun 25. [Epub ahead of print] 19. Jacobs S, Hunt R, Tarnow-Mordi W, Inder T, Davis P (2003) Cooling for
newborns with hypoxic ischaemic encephalopathy. Cochrane Database Syst Rev
CD003311. Neonatal Ed Jun 25. [Epub ahead of print] 20. Shah PS (2010) Hypothermia: a systematic review and meta-analysis of clinical
trials. Semin Fetal Neonatal Med 15: 238–246. 30. Austin T, Shanmugalingam S, Clarke P (2012) To cool or not to cool? Hypothermia treatment outside trial criteria. Available: http://fn.bmj.com/cgi/
doi/10.1136/archdischild-2012-302069. Accessed 25 March 2013. 21. Tagin MA, Woolcott CG, Vincer MJ, Whyte RK, Stinson DA (2012)
Hypothermia for Neonatal Hypoxic Ischemic Encephalopathy: An Updated
Systematic Review and Meta-analysis. Arch Pediatr Adolesc Med 166: 558–566. 31. Walsh W (2013) Pilot study of head cooling in preterm infants with hypoxic-
ischaemic encephalopathy. Available: http://clinicaltrials.gov/ct2/show/
NCT00620711. Accessed 11 April 2013. 22. Saliba E, Debillon T (2010) Hypothermia for hypoxic-ischemic encephalopathy
in fullterm newborns. Arch Pediatr 17: S67–77. 32. Tao JD, Mathur AM (2010) Using amplitude-integrated EEG in neonatal
intensive care. J Perinatol 30: S73–S81. 23. Strohm B, Hobson A, Brocklehurst P, Edwards AD, Azzopardi D (2011)
Subcutaneous fat necrosis after moderate therapeutic hypothermia in neonates. Pediatrics 128: e450–452. 33. Shellhaas RA, Soaita AI, Clancy RR (2007) Sensitivity of Amplitude-Integrated
Electroencephalography for Neonatal Seizure Detection. Pediatrics 120: 770–
777. 24. Pfister RH, Bingham P, Edwards EM, Horbar JD, Kenny MJ, et al. (2012) The
Vermont oxford neonatal encephalopathy registry: rationale, methods, and
initial results. BMC Pediatrics 12: 84. 34. Spitzmiller RE, Phillips T, Meinzen-Derr J, Hoath SB (2007) Amplitude-
Integrated EEG Is Useful in Predicting Neurodevelopmental Outcome in Full-
Term Infants With Hypoxic-Ischemic Encephalopathy: A Meta-Analysis. J Child Neurol 22: 1069–1078. 25. Amiel-Tison C (2002) Update of the Amiel-Tison neurologic assessment for the
term neonate or at 40 weeks corrected age. Pediatr Neurol 27: 196–212. 26. References N Engl J
Med 361: 1349–1358. 17. Shankaran S, Laptook A, Wright LL,Ehrenkranz RA, Donovan EF, et al. (2002)
Whole-body hypothermia for neonatal encephalopathy: animal observations as a
basis for a randomized, controlled pilot study in term infants. Pediatrics 110:
377–385. 9. Azzopardi D, Strohm B, Edwards AD, Halliday H, Juszczak E, et al. (2009)
Treatment of asphyxiated newborns with moderate hypothermia in routine
clinical practice: how cooling is managed in the UK outside a clinical trial. Arch
Dis Child Fetal Neonatal Ed 94: F260–264. December 2013 | Volume 8 | Issue 12 | e83742 December 2013 | Volume 8 | Issue 12 | e83742 7 PLOS ONE | www.plosone.org Hypothermia for Neonates Hypothermia for Neonates Groenendaal F, Casaer A, Dijkman KP, Gavilanes AW, de Haan TR et al. (2013) Introduction of hypothermia for neonates with perinatal asphyxia in the
Netherlands and Flanders. Neonatology. 104: 15–21. 35. Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, Mc Donald SA, et al. (2005) Whole-body hypothermia for neonates with hypoxic-ischemic encepha-
lopathy. N. Engl. J. Med. 353: 1574–1584. gy
27. Laptook A (2013) Late hypothermia for hypoxis-ischaemic encephalopathy. Available: http://clinicaltrials.gov/ct2/show/NCT00614744. Accessed 11 April
2013. December 2013 | Volume 8 | Issue 12 | e83742 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 8
|
https://openalex.org/W2512510278
|
https://durham-repository.worktribe.com/preview/1375045/19768.pdf
|
English
| null |
Social Media and Social Work: The Challenges of a New Ethical Space
|
Australian social work
| 2,016
|
cc-by
| 8,174
|
Dr Jennifer Boddy
Menzies Health Institute
School of Human Services and Social Work
Griffith University, Gold Coast Campus
Parklands Drive, Southport QLD 4222, Australia
Email: j.boddy@griffith.edu.au
Telephone: +61 7 5552 7396
Dr Lena Dominelli
Professor in the School of Applied Social Sciences
University of Durham, Durham, United Kingdom.
Email: lena.dominelli@durham.ac.uk
Telephone: + 44 191 334 2000 Dr Jennifer Boddy
Menzies Health Institute
School of Human Services and Social Work
Griffith University, Gold Coast Campus
Parklands Drive, Southport QLD 4222, Australia
Email: j.boddy@griffith.edu.au
Telephone: +61 7 5552 7396 Dr Lena Dominelli
Professor in the School of Applied Social Sciences
University of Durham, Durham, United Kingdom. Email: lena.dominelli@durham.ac.uk
Telephone: + 44 191 334 2000 Dr Lena Dominelli
Professor in the School of Applied Social Sciences
University of Durham, Durham, United Kingdom. Email: lena.dominelli@durham.ac.uk
Telephone: + 44 191 334 2000 1 1 Abstract Social media and other online technologies have transformed communication between social
workers and service users, with many practitioners engaging and working with clients
through social networking sites. While it is readily agreed that there are numerous ethical
issues associated with online practice, such as those related to confidentiality, dual
relationships, and boundary crossing, there is a lack of clarity about how to deal with such
issues. Consequently, this paper draws from a case example to develop a nuanced
understanding of ethical issues and ethical behaviour in online spaces. We argue that social
workers need to develop their knowledge of the complex interplay between discourses such
as those related to power, permanency, authorship, audience, embodiment, and
professionalism because these have understandings that underpin daily practice. Social
workers must also remain committed to ethical values and critical reflective practice. We
conclude with recommendations for education, research and practice. Key words: Social media, social networking, ethics, social work practice Key words: Social media, social networking, ethics, social work practice 2 2 Social work has only recently begun examining the use of social media and other
online technologies in social work practice. Online technologies have “crept” into social
work practice and revolutionised communication between practitioners and service users
(Mishna, Bogo, Root, Sawyer, & Khoury-Kassabri, 2012, p. 283). Social workers make use
of online, video, and telephone therapy, as well as text messaging, email and social
networking sites for connecting with clients and colleagues (Reamer, 2013). This
transformation of practice has raised a number of ethical issues. Reamer (2013) identifies
concerns related to confidentiality, privacy, informed consent, conflicts of interest, dual
relationships, boundary crossing, service termination, documentation, and research evidence
(or lack thereof) (see also Fange, Mishna, Zhang, Van Wert, & Bogo, 2014). While
practitioners have readily identified ethical issues with online mediums, they are not always
clear about how to deal with them (Mishna et al., 2012). Further, it would seem that many
social work students are unaware of the ethical issues and dilemmas that can arise in online
communication and the importance of maintaining professional behaviour and boundaries in
online spaces (Mukherjee & Clark, 2012). Social work professional associations have responded to concerns about online ethical
issues by preparing guidelines for use with social media and other technologies. Abstract For example,
the British Association of Social Work released a policy statement in 2012 that “encourages
the positive uses of social media, to which social workers should apply the values and
principles of the Code of Ethics” (The Policy Ethics and Human Rights Committee, 2012, p. 10). The Australian Association of Social Workers (AASW) have updated their practice
standards to state that social workers must identify “ethical considerations with respect to
using online communication and social media” (AASW, 2013a, p. 15) and published
guidelines on social networking and online service provision (AASW, 2013b, 2014). In the
United States, the National Association of Social Work (NASW) and the Association of 3 3 Social Work Boards (ASWB) set standards for technology use ten years ago (ASWB, 2005). These centred on cultural and technical competence, privacy and confidentiality, and
documentation and risk management. These guidelines have yet to be updated, despite the
significant changes in online communication since then. A major problem, according to
Voshel and Wesala (2015), is that “practice standards continue to lag far behind the rapid
growth of online social media” (p. 68) and leaves a gap to be filled. To date, scholars providing guidance on ethical issues in online practice arenas have
relied on existing, and sometimes dated, codes of ethics. This has meant that there is no
comprehensive contemporary discussion of the complexities and interrelationships between
social media, social work practice, and social work ethics. A more nuanced understanding of
ethics in online spaces is needed. Consequently, this article adds to the emerging body of
literature on social work, social media and ethics by highlighting broader issues pertaining to
social media and their intersection with social work values and practice realities. We begin by
highlighting the opportunities and dangers associated with social media, before drawing from
a case example (described below) to extrapolate professional issues inherent in social media. We conclude with suggestions for promoting social justice in online domains. Opportunities and dangers The growth of social media has come with huge benefits for individuals, groups, The growth of social media has come with huge benefits for individuals, groups,
organisations, communities, and businesses. People can now develop new friendships,
maintain old friendships, establish a small business, connect with others, and keep abreast of
research and current affairs more easily. Social media has allowed adopted children and
children in care to make contact with birth parents (Greenhow, 2015). Communication has
never been easier for a global audience within instantaneous reach, e.g., Social Work Without
Borders (see Social Dialogue, August 2015). Health departments, fire, police, ambulance, 4 4 and other essential services can quickly issue warnings to a wide audience through
information technologies (Alexander, 2014). Evidence also suggests that young men who
speak online to friends about personal problems are more likely to have higher levels of
mental wellbeing than those who do not (Best, Manktelow, & Taylor, 2014). Social media
can promote open dialogue with collaborative reflections (Friesen & Lowe, 2012),
democratic participation and engagement in politics (Bertot, Jaeger, & Hansen, 2012),
coordinate successful political action (see Shirkey, 2011), strengthen relationships (Ellison,
Steinfield, & Lampe, 2007), and be inclusive (Bertot et al., 2012). emocratic participation and engagement in politics (Bertot, Jaeger, & Hansen, 2012), However, there are dangers. While social media can empower individuals, it can also
empower trollers, stalkers, and predators, as numerous reports of paedophiles using social
media to access victims (Kim, Jeong, & Lee, 2010) or of children and young people being
bullied online (O'Keeffe & Clarke-Pearson, 2011) exemplify. The speed at which posts can
go viral can contagiously affect others in harmful ways (Fu, Cheng, Wong, & Yip, 2013). However, there are dangers. While social media can empower individuals, it can also
empower trollers, stalkers, and predators, as numerous reports of paedophiles using social
media to access victims (Kim, Jeong, & Lee, 2010) or of children and young people being
bullied online (O'Keeffe & Clarke-Pearson, 2011) exemplify. The speed at which posts can
go viral can contagiously affect others in harmful ways (Fu, Cheng, Wong, & Yip, 2013). Further, regimes have tightened their control on social media when political uprisings have
been unsuccessful (Shirkey, 2011). Social media has been used to promote terrorist acts and
disseminate rumours in disaster situations (Alexander, 2014). Opportunities and dangers Such misuse of these
communication tools have led to calls for detailed increased surveillance of citizens and their
online communications, with Edward Snowden revealing in 2013 that both Britain and the
United States had indulged in widespread surveillance of private communications. The challenge for social work is to use the benefits and opportunities which social
media enables, without causing harm. Using social media requires new ways of thinking
about and reflecting upon everyday activities. The following sections explore the
complexities of social media and assist social workers in developing a more nuanced
understanding of this area of practice. 5 5 Conceptualising social work, social media, values and ethics Social media complicates social work practice in a way not previously witnessed. It is no
longer possible to understand the impact of social media and the ethical issues that arise from
it in simple, binary or linear ways. As shown in Figure 1, the social contexts in which Social media complicates social work practice in a way not previously witnessed. It is no
longer possible to understand the impact of social media and the ethical issues that arise from
it in simple, binary or linear ways. As shown in Figure 1, the social contexts in which
communications occur are crucial in comprehending its usage. We highlight that social media
and social work practice occur in a neoliberal context which privileges technology, financial
power and a collapse of time and space (Virilio, 2000). Social workers must remain
committed to their ethical values (as stated in previous literature), and practice in a critically
reflective manner. Figure 1 indicates that social workers need to develop their knowledge of
the complex interplay between a range of discourses, such as those related to embodiment
and disembodiment, power and empowerment, permanence and impermanence, and underpin
their daily practice with these understandings. INSERT FIGURE 1 HERE We discuss each of these discourses through the case study below. It has been
compiled by drawing upon real-life examples shared online, research, and stories offered by
other people. 6 6 Case study: Mary, William and Adam parenting abilities and confidence, making her increasingly dependent on him. The
abuse escalates and Adam uses social media to distribute and sell abusive material to
people who pay increasing amounts for the degree of abuse inflicted on William. Mary’s friends online, including Mary’s former social worker, are concerned that This case study raises important questions for both social workers and users of social media
more generally, but especially parents of young children. These can be considered in terms
of: macro-level contexts; online ethical issues; and practice considerations. The ethical issues
inherent in social media and highlighted in this case study are influenced by concepts related
to abusive interactions, privacy, empowerment, authorship, permanence, embodiment,
professionalism, and consequences. Practitioners need a solid understanding of each of these
elements, along with a commitment to the values and ethics of the profession and exceptional
skills in critical thinking. We discuss these in turn below. This case study raises important questions for both social workers and users of social media
more generally, but especially parents of young children. These can be considered in terms
of: macro-level contexts; online ethical issues; and practice considerations. The ethical issues
inherent in social media and highlighted in this case study are influenced by concepts related
to abusive interactions, privacy, empowerment, authorship, permanence, embodiment, professionalism, and consequences. Practitioners need a solid understanding of each of these
elements, along with a commitment to the values and ethics of the profession and exceptional
skills in critical thinking. We discuss these in turn below. Case study: Mary, William and Adam Case study: Mary, William and Adam Mary is a 23 year-old, single parent mother, recently separated, and has given birth to
a son, William. Mary grew up in out-of-home foster care in a rural town, but moved to
the city when she turned 18. She has limited money, has no contact with her ex-
partner and father of William, and is socially isolated. She does, however, have a
strong network of friends on Facebook, which includes her former social worker, who
she connects with online frequently. Mary wants to show that she is a good mother and she does this, in part, by
posting lots of status updates, profile picture updates, and pictures which include both
herself and William. Mary is unconcerned about the safety risks posed by posting
photos online because she has set her security settings quite high. When William turns one, Mary posts a status update celebrating his birthday. Her close friend Emily shares this update with her networks and adds the comment
‘time to party’. Shortly afterwards, Mary receives a ‘friend’ request from Adam, who
is a friend of Emily’s on Facebook. Mary accepts the request because she trusts
Emily’s judgement about who she would connect with online and likes Adam’s
profile picture. Adam and Mary begin conversing online. When William is fourteen
months old, Mary and Adam run into each other at a park. Mary is unaware that Adam
has located Mary via a geotagging platform where Mary has ‘checked-in’ at her
location. Soon after, Mary and Adam start dating and two months later Adam moves
in. Mary is happy to be in a relationship with someone who is caring and she
appreciates how kind Adam is to William. 7
Over time Adam erodes Mary’s social networks and begins controlling her
online activities and face-to-face meetings with friends. Mary is unaware that Adam
has begun to sexually abuse William. At the same time, he is undermining Mary’s 7 parenting abilities and confidence, making her increasingly dependent on him. The
abuse escalates and Adam uses social media to distribute and sell abusive material to
people who pay increasing amounts for the degree of abuse inflicted on William. Mary’s friends online, including Mary’s former social worker, are concerned that
Mary’s engagement online diminishes overtime. They continue to post comments on
her Facebook page in an attempt to connect with her better, without success. Neoliberalism The growth of social media and online communication technologies have emerged in a
context of neoliberalism; an ideology grounded in the belief that market forces are the driving
principle in all social, political and economic decisions (Giroux, 2005). Neoliberalism results
in: the loss of public spaces, a diminution of government-funded institutions, blindness to
unregulated market competition, freedom for capitalists to move their assets around the globe,
interpersonal relationships based on market individualism (Bauman in Wallace & Pease,
2011), and shrinkage of time and space and acceleration of the speed with which things 8 8 happen (Virilio, 2000). According to Giroux (2005) “under neoliberalism everything either is
for sale or is plundered for profit” (p. 2). Products and services are designed to maximise
profits and minimise costs. Corporations dominate not only economics, but also social and
political life and produce commodified relationships, communication, and services
(Dominelli, 2007), with citizenship becoming a function of consumerism (Giroux, 2005). Neoliberalism dominates almost every area of people’s lives and “has changed the
relationship between the individual citizens and the state, individuals and their social and
physical environments” (Dominelli, 2007, p. 32). In many ways, capitalism has driven
technological progress (Nelson, 1990) and has had overwhelming influence on the creation
and use of social media. It has made social media ubiquitous and cheaply available
everywhere. And despite its potential for control over individuals, it enables people to
connect with large audiences quickly. It also provides opportunities for individuals to abuse
and exploit other individuals, especially sexually and financially. 9
In the case study, photographs of the abuse of William are disseminated online for
profit using a social networking site. This site, like many, allows for the commodification and
marketization of human suffering inherent in a neoliberal society. Many websites rely on
clickbait (a term used to describe online content that generates advertising income by enticing
web users to view the content). In the abuse of William, Adam sells abusive photos for profit. Such transactions expose how the free market drives demand for abusive material. Additionally, the secrecy offered by the web has enabled William’s abuser, Adam, to
maintain his privacy to avoid being found out, although he had to take the precaution, as
many perpetrators of sexual abuse do, of isolating Mary, betraying her trust, and making her
dependent upon him (Dominelli, 1989). Neoliberalism Many people trust social media sites to look after
their interests, yet with limited safeguards in place and in the context of a dominating
neoliberal culture, safety comes second to profit. Individuals are expected to take care of their
own security, with providers being reluctant to intervene quickly (O'Brien, 2014). While this In the case study, photographs of the abuse of William are disseminated online for
profit using a social networking site. This site, like many, allows for the commodification and
marketization of human suffering inherent in a neoliberal society. Many websites rely on
clickbait (a term used to describe online content that generates advertising income by enticing
web users to view the content). In the abuse of William, Adam sells abusive photos for profit. Such transactions expose how the free market drives demand for abusive material. 9
Such transactions expose how the free market drives demand for abusive material. Additionally, the secrecy offered by the web has enabled William’s abuser, Adam, to
maintain his privacy to avoid being found out, although he had to take the precaution, as
many perpetrators of sexual abuse do, of isolating Mary, betraying her trust, and making her
dependent upon him (Dominelli, 1989). Many people trust social media sites to look after
their interests, yet with limited safeguards in place and in the context of a dominating
neoliberal culture, safety comes second to profit. Individuals are expected to take care of their
own security, with providers being reluctant to intervene quickly (O'Brien, 2014). While this 9 may be changing, (e.g., the work with Facebook the National Society for the Prevention of
Cruelty to Children is doing in the UK), social media, allows people like Adam to empower
themselves with limited recourse for victim-survivors or their families. Social workers who
are aware of the potential for harm and exploitation that the web offers those wishing to
perpetrate violence against others can exercise vigilance and explore matters further if they
begin to suspect that an individual’s pattern of behaviour is changing without apparent
reason. In William’s case, the social worker could have asked to meet Mary, or gone to her
house to see what had happened when she stopped responding online. Embodiment and disembodiment Social media provides users with the ability to form communities, share information, connect
with others, and socialise (Bertot et al., 2012). Online relationships and interactions become
both embodied and contextualised (van Doorn, 2011). They are informed by and inform
offline relationships, behaviours and events. Essentially, material moves from physical spaces
to digital spaces and back again. Thus, ‘everyday (inter)actions are materialized in digital
space’ (van Doorn, 2011, p. 538). This can blur the boundaries between virtual reality and
physical reality, and create ‘lived-in spaces’ that acquire meaning and significance for
individual(s). However, while interactions online may be embodied with congruence between
mind and body, the user cannot see the reactions of others and is unable to get immediate
feedback from them. This produces an element of disembodiment associated with online
interactions and causes the user to be unclear about how another person will receive the
information that has been posted. It can be difficult to predict the outcome of a particular
comment. Thus, social media can create a sense of connection and disconnection
simultaneously. 10 Mary’s friends care deeply about her and actively seek to connect with her online. Their relationship with her is embodied. Yet, because the relationship is mediated by social
media and a digital or disembodied space, they are unable to transcend its limitations and
fully understand Mary’s circumstances and the abuse she is experiencing. Because Mary’s
suffering is invisible to them, they feel disempowered and unable to ask Mary what is
troubling her. Hence, an element of silencing accompanies the medium. Yet, while some
voices are silenced, others, such as Adam’s are amplified through their control of the media. The embodied nature of online interactions is exemplified in the manner in which Mary first
met Adam through online chats. However, the disembodied nature of online communications
means that many social media users will seek to meet outside of the digital realm, as Mary
and Adam did. Having established the basis of trust online, Mary did not have full access to
the signals that might have made her more wary of entering into a relationship. tended and unintended consequences Intended and unintended consequences
While there are often intended and unintended consequences for any actions taken, these may
become amplified online. Many of these relate to privacy, empowerment, or lack thereof
online, and permanency. Social media allows users to reach a large audience irrespective of
their intention to do so. This can be valuable when promoting positive change, but can also be
damaging. The presence of ‘digital dirt’, for example, can have unforeseeable negative
consequences, particularly for children and young people (O'Keeffe & Clarke-Pearson, 2011). 11
q
While there are often intended and unintended consequences for any actions taken, these may
become amplified online. Many of these relate to privacy, empowerment, or lack thereof
online, and permanency. Social media allows users to reach a large audience irrespective of
their intention to do so. This can be valuable when promoting positive change, but can also be
damaging. The presence of ‘digital dirt’, for example, can have unforeseeable negative
consequences, particularly for children and young people (O'Keeffe & Clarke-Pearson, 2011). Mary did not foresee how the disclosure of personal information online put her and her son at
risk of abuse because a knowledgeable user would be able to locate her. Furthermore, it is
unlikely that her friend Emily thought through the potential consequences of friending Adam
online. Social workers need to become more aware of unintended consequences of online
behaviour and exhibit greater consideration about how material may be received by the
intended (or unintended) audience and used to abuse people who are vulnerable. Social 11
Mary did not foresee how the disclosure of personal information online put her and her son at
risk of abuse because a knowledgeable user would be able to locate her. Furthermore, it is
unlikely that her friend Emily thought through the potential consequences of friending Adam
online. Social workers need to become more aware of unintended consequences of online
behaviour and exhibit greater consideration about how material may be received by the
intended (or unintended) audience and used to abuse people who are vulnerable. Social 11 11 workers need to be careful not to act unethically because they did not check someone or
something out. workers need to be careful not to act unethically because they did not check someone or
something out. Professionalism and non-professionalism
Many practitioners utilise social media to publicise professional services (Ahmed et al.,
2013). tended and unintended consequences Social media enhances their capacity for career building by marketing oneself through
self-branding to promote themselves as employable and professional (Gershon, 2014). This is
important for job-seekers, as many employers check a job applicant’s personal websites and
social media postings (Toten, 2014) and use social networking sites for recruitment
(Schawbel, 2012). Professionalism and non-professionalism y practitioners utilise social media to publicise professional services (Ahmed et al., However, there have been instances where employees, including some in the health
and social services, have lost their job due to social media misuse or privacy breaches. Many
practitioners have not considered the impact of their online material on service users
(Greyson, Kind, & Chretien, 2010). Their failure to do so can pose risks to them individually,
their profession, and service users (Bickhoff, 2014). For example, a social worker was
sanctioned by the Health and Care Professionals Council in the UK after a mother involved in
a court case searched for the social worker on the internet and found the social worker's
publicly available Facebook page contained a passage where she had described her glee at the
mother's children being removed (Stevenson, 2014). 12
In social work, it is often unclear what is permissible and what is not in online spaces. Mishna et al. (2012) refers to this as the ‘ethical grey zone’. In the case study, one of Mary’s
online friends is her former social worker, which in contexts like out-of-home foster care can
be important for service users where connections with former workers helps maintain
continuity and is valued by service users (Dominelli, 2005). However, in the case study, the
social worker may inadvertently become complicit in William’s abuse through inaction. She
failed to examine the reasons behind Mary’s reduced contact, and has missed her abuse as a 12 mother and young woman. Moreover, by not following up on Mary, the social workers misses
a potential opportunity to pick up on William’s abuse. The social worker’s inaction in the
nebulous spaces of online reality raises questions of culpability alongside issues about fitness
to practice. Social workers thus need to consider the implications of online behaviour
carefully and get the support of their professional associations to do so. Single and multiple authorships g
p
p
The boundaries between author and reader have become unclear with the rise of social media
(Zeng, Chen, Lusch, & Li, 2010). Its collaborative and participatory nature denies people of
sole authorship of their life stories (Bertot, Jaeger, & Grimes, 2010). Instead, these are often
developed through a compilation of the views of many people packaged as one profile, with
status updates and tweets being repeatedly shared, modified and reposted (Murthy, 2012). Thus “every new medium affects who and how many people can be the author of a
statement” (Gershon, 2014, p. 283). This can result in a lack of consent by specific authors
when there are different authors, and an expanding authorship which has no explicit limits. The original author often loses control of the material and may be unaware of what someone
might do with the information posted online. In Mary’s case, she posted a status update that
was reposted and embellished by her friend Emily that was subsequently read not only by
Mary’s intended audience, but by people in Emily’s online network, which included a child
abuser who was not known as such. Mary thus lost control about who viewed her post and
how it was conveyed. Material is repeatedly shared online by Mary and Adam about William. His life story, in a sense, is being authored and co-authored by others, a process in which he
has no input, raising questions not only about authorship, but about consent and power. The boundaries between author and reader have become unclear with the rise of social media
(Zeng, Chen, Lusch, & Li, 2010). Its collaborative and participatory nature denies people of
sole authorship of their life stories (Bertot, Jaeger, & Grimes, 2010). Instead, these are often
developed through a compilation of the views of many people packaged as one profile, with
status updates and tweets being repeatedly shared, modified and reposted (Murthy, 2012). Thus every new medium affects who and how many people can be the author of a
statement” (Gershon, 2014, p. 283). This can result in a lack of consent by specific authors
when there are different authors, and an expanding authorship which has no explicit limits. The original author often loses control of the material and may be unaware of what someone
might do with the information posted online. Public and private spaces 14
p
p
The boundaries between public and private spaces are blurred online (Strauß & Nentwich,
2013). Users of social media sites often have to agree to terms and conditions that allow for
surveillance, data mining, and target marketing, with applications (apps) retaining users
details, conversations, and material they have shared privately (Reyman, 2013). This blurring
of boundaries differs from that experienced in daily life routines when private woes are
turned into public issues so that they can be investigated and the personal domain can be
overtly politicised, as in the feminist slogan, the ‘personal is political’ (Dominelli, 2002). In
online transgressions of the private-public divide, it is done surreptitiously as a condition of
accessing a particular site or service, with social media users giving little thought to the terms
of agreement. Standards expected by one person sharing something privately can easily be
violated by another person who shares something publicly (Grodzinsky & Tavani, 2010), as
occurred to Mary, who assumed that her friend would only share materials with bona fide
friends that she trusted. According to Alexander (2014) this is “part of a broad trend towards
the gradual abandonment of personal discretion and increasing tendency to share intimate
details” (p. 728). The erosion of privacy remains largely invisible, while the maintenance of
privacy can be at the expense of others. For example, the parents of 15 year old Eric Rash
who committed suicide were denied access to his emails and Facebook accounts (Boyle,
2013) and had to resort to the courts to acquire permission to do so. Thus, there are
considerable challenges that social workers must be aware of related to privacy, security,
discretion, respect, data management, and accessibility. Mary believed her data was safe
online because she had established high privacy settings. Spaces which are often viewed as
private can be very public and technically knowledgeable individuals can subvert privacy
settings. Additionally, her profile picture was still publicly available, information she shared
was readily shared with others, and it is likely her online data would be retained for the
purposes of marketing, data mining, and other surveillance purposes. The blurring between The boundaries between public and private spaces are blurred online (Strauß & Nentwich,
2013). Single and multiple authorships In Mary’s case, she posted a status update that
was reposted and embellished by her friend Emily that was subsequently read not only by
Mary’s intended audience, but by people in Emily’s online network, which included a child
abuser who was not known as such. Mary thus lost control about who viewed her post and
how it was conveyed. Material is repeatedly shared online by Mary and Adam about William. His life story, in a sense, is being authored and co-authored by others, a process in which he
has no input, raising questions not only about authorship, but about consent and power. 13 Public and private spaces Public and private spaces Users of social media sites often have to agree to terms and conditions that allow for
surveillance, data mining, and target marketing, with applications (apps) retaining users
details, conversations, and material they have shared privately (Reyman, 2013). This blurring
of boundaries differs from that experienced in daily life routines when private woes are
turned into public issues so that they can be investigated and the personal domain can be
overtly politicised, as in the feminist slogan, the ‘personal is political’ (Dominelli, 2002). In
online transgressions of the private-public divide, it is done surreptitiously as a condition of
accessing a particular site or service, with social media users giving little thought to the terms
of agreement. Standards expected by one person sharing something privately can easily be
violated by another person who shares something publicly (Grodzinsky & Tavani, 2010), as
occurred to Mary, who assumed that her friend would only share materials with bona fide
friends that she trusted. According to Alexander (2014) this is “part of a broad trend towards
the gradual abandonment of personal discretion and increasing tendency to share intimate
details” (p. 728). The erosion of privacy remains largely invisible, while the maintenance of
privacy can be at the expense of others. For example, the parents of 15 year old Eric Rash
who committed suicide were denied access to his emails and Facebook accounts (Boyle,
2013) and had to resort to the courts to acquire permission to do so. Thus, there are
considerable challenges that social workers must be aware of related to privacy, security,
discretion, respect, data management, and accessibility. Mary believed her data was safe
online because she had established high privacy settings. Spaces which are often viewed as
private can be very public and technically knowledgeable individuals can subvert privacy
settings. Additionally, her profile picture was still publicly available, information she shared 14 public and private boundaries raises important questions: What could her former social
worker have done to alert Mary to these possible dangers when she became her ‘Facebook
friend’? Given that her formal professional relationship had ended, what responsibility did
she have for Mary, given her vulnerability as a mother of a young child? Did she have any
responsibility towards William, given child protection considerations? Where should the
professional boundary lie? Who will determine ensuing dilemmas, and how? Permanence and impermanence Social media carries with it both a sense of permanence and impermanence: permanence in
that users leave behind evidence of the sites they have visited and impermanence due to the
speed at which current information supersedes previous data. Users often have little say in
what information is retained permanently online. Once material is posted, it can stay online
indefinitely. Further, such posts are often made in real time (Bertot et al., 2012), making the
speed of the transfer of information as provided by contemporary telecommunications
technologies contribute to a kind of pollution known as a ‘grey ecology’. Virilio (2010)
argues that “the pollution of time and distance is much more severe… than the pollution of
material substances” (p. 13). The material posted online about William may well retain a place on the internet
throughout his lifetime and become permanent. Further, the haste in which posts are made by
Mary allow little time for reflection about unintended consequences. Actions taken online
have both immediate and long term effects and can be difficult to permanently remove. Social
workers need to be aware that discourses related to power, authorship, and consequences have
a time dimension. Actions one day can unwittingly affect the future, without the possibility of
redress. Practice considerations Criticality, values and ethics Power and disempowerment Social media can be empowering to users when it breaks down hierarchical structures
(Castells, 2009) and gives users a platform to broadcast their views to a potentially large
audience. It can also promote openness and transparency in government, reducing corruption
and allowing users to monitor government activity (Bertot et al., 2010). However, for those
who have little access to social media or limited control over the content, speed, and direction
of material posted online it can be disempowering (Marlin-Bennett, 2013). With little way of
vetting connections, social media users can be the target of criminals, marketers and
fraudsters (O'Keeffe & Clarke-Pearson, 2011). Social media providers take little
responsibility for protecting users from abuse. This raises serious issues. For example,
Greenhow (2015) describes how adoptive parents can resent their adopted children getting
into contact with birth parents through social media, and the potential danger of unwanted
contact. At the same time, some parents in her sample, felt this provided a wonderful
opportunity for children to develop good relationships with their birth parents. In the case
scenario, William has no control over the information – good or bad – posted about him. Social media lends itself to a form of ‘adultism’ (Dominelli, 1989) where adults exercise
power over children without their involvement or consent. Additionally, William’s human
rights, and the social justice due to him as a child have been deliberately violated by Adam. opportunity for children to develop good relationships with their birth parents. In the case
scenario, William has no control over the information – good or bad – posted about him. Social media lends itself to a form of ‘adultism’ (Dominelli, 1989) where adults exercise
power over children without their involvement or consent. Additionally, William’s human
rights, and the social justice due to him as a child have been deliberately violated by Adam. 15 Social work should promote the rights of disempowered people not only in face-to-face
interactions, but also in those occurring online. Criticality, values and ethics Criticality, values and ethics
In light of the intersecting discourses around power, privacy, embodiment, professionalism,
authorship and consequences, social workers must be critically reflective in their practice. 16 Critical reflective practice (Fook, 1999; Healy, 2000) and critical theories are useful in
understanding and unpacking diversity, and raising questions that might not be otherwise
considered (Dominelli, 2014). It will help ensure that social workers do not engage in
unethical practice inadvertently. While it is important that social workers hold onto core
values and principles related to human rights, social justice, integrity, competence, and
respect, this alone, is not enough. Social workers must be fully informed of the complexities
of online interactions and remain up-to-date on research in this field. Social workers must
also help citizens to have digital and ethical literacy and they should promote the rights of
disempowered people in not only face-to-face interactions, but also online ones. Conclusions: Implications for research, practice and education 17
Being well-informed and able to exercise one’s rights is a condition of citizenship (Dominelli,
2014). Social workers need to help citizens understand ethics and ask for the realisation of
their rights if social justice is to be implemented. How do these relate to online chats that
have repercussions far beyond their existence in ethereal space that, for example, can affect
one’s sense of wellbeing, the right to be free of abuse and violence, and one’s current or
future employment prospects? These issues are greater than one individual, and we would
argue that social work’s professional associations – nationally and globally – need to develop
comprehensive guidelines to assist social workers in this task. These should include guidance
on how to be critically reflective practitioners online and how to question or interrogate
taken-for-granted assumptions. Moreover, we suggest that professional associations engage
with employers to develop social media policies that do not put the burden of anticipating the
consequences only upon an individual practitioner. Responding to this is becoming necessary
especially for young people who are increasingly unlikely to communicate via traditional
media. The question of who becomes included and excluded arises as digital divides become
more pronounced in a market-place that asks for credit cards upfront for online purchases 17 including applications that facilitate communication. Finally, we argue that more research
into social media is needed to help social workers keep pace with rapidly changing
technologies. Limited research in this area means that being well-informed about rights to
communication technologies, their use and misuse are items requiring urgent attention. Research can provide a robust foundation for teaching social work students how to use online
resources in an ethical manner that promotes social justice. We argue that such teaching
should become mandatory in the social work curriculum, and could potentially be covered in
modules on values and ethics. Getting to this point might require regulatory bodies and
professional associations to set standards regarding their inclusion in all programmes of
study. Our suggestions are feasible, and we would argue, essential for social work practice in
the 21st century. including applications that facilitate communication. Finally, we argue that more research
into social media is needed to help social workers keep pace with rapidly changing
technologies. Limited research in this area means that being well-informed about rights to
communication technologies, their use and misuse are items requiring urgent attention. Conclusions: Implications for research, practice and education Research can provide a robust foundation for teaching social work students how to use online
resources in an ethical manner that promotes social justice. We argue that such teaching
should become mandatory in the social work curriculum, and could potentially be covered in
modules on values and ethics. Getting to this point might require regulatory bodies and
professional associations to set standards regarding their inclusion in all programmes of
study. Our suggestions are feasible, and we would argue, essential for social work practice in
the 21st century. 18 References AASW. (2013a). AASW practice standards. Retrieved 8 June 2015, from
www.aasw.asn.au/document/item/4551 AASW. (2013b). Ethics and practice guideline – social media information and communication
technologies: Part two – social networking and professional boundaries. Retrieved 2
June 2015, from http://www.aasw.asn.au/document/item/4674 AASW. (2014). Ethics and practice guideline – social media, information and communication
technologies: Part three – providing social work services online/remotely. Retrieved 2
June 2015, from http://www.aasw.asn.au/document/item/6473 Best, P., Manktelow, R., & Taylor, B.J. (2014). Social work and social media: Online help-
seeking and the mental well-being of adolescent males. British Journal of Social Work,
1-20. doi: 10.1093/bjsw/bcu130 Best, P., Manktelow, R., & Taylor, B.J. (2014). Social work and social media: Online help-
seeking and the mental well-being of adolescent males. British Journal of Social Work,
1-20. doi: 10.1093/bjsw/bcu130 19 19 Bickhoff, L. (2014). Smart nurses thoughtless posts on social media. ANMJ, 22(4), 31. Boyle, L. (2013, 19 February 2013). Grieving parents battle Facebook for access to 15-year-old
son's profile after he committed suicide. Daily Mail. Retrieved from
http://www.dailymail.co.uk/news/article-2280800/Facebook-bans-parents-accessing-
sons-profile-committed-suicide.html
Castells, M. (2009). The rise of the network society, the information age: Economy, society and
culture. Oxford, UK: : Blackwell. Dominelli, L. (1989). Betrayal of trust: A feminist analysis of power relationships in incest
abuse and its relevance for social work practice. British Journal of Social Work, 19, 291-
307. Dominelli, L. (2002). Feminist social work theory and practice. London: Palgrave-Macmillan. Dominelli, L. (2007). Contemporary challenges to social work education in the united kingdom. Australian Social Work, 60(1), 29-45. doi: 10.1080/03124070601166695
Dominelli, L. (2014). Critical theories: Reflecting on citizenship status and practices. In L. Dominelli & M. Moosa-Mitha (Eds.), Reconfiguring citizenship: Citizenship and
diversity within inclusive citizenship practices (pp. 253-267). Aldershot: Ashgate. Dominelli, L., Strega, S., Callahan, M., & Rutman, D. (2005). Endangered children:
Experiencing and surviving the state as failed parent and grandparent. British Journal of
Social Work, 35(7), 1123-1144. doi: 10.1093/bjsw/bch224
Ellison, N.B., Steinfield, C., & Lampe, C. (2007). The benefits of Facebook “friends”: Social
capital and college students’ use of online social network sites. Journal of Computer‐
Mediated Communication, 12(4), 1143-1168. doi: 10.1111/j.1083-6101.2007.00367.x
Fange, L., Mishna, F., Zhang, V.F., Van Wert, M., & Bogo, M. (2014). Social media and social
work education: Understanding and dealing with the new digital world. Social Work in
Health Care, 53, 800-814. doi: 10.1080/00981389.2014.943455 Bickhoff, L. (2014). Smart nurses thoughtless posts on social media. ANMJ, 22(4), 31. Boyle, L. (2013, 19 February 2013). References Grieving parents battle Facebook for access to 15-year-old
son's profile after he committed suicide. Daily Mail. Retrieved from
http://www.dailymail.co.uk/news/article-2280800/Facebook-bans-parents-accessing-
sons-profile-committed-suicide.html sons-profile-committed-suicide.html
Castells, M. (2009). The rise of the network society, the information age: Economy, society and
culture. Oxford, UK: : Blackwell. Dominelli, L. (1989). Betrayal of trust: A feminist analysis of power relationships in incest
abuse and its relevance for social work practice. British Journal of Social Work, 19, 291-
307. Dominelli, L. (2002). Feminist social work theory and practice. London: Palgrave-Macmillan. Dominelli, L. (2007). Contemporary challenges to social work education in the united kingdom. Australian Social Work, 60(1), 29-45. doi: 10.1080/03124070601166695
Dominelli, L. (2014). Critical theories: Reflecting on citizenship status and practices. In L. Dominelli & M. Moosa-Mitha (Eds.), Reconfiguring citizenship: Citizenship and
diversity within inclusive citizenship practices (pp. 253-267). Aldershot: Ashgate. Dominelli, L., Strega, S., Callahan, M., & Rutman, D. (2005). Endangered children:
Experiencing and surviving the state as failed parent and grandparent. British Journal of
Social Work, 35(7), 1123-1144. doi: 10.1093/bjsw/bch224 Castells, M. (2009). The rise of the network society, the information age: Economy, society and
culture. Oxford, UK: : Blackwell. Dominelli, L. (1989). Betrayal of trust: A feminist analysis of power relationships in incest
abuse and its relevance for social work practice. British Journal of Social Work, 19, 291-
307. Ellison, N.B., Steinfield, C., & Lampe, C. (2007). The benefits of Facebook “friends”: Social
capital and college students’ use of online social network sites. Journal of Computer‐
Mediated Communication, 12(4), 1143-1168. doi: 10.1111/j.1083-6101.2007.00367.x
Fange, L., Mishna, F., Zhang, V.F., Van Wert, M., & Bogo, M. (2014). Social media and social
work education: Understanding and dealing with the new digital world. Social Work in
Health Care, 53, 800-814. doi: 10.1080/00981389.2014.943455 20 Fook, J. (1999). Critical reflectivity in education and practice. In B. Pease & J. Fook (Eds.),
Transforming social work practice: Modern, critical perspectives (pp. 195-207). London: Routledge. Friesen, N., & Lowe, S. (2012). The questionable promise of social media for education:
Connective learning and the commercial imperative. Journal of Computer Assisted
Learning, 28(3), 83-194. Fu, K.-w., Cheng, Q., Wong, P.W.C., & Yip, P.S.F. (2013). Responses to a self-presented
suicide attempt in social media: A social network analysis. Crisis, 34(6), 406-412. doi:
10.1027/0227-5910/a000221 10.1027/0227-5910/a000221 Gershon, H. (2014). Selling your self in the united states. Political and Legal Anthropology
Review, 37(2), 281-295. doi: 10.1111/plar.12075 Giroux, H.A. (2005). The terror of neoliberalism: Rethinking the significance of cultural
politics. College Literature, 32(1), 1-19. Greenhow, S. (2015). Chatting online with my other mother: Adoptive family views and
experiences of the use of traditional and technological forms of post-adoption contact. (PhD Thesis), Durham University, Durham. Greyson, S.R., Kind, T., & Chretien, K.C. (2010). Online professionalism and the mirror of
social media. J Gen Intern Med, 25(11), 1227-1229. doi: 10.1007/s11606-010-1447-1
Grodzinsky, F.S., & Tavani, H.T. (2010). Applying the ‘‘contextual integrity’’ model of privacy
to personal blogs in the blogosphere. International Journal of Internet Research Ethics,
3, 38-47. Healy, K. (2000). Social work practices: Contemporaryperspectives on change. London: Sage. Kim, W., Jeong, O.-R., & Lee, S.-W. (2010). On social web sites. Information Systems, 35, 215-
236. doi: 10.1016/j.is.2009.08.003 Marlin-Bennett, R. (2013). Embodied information, knowing bodies, and power. Millennium:
Journal of International Studies, 41(3), 601-622. doi: 10.1177/0305829813486413 21 Mishna, F., Bogo, M., Root, J., Sawyer, J.-L., & Khoury-Kassabri, M. (2012). ‘‘It just crept in’’:
The digital age and implications for social work practice. Clinical Social Work Journal,
40, 277-286. doi: 10.1007/s10615-012-0383-4
Mukherjee, D., & Clark, J. (2012). Students’ participation in social networking sites:
Implications for social work education. Journal of Teaching in Social Work, 32, 161-
173. Murthy, D. (2012). Towards a sociological understanding of social media: Theorizing twitter. Sociology, 46(6), 1059-1073. doi: 10.1177/0038038511422553
Nelson, R.R. (1990). Capitalism as an engine of progress. Research Policy, 19(3), 193-214. doi:
10.1016/0048-7333(90)90036-6
O'Brien, W. (2014). Australia’s digital policy agenda: Adopting a children's rights approach. The International Journal of Children's Rights, 22(4), 748-775. O'Keeffe, G.S., & Clarke-Pearson, K. (2011). Clinical report—the impact of social media on
children, adolescents, and families. Pediatrics, 127(4), 800-804. doi: 10.1542/peds.2011-
0054
Reamer, F.G. (2013). Social work in a digital age: Ethical and risk management challenges. 58(2), 163-172. doi: 10.1093/sw/swt003
Reyman, J. (2013). User data on the social web: Authorship, agency, and appropriation. College
English, 513-533. Schawbel, D. (2012). How recruiters use social networks to make hiring decisions now. Time
online. http://business.time.com/2012/07/09/how-recruiters-use-social-networks-to-
make-hiring-decisions-now/
Shirkey, C. (2011). The political power of social media: Technology, the public sphere, and Mishna, F., Bogo, M., Root, J., Sawyer, J.-L., & Khoury-Kassabri, M. (2012). ‘‘It just crept in’’:
The digital age and implications for social work practice. Clinical Social Work Journal,
40, 277-286. doi: 10.1007/s10615-012-0383-4 en, W. 10.1027/0227-5910/a000221 (2014). Australia’s digital policy agenda: Adopting a children's rights approach. Schawbel, D. (2012). How recruiters use social networks to make hiring decisions now. Time
online. http://business.time.com/2012/07/09/how-recruiters-use-social-networks-to-
make-hiring-decisions-now/ Shirkey, C. (2011). The political power of social media: Technology, the public sphere, and
political change. Foreign Affairs, 90(1), 28-41. Stevenson, L. (2014). Social work sanctioned over Facebook posts reflects on feeling abandoned
amidst a media storm. Retrieved 8 December 2015, from 22 http://www.communitycare.co.uk/2014/10/14/social-worker-sanctioned-facebook-posts-
reflects-feeling-abandoned-amidst-media-storm/
Strauß, S., & Nentwich, M. (2013). Social network sites, privacy and the blurring boundary
between public and private spaces. Science and Public Policy, 40(6), 724-732. doi:
10.1093/scipol/sct072
The Policy Ethics and Human Rights Committee. (2012). BASW social media policy. Retrieved 2 June 2015, from cdn.basw.co.uk/upload/basw_34634-1.pdf
Toten, M. (2014). Social media posts jeopardise job prospects. Workplace info news and info
for australian hr/ir professionals. from
http://workplaceinfo.com.au/recruitment/analysis/social-media-postings-discourage-
hirers#.VGx93TSUd8E
van Doorn, N. (2011). Digital spaces, material traces: How matter comes to matter in online
performances of gender, sexuality and embodiment. Media, Culture and Society, 33(4),
531-547. doi: 10.1177/0163443711398692
Virilio, P. (2000). The Information Bomb (C. Turner, Trans.) New York: Verso Books. Virilio, P. (2010). Grey ecology (D. Burk, Trans.) New York: Atropos. Voshel, E.H., & Wesala, A. (2015). Social media and social work ethics: Determining best
practices in an ambiguous reality. Journal of Social Work Values and Ethics, 12(1), 67-
76. Wallace, J., & Pease, B. (2011). Neoliberalism and australian social work: Accommodaiton or
resistance? Journal of Social Work, 11(2), 132-142. doi: 10.1177/1468017310387318
Zeng, D., Chen, H., Lusch, R., & Li, S.-H. (2010). Social media analytics and intelligence. IEEE
Computer Society, 13-16. http://www.communitycare.co.uk/2014/10/14/social-worker-sanctioned-facebook-posts-
reflects-feeling-abandoned-amidst-media-storm/ http://www.communitycare.co.uk/2014/10/14/social-worker-sanctioned-facebook-post
reflects-feeling-abandoned-amidst-media-storm/ Strauß, S., & Nentwich, M. (2013). Social network sites, privacy and the blurring boundary
between public and private spaces. Science and Public Policy, 40(6), 724-732. doi:
10.1093/scipol/sct072 http://workplaceinfo.com.au/recruitment/analysis/social-media-postings-discourage-
hirers#.VGx93TSUd8E van Doorn, N. (2011). Digital spaces, material traces: How matter comes to matter in online
performances of gender, sexuality and embodiment. Media, Culture and Society, 33(4),
531-547. doi: 10.1177/0163443711398692 Virilio, P. (2000). The Information Bomb (C. Turner, Trans.) New York: Verso Books. Virilio, P. (2010). Grey ecology (D. Burk, Trans.) New York: Atropos. Voshel, E.H., & Wesala, A. (2015). Social media and social work ethics: Determining best
practices in an ambiguous reality. Journal of Social Work Values and Ethics, 12(1), 67-
76. Wallace, J., & Pease, B. (2011). Neoliberalism and australian social work: Accommodaiton or
resistance? Journal of Social Work, 11(2), 132-142. 10.1027/0227-5910/a000221 doi: 10.1177/1468017310387318
Zeng, D., Chen, H., Lusch, R., & Li, S.-H. (2010). Social media analytics and intelligence. IEEE
Computer Society, 13-16. Wallace, J., & Pease, B. (2011). Neoliberalism and australian social work: Accommodaiton or
resistance? Journal of Social Work, 11(2), 132-142. doi: 10.1177/1468017310387318
Zeng, D., Chen, H., Lusch, R., & Li, S.-H. (2010). Social media analytics and intelligence. IEEE
Computer Society, 13-16. 23
|
https://openalex.org/W2776193549
|
https://europepmc.org/articles/pmc5804115?pdf=render
|
English
| null |
Use of High-Flow Nasal Cannula Oxygen Therapy in a Pregnant Woman with Dermatomyositis-Related Interstitial Pneumonia
|
Case reports in critical care
| 2,017
|
cc-by
| 2,909
|
1. Introduction patient had previously undergone three vaginal deliveries. A
review of family history revealed that the patient’s paternal
grandmother had rheumatoid arthritis and the patient’s
father had unspecified IP. The patient first experienced res-
piratory distress in her 28th week of gestation; her condition
deteriorated two weeks later, and she was transported to our
hospital via ambulance. Upon admission, the patient was
lucid and afebrile (36.5∘C). The respiratory and hemody-
namic levels are revealed in Table 1. She had blood pressure
of 88/49 mmHg, a heart rate of 86 bpm, a respiratory rate of
18 breaths/minute, and peripheral oxygen saturation (SpO2)
on room air of 90%. Fine crackles were noted in both lower
lung fields. High-flow nasal cannula (HFNC) oxygen therapy is widely
used in the management of acute respiratory failure and also
has applications in cases with acute exacerbation of interstitial
pneumonia (IP) [1–3]. Although pregnant patients with
IP rarely develop concurrent complications of polymyositis
(PM) or dermatomyositis (DM), the prompt diagnosis of
PM/DM in these patients is critical due to the high risk of
potentially fatal outcomes to both the mother and the fetus
[4–6]. This case report describes the use of HFNC oxygen
therapy without intubation in a 33-year-old pregnant woman
who developed progressive IP complicated by DM at 28
weeks of gestation. The patient was successfully treated with
combination immunosuppressive therapy. Laboratory examination revealed slight elevations in
white blood cell count (11,900/𝜇l), serum C-reactive protein
concentration (2.65 mg/dl), and aldolase level (7.1 U/l). Serum
KL-6 level was highly elevated at 986 U/ml. An arterial blood
gas test showed poor oxygenation with arterial oxygen partial
pressure (PaO2) on room air of 61.7 mmHg. Correspondence should be addressed to Takeshi Umegaki; umegakit@hirakata.kmu.ac.jp Correspondence should be addressed to Takeshi Umegaki; umegakit@hirakata.kmu.ac.jp Received 28 July 2017; Revised 3 December 2017; Accepted 17 December 2017; Published 31 December 2017 ved 28 July 2017; Revised 3 December 2017; Accepted 17 December 2017; Published 31 December 2017 Received 28 July 2017; Revised 3 December 2017; Accepted 17 December 2017; Published 31 December 2017
Academic Editor: Mabrouk Bahloul Copyright © 2017 Tomohiro Shoji 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. A 33-year-old pregnant woman was referred to our hospital with respiratory distress at 30 weeks of gestation. Chest computed
tomography (CT) scans revealed pulmonary infiltrates along the bronchovascular bundles and ground-glass opacities in both lungs. Despite immediate treatment with steroid pulse therapy for suspected interstitial pneumonia, the patient’s condition worsened. Respiratory distress was slightly alleviated after the initiation of high-flow nasal cannula (HFNC) oxygen therapy (40 L/min,
FiO2 40%). We suspected clinically amyopathic dermatomyositis (CADM) complicating rapidly progressive refractory interstitial
pneumonia. In order to save the life of the patient, the use of combination therapy with immunosuppressants was necessary. The patient underwent emergency cesarean section and was immediately treated with immunosuppressants while continuing
HFNC oxygen therapy. The neonate was treated in the neonatal intensive care unit. The patient’s condition improved after 7
days of hospitalization; by this time, she was positive for myositis-specific autoantibodies and was diagnosed with interstitial
pneumonia preceding dermatomyositis. This condition can be potentially fatal within a few months of onset and therefore requires
early combination immunosuppressive therapy. This case demonstrates the usefulness of HFNC oxygen therapy for respiratory
management as it negates the need for intubation and allows for various treatments to be quickly performed. Hindawi
Case Reports in Critical Care
Volume 2017, Article ID 4527597, 5 pages
https://doi.org/10.1155/2017/4527597 Hindawi
Case Reports in Critical Care
Volume 2017, Article ID 4527597, 5 pages
https://doi.org/10.1155/2017/4527597 2. Case Report A 33-year-old pregnant woman was admitted to our hospital
due to respiratory distress at 30 weeks of gestation. The 2 Case Reports in Critical Care Table 1: The respiratory and hemodynamic levels from hospital admission to ICU discharge. Table 1: The respiratory and hemodynamic levels from hospital admission to ICU discharge. Variables
Oxygen therapy
SpO2 (%)
PaO2 (mmHg)
Respiratory rate
(min−1)
Systolic blood
pressure
(mmHg)
Hospital admission
Room air
90
61.7
18
88
ICU admission
HFNC
40 L/min, FiO2
0.40
94
64.5
28
111
ICU day 2
HFNC
40 L/min, FiO2
0.40
95
73.5
19
94
ICU day 3
HFNC
40 L/min, FiO2
0.40
95
73.3
17
124
ICU day 4
HFNC
40 L/min, FiO2
0.40
96
89.3
17
122
ICU: intensive care unit; SpO2: oxygen saturation of peripheral artery; PaO2: partial pressure of arterial oxygen; HFNC: high-flow nasal cannula; FiO2: fraction
of inspiratory oxygen. ICU: intensive care unit; SpO2: oxygen saturation of peripheral artery; PaO2: partial pressure of arterial oxygen; HFNC: high-flow nasal cannula; FiO2: fraction
of inspiratory oxygen. (a)
(b)
Figure 1: Chest CT scans showing the patient’s middle (a) and lower (b) lung fields upon admission. Bilateral pulmonary infiltrates along the
peripheral bronchovascular bundles and ground-glass opacities with a panlobular distribution were observed. (b) (a) (b) (a) Figure 1: Chest CT scans showing the patient’s middle (a) and lower (b) lung fields upon admission. Bilateral pulmonary infiltrates along the
peripheral bronchovascular bundles and ground-glass opacities with a panlobular distribution were observed. treatment, the patient’s condition worsened on the second
day of hospitalization, and she developed orthopnea (grade
V based on the Hugh-Jones classification). As a result, HFNC
oxygen therapy was initiated at 30 L/min with a fraction of
inspired oxygen (FiO2) of 0.30 according to the instructions
of the intensivists and anesthesiologists. However, this did
not improve respiratory distress with SpO2 remaining at 90%. HFNC oxygen parameters were increased to 40 L/min with
FiO2 at 0.40, and respiratory distress began to improve (SpO2:
92–94%). Although the patient had eczema and ulceration on the
dorsal surface of both hands on the first day of hospitalization,
she did not present with Gottron’s sign or muscle weakness,
which are characteristic of DM. Chest computed tomography
(CT) scans (Figure 1) revealed pulmonary infiltrates along
the bronchovascular bundles and panlobular ground-glass
opacities in both lungs. N-terminal (NT) pro-B-type natri-
uretic peptide (BNP) level was at 258.8 pg/ml without renal
dysfunction. 2. Case Report Cardiac dysfunction was not revealed except
for slight dilatation of the left ventricle. The differential
diagnosis included idiopathic IP and IP complicated by a
collagen disease such as DM. Due to the rapid progression of
respiratory distress within a short period of time, the patient
was given intravenous methylprednisolone pulse therapy
(1 g/day) from the first day of hospitalization. Despite this Due to the rapid disease progression and resistance to
steroid treatment, we suspected IP complicated by PM/DM or
clinically amyopathic DM (CADM), which is a form of DM
without overt signs of myositis. Accordingly, we deemed it
necessary to begin immunosuppressive therapy. At 30 weeks Case Reports in Critical Care 3 (a)
(b)
Figure 2: Chest CT scans showing the patient’s middle (a) and lower (b) lung fields after two months from the initiation of treatment. The
pulmonary infiltrates had disappeared. (b) (a) (b) (a) Figure 2: Chest CT scans showing the patient’s middle (a) and lower (b) lung fields after two months from the initiation of treatment. The
pulmonary infiltrates had disappeared. of gestation, the fetal body weight was over 1500 g, and it was
determined that the neonate could be treated at the neonatal
intensive care unit after delivery. HFNC oxygen therapy
was continued, and an emergency cesarean section without
use of tocolytics was performed under spinal anesthesia. The neonate weighed 1550 g, and the Apgar scores at one
minute and five minutes after birth were 8 and 9, respectively. Tracheal intubation was not required during the procedure. A chest X-ray indicated that the pulmonary infiltrates had
spread further. Due to this exacerbation of IP, we began
treatment with ciclosporin (0.2 g/day) from the third day of
hospitalization. On the following day, we observed newly
formed heliotrope rash on both upper eyelids and keratotic
rash along the surface of the fingers of both hands. Chest CT
scans confirmed that the infiltrates had expanded since the
patient was admitted, and cyclophosphamide pulse therapy
(1 g/month) was added to the patient’s regimen on the fifth
day of hospitalization. that time, the patient complained of polyarthralgia. Together
with the other symptoms of heliotrope rash, elevated aldolase
level, elevated C-reactive protein concentration, and positive
titers for anti-ARS antibodies, the inclusion of arthralgia
fulfilled the diagnostic criteria for DM as stipulated by Japan’s
Ministry of Health, Labour and Welfare based on Tanimoto et
al. [7]. 2. Case Report The final diagnosis was IP preceding DM with delayed
manifestation of specific cutaneous findings without overt
signs of myositis. References [1] Y. Horio, T. Takihara, K. Niimi et al., “High-flow nasal cannula
oxygen therapy for acute exacerbation of interstitial pneumo-
nia: A case series,” Respiratory Investigation, vol. 54, no. 2, pp. 125–129, 2016. It should be noted that the use of immunosuppressants
for the treatment of IP does not ensure rapid improvement in
patient condition. In a similar case report, a pregnant woman
at 16 weeks of gestation had developed IP preceding PM and
was treated with a combination of steroid pulse therapy and
tacrolimus [11]. Due to that patient’s worsening respiratory
condition, the pregnancy was terminated in the 21st week
of gestation to save the mother. Cyclophosphamide pulse
therapy was subsequently added to the treatment regimen,
and the patient began to show signs of improvement. Cases
of IP complicated by PM/DM or CADM in pregnant women
are extremely rare. Therefore, it remains unclear if the
use of combination immunosuppressive therapy (including
cyclophosphamide) would produce quick therapeutic effects
in cases without termination of pregnancy. [2] H. Y. Lee, C. K. Rhee, and J. W. Lee, “Feasibility of high-flow
nasal cannula oxygen therapy for acute respiratory failure in
patients with hematologic malignancies: A retrospective single-
center study,” Journal of Critical Care, vol. 30, no. 4, pp. 773–777,
2015. [3] J. P. Frat, A. W. Thille, A. Mercat et al., “High-flowoxygen-
through nasal cannula inacutehypoxemicrespiratory failure,”
New England Journal of Medicine, vol. 372, no. 23, pp. 2185–2196,
2015. [4] B. A. Rosenzweig, S. Rotmensch, S. P. Binette, and M. Phillippe,
“Primary idiopathic polymyositis and dermatomyositis compli-
cating pregnancy: diagnosis and management,” Obstetrical &
Gynecological Survey, vol. 44, no. 3, pp. 162–170, 1989. Cardiac involvement has been reported in patients with
DM, and the incidence has reached as high as 45.7% [12]. Moreover, interstitial pneumonia has been reported as one of
the major predictive factors of cardiac dysfunction in patients
with DM [12]. Left ventricular diastolic dysfunction is an
early feature of cardiac involvement in patients with PM/DM
[13], and cardiac involvement is a common cause of death
[14]. This case has not clinically revealed cardiac dysfunction,
but diastolic dysfunction might have potentially progressed
because of elevation of NT-pro BNP, slight dilatation of the
left ventricle, and alveolar syndrome with air bronchogram
on the CT chest. HFNC might have suitably applied positive
end expiratory pressure [15]. 3. Discussion This form of respiratory management should therefore
be considered for other similar cases in the future.h The association between the prognosis of IP patients with
DM and the degree of myositis disease activity has been previ-
ously documented, and the early use of immunosuppressants 4 Case Reports in Critical Care should be employed in cases with rapidly progressive IP [5]. When refractory IP is complicated by PM/DM or CADM,
the pulmonary tissue may become irreversibly damaged. As
a result, the condition may become resistant to combination
immunosuppressive therapy and eventually lead to death
after only several months [5, 9, 10]. As our patient’s respiratory
condition continued to worsen despite immediate steroid
pulse therapy, we suspected refractory IP complicated by
PM/DM or CADM, and the early use of immunosuppressive
therapy was deemed necessary. While the diagnosis of DM
was made later, the possibility of refractory IP preceding
PM/DM or CADM prompted us to consider the early use of
combination immunosuppressive therapy. therapy and immunosuppressants. HFNC oxygen therapy is a
useful respiratory management method that negates the need
for intubation and allows for greater freedom of treatment
and patient comfort. Conflicts of Interest The authors declare that there are no conflicts of interest
regarding the publication of this article. References HFNC oxygen therapy was
seamlessly provided without interruption throughout the
patient’s treatment in the intensive care unit, the operating
theater, and the general ward and during transfers between
these units. As the pregnancy had progressed to the point
where the baby could be treated in the neonatal intensive
care unit after delivery, the nonuse of intubation allowed the
patient to be quickly transitioned from the cesarean section
to immunosuppressive therapy. This way, we were able to save
both the mother and the child. [5] Y. Nawata, K. Kurasawa, K. Takabayashi et al., “Corticos-
teroid resistant interstitial pneumonitis in dermatomyosi-
tis/polymyositis: Prediction and treatment with cyclosporine,”
The Journal of Rheumatology, vol. 26, no. 7, pp. 1527–1533, 1999. [6] M. Fathi and I. E. Lundberg, “Interstitial lung disease
in polymyositis and dermatomyositis,” Current Opinion in
Rheumatology, vol. 17, no. 6, pp. 701–706, 2005. [7] K. Tanimoto, K. Nakano, S. Kano et al., “Classification criteria
for polymyositis and Dermatomyositis,” The Journal of Rheuma-
tology, vol. 22, no. 4, pp. 668–674, 1995. [8] J. R. Masclans, P. P´erez-Ter´an, and O. Roca, “The role of high
flow oxygen therapy in acute respiratory failure,” Medicina
Intensiva, vol. 39, no. 8, pp. 505–515, 2015. [9] P. Gerami, J. M. Schope, L. McDonald, H. W. Walling, and R. D. Sontheimer, “A systematic review of adult-onset clinically
amyopathic dermatomyositis (dermatomyositis sin´e myositis):
a missing link within the spectrum of the idiopathic inflam-
matory myopathies,” Journal of the American Academy of
Dermatology, vol. 54, no. 4, pp. 597–613, 2006. [10] R. D. Sontheimer and S. Miyagawa, “Potentially fatal interstitial
lung disease can occur in clinically amyopathic dermatomyosi-
tis,” Journal of the American Academy of Dermatology, vol. 48,
no. 5, pp. 797-798, 2003. [11] R. Okad, Y.-S. Miyabe, S. Kasai et al., “Successful treatment
of interstitial pneumonia and pneumomediastinum associated
with polymyositis during pregnancy with a combination of
cyclophosphamide and tacrolimus: A case report,” Japanese
Journal of Clinical Immunology, vol. 33, no. 3, pp. 142–148, 2010. 3. Discussion This case provided valuable findings that the use of HFNC
oxygen therapy was able to contribute to the alleviation of
respiratory distress in rapidly progressive IP. To the best of
our knowledge, this report describes the first case of rapidly
progressive IP where hypoxia was successfully prevented in
both the mother and the fetus without intubation. y
p
With HFNC oxygen therapy (40 L/min, FiO2: 0.40),
respiratory distress was alleviated (SpO2: 93–96%). Three
days after the cesarean section (fifth day of hospitalization),
the patient was transferred from the intensive care unit to
a general ward. On the seventh day of hospitalization, the
results of analysis of a blood sample taken on the day of
admission showed that the patient had positive titers for
autoantibodies against aminoacyl tRNA synthetase (ARS),
including anti-Jo-1 antibodies. Due to the presence of anti-
ARS antibodies, we excluded the possibility of CADM. The patient’s respiratory condition further improved, and
the parameters of HFNC oxygen therapy were reduced to
30 L/min with FiO2 at 0.30. A chest CT scan taken after
18 days of hospitalization indicated a new case of pneumo-
mediastinum, but the interstitial shadows in the lung field
had substantially receded. The patient was discharged after
47 days of hospitalization. Following 2 months of treatment,
chest CT scans showed that the pneumomediastinum and
the interstitial shadows had disappeared (Figure 2). During It has been reported that the use of HFNC oxygen therapy
in acute respiratory failure cases did not result in lower
intubation rates relative to oxygen therapy delivered through
a face mask and noninvasive positive-pressure ventilation,
but it was associated with more ventilator-free days and a
higher survival rate [3]. In addition, HFNC oxygen therapy
allows for patients to eat, drink, and move around without
the need to interrupt treatment [8]. Our case did not require
intubation throughout the cesarean section procedure and
immunosuppressive therapy, which allowed her to have
meals, converse with others, and interact with her child. In
addition to alleviating respiratory distress, the use of HFNC
oxygen therapy may have reduced the patient’s feelings of
anxiety and improved her quality of life during hospitaliza-
tion. 4. Conclusions This case report describes the successful use of HFNC oxygen
therapy for respiratory management in a pregnant patient
who developed rapidly progressive IP complicated by DM. Both the mother and the child were saved. It is necessary to
quickly treat such cases with a combination of steroid pulse [12] C. Zuo, X. D. Wei, Y. L. Ye et al., “Risk factors associated
with cardiac involvement in patients with dermatomyosi-
tis/polymyositis,” Sichuan DaXueXueBao YiXue Ban, vol. 44, no. 5, pp. 801–804, 2013. Case Reports in Critical Care 5 [13] Z. Lu, Q. Wei, Z. Ning, Z. Qian-Zi, S. Xiao-Ming, and W. Guo-
Chun, “Left ventricular diastolic dysfunction - early cardiac
impairment in patients with polymyositis/dermatomyositis: a
tissue doppler imaging study,” The Journal of Rheumatology, vol. 40, no. 9, pp. 1572–1577, 2013. [14] Z. Lu, W. Guo-Chun, M. Li, and Z. Ning, “Cardiac involvement
in adult polymyositis or dermatomyositis: a systematic review,”
Clinical Cardiology, vol. 35, no. 11, pp. 686–691, 2012. [15] A. Corley, L. R. Caruana, A. G. Barnett, O. Tronstad, and J. F. Fraser, “Oxygen delivery through high-flow nasal cannulae
increase end-expiratory lung volume and reduce respiratory
rate in post-cardiac surgical patients,” British Journal of Anaes-
thesia, vol. 107, no. 6, pp. 998–1004, 2011.
|
https://openalex.org/W2888456635
|
https://zenodo.org/records/1408293/files/JHR_article_26976.pdf
|
English
| null |
A taxonomic account of the genus Labus de Saussure, 1867 (Hymenoptera, Vespidae, Eumeninae) with descriptions of three new species
|
Journal of Hymenoptera Research/Journal of Hymenoptera research
| 2,018
|
cc-by
| 8,193
|
A taxonomic account of the genus Labus de Saussure, 1867...
23
JHR 65: 23–46 (2018)
doi: 10.3897/jhr.65.26976
http://jhr.pensoft.net
RESEARCH ARTICLE A taxonomic account of the genus Labus de Saussure, 1867... 23
JHR 65: 23–46 (2018)
doi: 10.3897/jhr.65.26976
http://jhr.pensoft.net
RESEARCH ARTICLE A
JHR 65: 23–46 (2018)
doi: 10.3897/jhr.65.26976
http://jhr.pensoft.net Abstracth Abstract
Three new species, namely Labus edentatus sp. n. from China, L. sparsipunctus sp. n. from Thailand and
L. robustus sp. n. from Indonesia are described and illustrated. Both L. amoenus van der Vecht, 1935 and
L. pusillus van der Vecht, 1963 are newly recorded from China and Vietnam, and L. angularis van der Vecht,
1935 is also firstly recorded from China. An updated key to the world species of the genus Labus is provided. A taxonomic account of the genus Labus de Saussure,
1867 (Hymenoptera, Vespidae, Eumeninae)
with descriptions of three new species Ting-Jing Li1, James M. Carpenter2 1 Chongqing Key Laboratory of Vector Insects, Chongqing Key Laboratory of Animal Biology, Institute of Insect
and Molecular Biology, Chongqing Normal University, Chongqing 401331, China 2 Division of Invertebrate
Zoology, American Museum of Natural History, Central Park West at 79th Street, New York, NY 10024, USA Corresponding author: James M. Carpenter (carpente@amnh.org) Academic editor: M. Ohl | Received 1 June 2018 | Accepted 7 August 2018 | Published 27 August 2018
http://zoobank.org/CA564E05-1B39-449D-B854-B0C133539BFC Citation: Li T-J, Carpenter JM (2018) A taxonomic account of the genus Labus de Saussure, 1867 (Hymenoptera,
Vespidae, Eumeninae) with descriptions of three new species. Journal of Hymenoptera Research 65: 23–46. https://doi. org/10.3897/jhr.65.26976 Introduction The genus Labus de Saussure is an Oriental genus containing 13 species (Girish Kumar
et al. 2014) each of which is relatively slender with a petiolate metasoma and body length
mainly 6.0–8.0 mm. De Saussure (1855, 1867), van der Vecht (1935, 1963), Giordani
Soika (1960, 1973, 1986, 1991), Gusenleitner (1988), and Girish Kumar et al. (2014)
made important contributions to the taxonomy of the genus. During this study of the
genus Labus collections from Vietnam, Thailand, Malaysia, China, Philippines, Sri Lanka
and Indonesia which are deposited in American Museum of Natural History in New York,
three new species are confirmed by comparison with known species. In addition, both L. amoenus van der Vecht, 1935 and L. pusillus van der Vecht, 1963 are newly recorded from
China and Vietnam, and L. angularis van der Vecht, 1935 is also firstly recorded from
China. In the present paper, these three new species are described and illustrated in detail,
and some main characters of other known species are provided with some related figures. In addition, an updated key to the world species of Labus is also given. Keywords Hymenoptera, Vespidae, Eumeninae, Labus, new species Copyright Ting-Jing Li, James M. Carpenter. This is an open access article distributed under the terms of the Creative Commons Attribution License
(CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 24 Taxonomy Genus Labus de Saussure, 1867 Materials and methods The specimens examined are deposited in the American Museum of Natural History
(USA), Chongqing Normal University (China), Queen Sirikit Botanic Garden (Thai
land), and Yunnan Agricultural University (China), respectively. Descriptions and
measurements were made under a stereomicroscope (Nikon SMZ1500), and all figures
were taken with Microptics-USA/Visionary Digital photomicrographic system devel
oped by Roy Larimer and multiple layers stacked using Helicon Focus. The ratios used
throughout the descriptions were measured in the same magnification of the stereomi
croscope. All measurements were taken as the maximal length of body parts measured. Body length was measured from the anterior margin of the head to the posterior mar
gin of metasomal tergum 2. For the density description of punctures, “sparsely” means
that interspaces are larger than punctures diameter, “moderately” means equal to the
diameter, and “densely” means less than the diameter. Terminology principally follows
van der Vecht (1935, 1963) and Girish Kumar et al. (2014). The abbreviations used in the text are shown as follows: A1
for antennal segment 1,
A2
for antennal segment 2,
T1
for metasomal tergum 1,
T2
for metasomal tergum 2,
S1
for metasomal sternum 1,
S2
for metasomal sternum 2, and so on. AMNH American Museum of Natural History, New York
CQNU Chongqing Normal University, Chongqing
QSBG
Queen Sirikit Botanic Garden, Chiang Mai
YNAU
Yunnan Agricultural University, Kunming A taxonomic account of the genus Labus de Saussure, 1867... 25 Genus Labus de Saussure, 1867 Labus de Saussure, 1867: 3; van der Vecht 1935: 157; 1963: 5; Gusenleitner 1988:
174–177, 188–198; Girish Kumar et al. 2014: 29–30. Type species. Labus spiniger de Saussure, 1867, by subsequent designation of Bingham
(1897). Type species. Labus spiniger de Saussure, 1867, by subsequent designation of Bingham
(1897). Type species. Labus spiniger de Saussure, 1867, by subsequent designation of Bingham
(1897). Diagnosis. Body slender; in female frons with fovea in front of the anterior ocel
lus; tegula not exceeding parategula posteriorly; pronotum usually without normal
transverse carina; metanotum dentiform mesally; submarginal carina of propodeum
produced posteriorly; propodeal valvula elongate, rectangular; second submarginal cell
of forewing forming obtuse angle basally; midtibia with one spur; metasoma petiolate,
petiole abruptly swollen apically (Girish Kumar et al. 2014). Distribution. Oriental Region. Labus edentatus Li & Carpenter, sp. n. http://zoobank.org/428573BE-C548-4186-B67A-50DC96182E51
Figs 1–5 Material examined. Holotype, ♀, China: Hongkong, Hung Fa Leng, 50Q KK 108
854, 435m, 16.IV–16.V. 2014, Yiu Vor, HFL-Moo1.F.Hy.9, deposited in AMNH. Description. Female (Figs 1–2): body length 7.0 mm. Black, with the following
parts yellow: a narrow and transverse band of clypeus basally (Fig. 2), a widely inter
rupted band on pronotum anteriorly (interruption less than each marking), a small
spot on upper part of mesepisternum; outer margin of tegula anteriorly and posteri
orly, parategula, two transverse spots on scutellum posteriorly, apical lamellae of pro
podeum, a small apical spot of fore femur inside, a large spot at outer side of fore and
mid tibiae, a small spot on hind tibiae basally, narrow apical bands of T1–T2 and S2;
apex of mandible, A12 beneath, and fore and mid tibiae dark ferruginous with excep
tion of yellow spots. Wings slightly infuscated. Head. In front view head (Fig. 2) slightly wider than high, its sides rounded; cl
ypeus sparsely punctate, clypeal width 1.35× length, weakly convex at basal half, an
terior portion less produced and with wide emargination in the middle, clypeal total
width 4.6× its apical width, apical width 2× emargination depth, its teeth short and
blunt; frons convex and densely punctate, inter-antennal carina continued on lower
part of frons, frontal fovea deep, almost circular and distinctly defined (Fig. 3). i
Mesosoma. Anterior angles of pronotum projecting only slightly, pronotal trans
verse carina obsolete; punctures on pronotum, mesoscutum, mesepisternum and
scutellum slightly denser and their interspaces duller than those on frons; metanotum
with an acute tubercle in the middle; propodeum posteriorly on each side without a
tooth above the apical spine formed by the submarginal carina (Fig. 5); dorsal area of T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 26 Figures 1–5. Labus edentatus sp. Type species. Labus spiniger de Saussure, 1867, by subsequent designation of Bingham
(1897). n., holotype 1 habitus (dorsal view), ♀ 2 head in frontal view, ♀ 3 frontal
fovea, ♀ 4 metasomal petiole, ♀ 5 propodeum in dorsal view, ♀. Figures 1–5. Labus edentatus sp. n., holotype 1 habitus (dorsal view), ♀ 2 head in frontal view, ♀ 3 frontal
fovea, ♀ 4 metasomal petiole, ♀ 5 propodeum in dorsal view, ♀. A taxonomic account of the genus Labus de Saussure, 1867... 27 propodeum sparsely punctate and the interspaces between punctures coriaceous, pos
terior area with dense setae, lateral area obviously coriaceous. Metasoma. Metasomal petiole (Fig. 4) long and moderately slender, total length
of petiole 6.19× basal width and 2.6× apical one, swollen part of metasomal petiole
slightly longer than (1.07×) half of the length of the petiole, linear part of petiole ru
gosely punctate, swollen part coriaceous and with scattered minute punctures; each of
T2 and S2 with an apical translucent lamella, and microscopically sculptured, sparsely
covered with extremely minute and shallow punctures. Distribution. China (Hongkong). Distribution. China (Hongkong).h Remarks. This species resembles L. pusillus van der Vecht, 1963 by propodeum pos
teriorly on each side without a tooth above the apical spine formed by the submarginal
carina and swollen part of metasomal petiole as long as or longer than half of the length
of the petiole. It differs from L. pusillus and all other members of the genus by the follow
ing character combination: the median portion of clypeus less produced, its emargina
tion wide medially and lateral teeth slightly blunter (Fig. 2), frontal fovea (Fig. 3) much
larger than the anterior ocellus and bigger than that in L. pusillus (Fig. 37).hi Etymology. The specific name edentatus is derived from two Latin words: e- and
dentatus, referring to propodeum posteriorly on each side without a tooth above the
apical spine formed by the submarginal carina. Labus sparsipunctus Li & Carpenter, sp. n. http://zoobank.org/51004A96-11AB-4746-B93D-AB461BA2D5F9
Figs 6–12 Material examined. Holotype, ♀, Thailand, Loei Phu Ruea NPPhaLonNoi,
17°30.502'N, 101°20.868'E, 1343m, Pan traps, 5–6.III.2007, Patikhom Tum
tip, T2297, deposited in QSBG; 1♂, Thailand, Loei Phu Ruea NPPhaLonNoi,
17°30.502'N, 101°20.868'E, 1343m, Malaise trap, 12–19.III.2007, Patikhom Tum
tip, T2307, deposited in AMNH. Description. Female (Figs 6, 8): body length 6.0 mm. Black, with the follow
ing parts yellow: a curved and transverse band of clypeus basally (Fig. 8), a widely
interrupted band on pronotum anteriorly (interruption less than each marking), a
small spot on upper part of mesepisternum, outer margin of tegula, parategula, two
transverse and nearly rectangular spots on scutellum posteriorly, apical lamellae of pro
podeum, small apical spots of fore and mid femora inside, outer side of fore and mid
tibiae, basal half at outer side of hind tibiae, narrow apical bands of T1–T2 and S2;
apex of mandible, inside part of tegula, fore and mid tibiae with exception of yellow
spots, and tarsi dark ferruginous. Wings slightly infuscated. Head. Head (Fig. 8) in front view wide almost as long as high, its sides rounded;
clypeus sparsely setose and punctate, interspaces between punctures obviously shiny, T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 28 Figures 6–12. Labus sparsipunctus sp. n. Distribution. China (Hongkong). 6 habitus of holotype (dorsal view), ♀ 7 habitus of paratype
(dorsal view), ♂ 8 head of holotype (frontal view), ♀ 9 frontal fovea of holotype, ♀ 10 head of paratype
(frontal view), ♂ 11 propodeum of paratype (dorsal view), ♂ 12 metasomal petiole of paratype, ♂. Figures 6–12. Labus sparsipunctus sp. n. 6 habitus of holotype (dorsal view), ♀ 7 habitus of paratype
(dorsal view), ♂ 8 head of holotype (frontal view), ♀ 9 frontal fovea of holotype, ♀ 10 head of paratype
(frontal view), ♂ 11 propodeum of paratype (dorsal view), ♂ 12 metasomal petiole of paratype, ♂. A taxonomic account of the genus Labus de Saussure, 1867... 29 clypeal width 1.25× length, weakly convex at basal half, anterior and median portion
narrowly produced (clypeal total width 7.0× its apical width) and with U-formed
emargination, lateral teeth acute, emargination width 1.25× its depth; frons con
vex and sparsely punctate and interspaces between punctures obviously shiny, inter-
antennal carina continued on lower part of frons, frontal fovea deep, elliptical and
defined (Fig. 9). Mesosoma. Anterior angles of pronotum projecting slightly, pronotal anterior and
transverse carina obsolete, punctures on pronotum dense, interspaces less than punc
tures, mesoscutum and scutellum sparsely punctate, interspaces more than punctures,
mesepisternum dull and coriaceous, with sparse punctures; metanotum with a small
and blunt tubercle in the middle; propodeum posteriorly on each side without a tooth
above the apical spine formed by the submarginal carina (Fig. 11); dorsal area of pro
podeum sparsely punctate and the interspaces between punctures shiny, posterior area
with dense setae, lateral area dull and obviously coriaceous. Metasoma. Metasomal petiole relatively long and slender, total length of petiole
7.53× its basal width and 2.76× apical one, swollen part of metasomal petiole almost
as long as half of the length of the petiole and shiny with scattered minute punctures;
linear part of petiole slightly rugosely punctate; each of T2 and S2 with an apical trans
lucent lamella, and sparsely covered with extremely minute and shallow punctures. Male (Figs 7, 10, 12). Body length 7.0 mm. Sculpture, punctuation, setae, and col
oration as in female except as follows: clypeus except anterior margin (Fig. Labus robustus Li & Carpenter, sp. n. Material examined. Holotype, ♂, Indonesia, W. Java, Kereteg, 10.VI.1965, J.E. Lu
kavsky, deposited in AMNH. Material examined. Holotype, ♂, Indonesia, W. Java, Kereteg, 10.VI.1965, J.E. Lu
kavsky, deposited in AMNH. Description. Male (Figs 13–14): body length 7.0 mm. Body black, with the fol
lowing parts yellow: clypeus except anterior margin (Fig. 14), basal part of mandible,
two anterior angles of pronotum; a small spot on upper part of mesepisternum; outer
margins of tegula, parategula; two transverse nearly elliptical spots on scutellum
posteriorly; apical lamellae of propodeum, small apical spots of fore and mid femora
inside, outer sides of fore and mid tibiae, basal half at outer side of hind tibiae, nar
row apical bands of T1–T2 and S2; apex of mandible, and fore and mid tibiae with
exception of yellow spots dark ferruginous; A13 yellowish brown. Wings slightly
infuscated. Head. Head in front view (Fig. 14) wide slightly longer than (1.09×) high, its sides
somewhat flattened; clypeus sparsely setose and shiny, punctures indistinct, clypeal
width as long as length, weakly convex at basal half, anterior and median portion
produced and with U-formed emargination, the emargination width 1.75× its depth
and teeth acute, clypeal total width 4.86× its apical width; frons convex, coarsely and
densely punctate, and with a longitudinal furrow in the middle, inter-antennal carina
not continued on lower part of frons. Mesosoma. Anterior angles of pronotum projecting moderately, pronotal ante
rior and transverse carina present, punctures on pronotum, mesoscutum and scutel
lum similar to those on frons; mesepisternum coarsely punctate and punctures slightly
denser than that of other parts; metanotum with a small tubercle in the middle; propo
deum posteriorly on each side with a moderate tooth above the apical spine formed by
the submarginal carina (Fig. 15); dorsal area of propodeum sparsely punctate and the
interspaces between punctures coriaceous, posterior area with dense setae, lateral area
dull, coarse, and slightly rugose. Metasoma. Metasomal petiole (Fig. 16) short and stout, total length of petiole
4.32× basal width and 2.16× apical width, swollen part of metasomal petiole as long as
half of the length of the petiole and shiny with scattered minute punctures; linear part
of petiole distinctly rugosely punctate and relatively wider and stouter than other spe
cies of the genus excluding L. postpetiolatus Gusenleitner; T2 and S2 with apical trans
lucent lamellae, and sparsely covered with extremely minute and shallow punctures. Distribution. Indonesia (Java).h Remarks. Distribution. China (Hongkong). 10), scape
ventrally and mandible except apex yellow; two apical flagellomeres yellowish brown;
spots on legs larger than those in female; clypeal width 1.23× its length, total width
7.4× apical width, apical width 2.5× emargination depth, emargination narrower and
shallower than that in female; A13 backward reaching basal margin of A11; punctures
on frons, pronotum and mesoscutum relatively denser than those in female; linear part
of petiole (Fig. 12) narrower and longer than female, total length of petiole 8.07× its
basal width and 2.10×apical one, swollen part less than (0.8×) half of the length of the
petiole; other characters same as those in female. Distribution. Thailand.h Remarks. This species is allied to L. clypeatus van der Vecht, 1935 from Indonesia,
with which it has the following characters in common: propodeum posteriorly on each
side without a tooth above the apical spine formed by the submarginal carina, and
swollen part of metasomal petiole less than half of the length of the petiole (Fig. 12). It differs from L. clypeatus and all other members of the genus by the following char
acter combination: frons sparsely punctate and more shiny between these punctures
in female (Fig. 8), anterior and median portion of clypeus narrowly produced (Figs. 8,
10), and total length of metasomal petiole at most 8.07× its basal width, whereas in L. clypeatus the petiole length more than 9× basal width (van der Vecht 1935). Etymology. The specific name sparsipunctus is derived from two Latin words: spar
sus and punctus, referring to frons sparsely punctate in female. T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 30 Etymology. The specific name is derived from one Latin word: robustus, referring
to metasomal petiole stout. Labus robustus Li & Carpenter, sp. n. This species resembles L. postpetiolatus Gusenleitner, 1988 from Thai
land by metasomal petiole short and stout, its total length about 4.5× basal width, and
its linear part distinctly rugosely punctate (Figs 16, 19). It differs from L. postpetiolatus
and all other members of the genus by the following character combination: the tooth
above the apical spine of propodeum comparatively blunter, lower, and closer to the A taxonomic account of the genus Labus de Saussure, 1867... 31 Figures 13–19. 13–16 Labus robustus sp. n., holotype 13 habitus in dorsal view, ♂ 14 Head in frontal
view, ♂ 15 propodeum in lateral view, ♂ 16 metasomal petiole, ♂ 17–19 Labus postpetiolatus Gusen
leitner, 1988. 17 habitus in dorsal view, ♀ 18 propodeum in lateral view, ♀ 19 metasomal petiole, ♀. Figures 13–19. 13–16 Labus robustus sp. n., holotype 13 habitus in dorsal view, ♂ 14 Head in frontal
view, ♂ 15 propodeum in lateral view, ♂ 16 metasomal petiole, ♂ 17–19 Labus postpetiolatus Gusen
leitner, 1988. 17 habitus in dorsal view, ♀ 18 propodeum in lateral view, ♀ 19 metasomal petiole, ♀. apical spine (Fig. 15); swollen part of metasomal petiole sparsely punctate (Fig. 16), in
L. postpetiolatus punctures more densely punctate (Fig. 19). Etymology. The specific name is derived from one Latin word: robustus, referring
to metasomal petiole stout. 32 T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) Figures 20–24. 20–22 Labus amoenus van der Vecht, 1935. 20 head in frontal view, ♀ 21 Head in
frontal view, ♂ 22 frontal fovea, ♀ 23–24 Labus angularis van der Vecht, 1935. 23 habitus, ♀ 24 head
in frontal view, ♀. Figures 20–24. 20–22 Labus amoenus van der Vecht, 1935. 20 head in frontal view, ♀ 21 Head in
frontal view, ♂ 22 frontal fovea, ♀ 23–24 Labus angularis van der Vecht, 1935. 23 habitus, ♀ 24 head Figures 20–24. 20–22 Labus amoenus van der Vecht, 1935. 20 head in frontal view, ♀ 21 Head in
frontal view, ♂ 22 frontal fovea, ♀ 23–24 Labus angularis van der Vecht, 1935. 23 habitus, ♀ 24 head
in frontal view, ♀. Labus amoenus van der Vecht, 1935
Figs 20–22 Vietnam (new record), China (new record); India; Laos; Malaysia;
Singapore; Indonesia. Labus amoenus van der Vecht, 1935
Figs 20–22 Figs 20–22 Figs 20–22 Labus armatus Cameron, 1900: 536; Dalla Torre 1904: 13; Kohl 1907: 242; van der
Vecht 1935: 159; Gusenleitner 2011: 1358. Junior secondary homonym of Labus
armatus (Gribodo, 1891). Labus amoenus van der Vecht, 1935: 159, 161, 162; 1963: 9; Gusenleitner 1988: 189,
194; Girish Kumar et al. 2014: 29–37. Labus amoenus van der Vecht, 1935: 159, 161, 162; 1963: 9; Gusenleitner 1988: 189,
194; Girish Kumar et al. 2014: 29–37. Material examined. 8♀♀2♂♂, Vietnam, Nghē An Prov. Khe Bo 19°3'N, 104°43'E,
123m, 25–28.IV.1998, James M. Carpenter (AMNH); 1♂, Vietnam: Hà Giang Prov. Cao Bō, Tham Ve River 22°45'N, 104°55'E, 295m, 24–25.IV.2000, James M. Car A taxonomic account of the genus Labus de Saussure, 1867... 33 penter (AMNH); 2♀♀2♂♂, China, Yunnan Prov., Puer City, Lancang County, Mulan
Town, 27.VII.2014, Pan Huang (CQNU); 2♂♂, Malaysia, Johor Panti Forest Reserve
01°51.45'N, 103°53.10'E, 23m, 1. VI. 2009, James M. Carpenter (AMNH); 2♀♀,
Malaysia, Johor Kota Tinggi Waterfalls, 01°49,45'N, 103°50.02'E, 16m, 1.VI.2009,
James M. Carpenter (AMNH); 1♂, Malaysia, Johor Panti Nature Park, 01°47.16'N,
103°56.33'E, 27m, 30.VII.2009, James M. Carpenter (AMNH); 1♂, Thailand, Phet
chabun Khao Kho NP decid. Forest 16°39.587'N, 101°08.134'E, 220m, Malaise trap,
26.II–5.III.2007, Somchai Chachumnan & Saink Singtong leg. (AMNH); 1♂, Thai
land, Phetchabun Khao Kho NP decid. Forest, 16°32.539'N, 101°2.483'E,524m, Ma
laise trap, 12–19.XII.2006, Somchai Chachumnan & Saink Singtong leg. (AMNH);
1♂, Thailand, Ratchathani Pha Taem NPE. of ThungLuang Dipt. for 15°39.989'N,
105°30.468'E, 238m, Malaise trap, 1–7.I.2007, Thongkam & Pakdee leg. (AMNH); 1♀,
Thailand, Chaiyaphum TatToneNP Nrsry at hd.wtr., 15°58,344'N, 102°2.169'E, 257m,
Malaise trap, 19–26.II.2007, Tawit Jaruphan & Orawan Budsawong leg. (AMNH); 1♂,
Singapore, under bank Felled trees New Road by Racecourse, 25.IV.1976 (AMNH). Diagnosis. Body length 7.0–8.0 mm; head (Figs 20–21) in frontal view slightly
longer than wide, its sides somewhat flattened; in female, frontal fovea (Fig. 22) el
liptical; punctures on clypeus sparse and indistinct; clypeus in female basally with a
yellow transverse band and in male almost totally yellow in some specimens; anterior
angles of pronotum projecting strongly outwards; the eraised area of the scutellum
with slightly projecting posterior angles; propodeum posteriorly on each side with a
moderate tooth above the apical spine formed by the submarginal carina; metasomal
petiole long and slender, total length of petiole 7.93× its basal width and 3.31× api
cal one; swollen part of metasomal petiole less than (0.89×) half of the length of the
petiole and irregularly punctate. Distribution. Labus clypeatus van der Vecht, 1935 Labus clypeatus van der Vecht, 1935: 162, 166–167; Gusenleitner 1988: 188, 193;
Girish Kumar et al. 2014: 36; Nguyen et al. 2014: 11. Labus clypeatus van der Vecht, 1935: 162, 166–167; Gusenleitner 1988: 188, 193;
Girish Kumar et al. 2014: 36; Nguyen et al. 2014: 11. Material examined. No specimens examined. Material examined. No specimens examined. Diagnosis. Clypeus somewhat shorter, the median portion less produced, its teeth
slightly blunter; spine of metanotum somewhat blunter; posterior excavation of pro
podeum not margined above, and propodeum posteriorly on each side without a tooth
above the apical spine formed by the submarginal carina; apical lamellae sharp; meta
somal petiole slender, its total length more than 9× basal width, the swollen part more
than 3× as wide as the basal linear part, about 2/3× as long as the lineal part and more
gradually swollen than in L. spiniger de Saussure, 1867; punctuation the same as in L. spiniger (van der Vecht 1935). Distribution. Vietnam; Indonesia (Java). Labus exiguus (de Saussure, 1855) Eumenes exiguus de Saussure, 1855: 150; Dalla Torre 1904: 22. Labus exigua: Dover, 1925: 291; van der Vecht 1935: 158; Girish Kumar et al. 2014: 36. Eumenes exiguus de Saussure, 1855: 150; Dalla Torre 1904: 22. Labus exigua: Dover, 1925: 291; van der Vecht 1935: 158; Girish Kumar et al. 2014: 36. g
Labus exigua: Dover, 1925: 291; van der Vecht 1935: 158; Girish Kumar et al. 2014: 36. Labus angularis van der Vecht, 1935
Figs 23–24 Labus angularis van der Vecht, 1935: 161, 164; Gusenleitner 1988: 174, 189, 190;
Madl 1995: 398; Gusenleitner 2006: 687; Girish Kumar et al. 2014: 29–37. Material examined. 2♀♀, China, Yunnan Prov., Xishuangbanna City, Mengla Coun
ty, Shangyong Town, Longmen Village, 2011.VIII.3–5, Yong Zhou (CQNU). Material examined. 2♀♀, China, Yunnan Prov., Xishuangbanna City, Mengla Coun
ty, Shangyong Town, Longmen Village, 2011.VIII.3–5, Yong Zhou (CQNU). Diagnosis. Head (Fig. 24) in frontal view approximately as long as wide, its sides
rounded; anterior angles of pronotum projecting only slightly; median area of scutel
lum rounded posteriorly; propodeum posteriorly on each side with a moderate tooth
above the apical spine formed by the submarginal carina; metasomal petiole long and
slender, total length of petiole 8.3× basal width and 3.07× apical one, swollen part of
metasomal petiole less than half of the length of the petiole (Fig. 23).h Distribution. China (new record); India; Myanmar; Thailand; Indonesia Distribution. China (new record); India; Myanmar; Thailand; Indonesia. 34 T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) Material examined. No specimens examined. Material examined. No specimens examined.h Diagnosis. The original description of the species gave insufficient detail to dis
tinguish it from other species. Head wider than long, without indentation; metasomal
petiole punctate and without bulging, widening a little back and forth, then truncated
right to its articulation, and carrying two insensitive tubercles in front of its middle; S2
bell-shaped, but very short, appreciably wider than long, strongly swollen above and
below, and offering at its posterior edge a strong duplication of the teguments; second
cubital cell strongly narrowed towards the radial and prolonged at its posterior border,
but at its external angle; antennal hooks very small; scape yellow beneath; pronotum
anteriorly with a yellow, narrow, perfectly regular and medianly interrupted band, and
each of T1–T2 and S2 with a yellow apical band; legs ferrugineous, with some parts of
femora and mid tibia black (de Saussure 1855; van der Vecht 1935). Distribution. Malaysia; Singapore; China: Hong Kong. Distribution. Malaysia; Singapore; China: Hong Kong. Material examined. No specimens examined. Labus humbertianus de Saussure, 1867: 4; Bingham 1896: 448, 1897: 349; van der Vecht
1935: 158; 1963: 5; Gusenleitner 1988: 189, 192; Girish Kumar et al. 2014: 30–32. Labus humbertianus de Saussure, 1867 Labus humbertianus de Saussure, 1867: 4; Bingham 1896: 448, 1897: 349; van der Vecht
1935: 158; 1963: 5; Gusenleitner 1988: 189, 192; Girish Kumar et al. 2014: 30–32. Labus humbertianus de Saussure, 1867: 4; Bingham 1896: 448, 1897: 349; van der Vecht
1935: 158; 1963: 5; Gusenleitner 1988: 189, 192; Girish Kumar et al. 2014: 30–32. Material examined. No specimens examined. Material examined. No specimens examined. A taxonomic account of the genus Labus de Saussure, 1867... 35 Diagnosis. Body black, with yellowish white and brown markings. Yellowish white
markings are as follows: anterior margin of horizontal portion of pronotum, parat
egula, at apical lamellar spine of the propodeum, fore tibiae on outer side, mid tibiae
with a small yellow mark at base, a narrow thin transverse band at the posterior apex
of T1, and a thin transverse band on T2 and S2. Brown markings are as follows: tegula
(almost brownish black) except a small yellow mark at apex, propodeal valvula, and
tarsi (partly); wings rather strongly infuscated; head and thorax densely and coarsely
punctate; frontal fovea large, distinctly larger than the anterior ocellus, concavity of
fovea divided by a blunt and low median ridge; pronotal spine rather short and conical;
propodeum on each side with a long and sharp tooth above the apical lamellar spine;
petiole coarsely and densely punctate, except on the swollen part which is sparsely and
more finely punctate; swollen part as long as half of the length of the petiole (van der
Vecht 1963; Girish Kumar et al., 2014). Distribution. India; Sri Lanka; Myanmar. Distribution. India; Sri Lanka; Myanmar. Labus lofuensis Giordani Soika, 1973
Figs 25–31 Labus lofuensis Giordani Soika, 1973: 99; Gusenleitner 1988: 189, 193; Girish Kumar
et al. 2014: 36; Nguyen et al. 2014: 11. Material examined. 1♀1♂, China, Hainan Prov.,Ta Hau, 4. VII. 1935, L. Gressit
(AMNH). Diagnosis. Body (Figs 25–26) length 6.5–8.0 mm; head (Figs 27, 31) in frontal
view slightly longer than wide, its sides flattened; in female, frontal fovea much deeper,
distinctly defined, and rounded (Fig. 28); propodeum posteriorly on each side with
a moderate tooth above the apical spine formed by the submarginal carina (Fig. 29);
metasomal petiole long and slender (Fig. 30), total length of petiole 7.2× basal width
and 2.57× apical width, swollen part slightly less than (0.98×) half the length of the
petiole and with uniform punctures. Distribution. China (first record from Hainan); Vietnam. Labus philippinensis Giordani Soika, 1986
Figs 32–35 Figs 32–35 Labus philippinensis Giordani Soika, 1986: 78; Gusenleitner 1988: 196; Girish Kumar
et al. 2014: 36. Labus philippinensis Giordani Soika, 1986: 78; Gusenleitner 1988: 196; Girish Kumar
et al. 2014: 36. Material examined. 2♂♂, Philipphines, Laguna, I.R.R. I.Univ. Phil. Los Banos,
13.III.1980, J.Kojima (AMNH); 1♂, Philipphines, Bataan Prov. Luzon, Dinalupihan
Municipality, 5.5 mi. w. of Culo, 16.IX.1945, P.I. Richard Dow (AMNH); 1♀, Philip T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 36 Figures 25–31. Labus lofuensis Giordani Soika, 1973. 25 habitus, ♀ 26 habitus, ♂ 27 head in frontal
view, ♀ 28 frontal fovea, ♀ 29 propodeum in lateral view, ♂ 30 metasomal petiole, ♂ 31 head in frontal
view, ♂. Figures 25–31. Labus lofuensis Giordani Soika, 1973. 25 habitus, ♀ 26 habitus, ♂ 27 head in frontal
view, ♀ 28 frontal fovea, ♀ 29 propodeum in lateral view, ♂ 30 metasomal petiole, ♂ 31 head in frontal
view, ♂. phines, Pampanga Prov. Luzon, Road west of Ft. Stotsepburg, 4.X.1945, P.I. Richard
Dow (AMNH). Diagnosis. Body (Figs 32–33) length 6.5–7.5 mm; clypeus distinctly punctate
(Figs 34–35); head in frontal view as long as wide, its sides rounded (Figs 34–35); pro
podeum posteriorly on each side with a moderate tooth above the apical spine formed
by the submarginal carina; petiole long and slender, total length of petiole 7.88× basal Diagnosis. Body (Figs 32–33) length 6.5–7.5 mm; clypeus distinctly punctate
(Figs 34–35); head in frontal view as long as wide, its sides rounded (Figs 34–35); pro
podeum posteriorly on each side with a moderate tooth above the apical spine formed
by the submarginal carina; petiole long and slender, total length of petiole 7.88× basal A taxonomic account of the genus Labus de Saussure, 1867... 37 Figures 32–35. Labus philippinensis Giordani Soika, 1986. 32 habitus, ♀ 33 habitus, ♂ 34 head in
frontal view, ♀ 35 head in frontal view, ♂. Figures 32–35. Labus philippinensis Giordani Soika, 1986. 32 habitus, ♀ 33 habitus, ♂ 34 head in
frontal view, ♀ 35 head in frontal view, ♂. width and 2.67× apical width, swollen part of metasomal petiole much less (0.83×)
than half of the length of the petiole. Distribution Philippines width and 2.67× apical width, swollen part of metasomal petiole much less (0.83×)
than half of the length of the petiole. Distribution. Philippines. Distribution. Philippines. Distribution. Thailand. Labus pusillus van der Vecht, 1963
Figs 36–41 China (new record); Vietnam (new record); India; Sri Lanka; Nepa Labus rufomaculatus van der Vecht, 1963: 8; Gusenleitner 1988: 195; Girish Kumar
et al. 2014: 36. Labus postpetiolatus Gusenleitner, 1988
Figs 17–19 Labus postpetiolatus Gusenleitner, 1988: 174; Girish Kumar et al. 2014: 36. Labus postpetiolatus Gusenleitner, 1988: 174; Girish Kumar et al. 2014: 36. Material examined. 1♂, Thailand, Chiang Mai Prov., Chiang Dao Farm, 24–25. IX.1999, M.I. Wibawa (AMNH). Diagnosis. Body (Fig. 17) length 7.0 mm; propodeum (Fig. 18) posteriorly on
each side with a sharply acute and high tooth above the apical spine formed by the sub
marginal carina, the tooth far away from apical spine; linear part of metasomal petiole
(Fig. 19) short and stout, and distinctly rugosely punctate, total length of petiole 4.51×
basal width and 2.03× apical width, swollen part of metasomal petiole longer than half
of the length of the petiole and densely punctate.h Distribution. Thailand. T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 38 Labus pusillus van der Vecht, 1963
Figs 36–41 Labus pusillus van der Vecht, 1963: 6; Gusenleitner 1987: 255; 1988: 188, 191; 2006:
687; Girish Kumar et al. 2014: 30–34. Labus pusillus van der Vecht, 1963: 6; Gusenleitner 1987: 255; 1988: 188, 191; 2006:
687; Girish Kumar et al. 2014: 30–34. Material examined. 1♀1♂, Sri Lanka, Central Pro., Kandy Dist. Vic. Randenigala
Rantembe Sanct. 07°13'N, 80°57'E, 13.VIII.1999, MT M. & J. Wasbauer (AMNH);
1♀, Sri Lanka, Central Prov., Kandy Dist. U. Peradeniya Gannoruwa Forest Area,
250 m, 07°17'N, 80°36'E, 13–14.VIII.1999, MT M. & J. Wasbauer (AMNH); 1♀,
Sri Lanka, Central Prov., Kandy Dist. U. Peradeniya Hantana Mt., 7°15'N, 80°37'E,
10–14.VIII.1999, MT M. & J. Wasbauer (AMNH); 1♂, Sri Lanka, Nilgala Uva Prov.,
1–14. VII. 1968, P. B. Karunaratne (AMNH); 1♀1♂, Vietnam, Nghē An Prov., Khe
Bo, 19°03'N, 104°43'E, 123 m, 25–28.IV.1998, James M. Carpenter (AMNH); 1♀,
Vietnam, Ha Giang Prov., Du Gia commune, 22°54'N, 105°14'E, 680 m, 29–30. IV.2000, James M. Carpenter (AMNH); 1♂, India: Cherangode, Nilgiri Hills 3500,
V, 1950, P. S. Nathan; 1♀, Sri Lanka, Colomlo; 2♀1♂, China, Yunnan Prov., Lijiang
City, Yulong County, Shudi valley, 4. V. 2004, Ting-Jing Li (CQNU); 1♀, China,
Yunnan Prov., Kunming City, Ciba Town, Helongtan mountain, 19.VI.2002, Peng
Wang (CQNU); 1♂, China, Yunnan Prov., Dali City, Gucheng Town, Yitasi, 19.VIII. 2003, Ming Luo (YNAU); 1♂, China, Yunnan Prov., Puer City, Jingdong County,
nearby Chuan river, 28.IV.2004, Kai Wu (YNAU); 1♀, China, Yunnan Prov., Wen
shan City, Qiubei County, Liulangdong, 3.V.2004, Peng Wang (CQNU); 1♀, China,
Yunnan Prov., Puer City, Jingdong County, Xujiaba, 30.IV.2005, Li Ma (YNAU);
1♀, China, Yunnan Prov., Baoshan City, Lujiang Ba, Pumanshao, 17.VII.2006, Rui
Zhang (YNAU); 2♀♀2♂♂, China, Sichuan Prov., Panzhihua City, Miyi County, Bai
ma, 29.VII.2011, Ting-Jing Li (CQNU); 1♀, China, Sichuan Prov., Liangshan City,
Dechang County, Yonglang Town, 1.VIII.2011, Tingjing Li (CQNU). g
y
g
g
gj g
Diagnosis. Body (Figs. 36, 39) length 6.0–7.5 mm; the median portion of clypeus
(Figs 37–38) slightly more produced, its emargination narrower medially and lateral
teeth sharper; propodeum posteriorly on each side without a tooth above the apical
spine formed by the submarginal carina (Fig. 40); the linear part of metasomal petiole
(Fig. 41) moderately long and slender, and slightly rugosely punctate, total length of
petiole 6.53× basal width and 2.45× apical width, swollen part of metasomal petiole
longer than (1.06×) half of the length of the petiole; in some specimens swollen part of
metasomal petiole apically ferrugineous. Distribution. Labus rufomaculatus van der Vecht, 1963 39 A taxonomic account of the genus Labus de Saussure, 1867... 39 Figures 36–41. Labus pusillus van der Vecht, 1963. 36 habitus, ♀ 37 head in frontal view, ♀ 38 head in
frontal view, ♂ 39 habitus, ♂ 40 propodeum in dorsal view, ♀ 41 metasomal petiole, ♀. Figures 36–41. Labus pusillus van der Vecht, 1963. 36 habitus, ♀ 37 head in frontal view, ♀ 38 head in
frontal view, ♂ 39 habitus, ♂ 40 propodeum in dorsal view, ♀ 41 metasomal petiole, ♀. Material examined. No specimens examined. Diagnosis. Closely allied to L. amoenus van der Vecht, 1935 with the following
characters in common: head higher than wide, the sides slightly flattened; anterior
lateral angles of pronotum each produced into a sharp spine; metanotum with sharp
median tooth; propodeum, as seen in profile, with short blunt tooth above the apical T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 40 tooth; metasomal petiole relatively long and slender (Van der Vecht 1935, fig. ia); the
morphological characters separating the two species are as follows: frontal fovea deeper
and slightly smaller, circular or even a little wider than long, distinctly defined; frons
not carinate; median part of scutellum regularly rounded posteriorly, without promi
nent angles; punctures on head and thorax very slightly coarser than L. amoenus; body
black, without yellow spots, and with the following parts red: an ill-defined transverse
spot at base of clypeus, a rather wide transverse band at anterior margin of horizontal
surface of pronotum, a small spot in upper part of mesepisternum below tegulae, a
transverse band on scutellum, interrupted by the black median groove, and the greater
part of the metasomal petiole (van der Vecht 1963). Distribution. Indonesia. Distribution. Indonesia. Labus spiniger de Saussure, 1867
Figs 42–46 Labus spiniger de Saussure, 1867: 4; van der Vecht 1935: 162, 165; 1963: 10; Gusen
leitner 1988: 189, 191; Girish Kumar et al. 2014: 36. Labus spiniger de Saussure, 1867: 4; van der Vecht 1935: 162, 165; 1963: 10; Gusen
leitner 1988: 189, 191; Girish Kumar et al. 2014: 36. Material examined. 1♀, China, Hainan Prov., Ta Hau, 04.VII.1935, L. Gressitt
(AMNH); 1♂, China, Hainan Prov., Ta Hian, 19.VI.1935, L. Gressit (AMNH). Material examined. 1♀, China, Hainan Prov., Ta Hau, 04.VII.1935, L. Gressitt
(AMNH); 1♂, China, Hainan Prov., Ta Hian, 19.VI.1935, L. Gressit (AMNH). Diagnosis. Frons coarsely and densely punctate (Figs. 43, 45), inter-antennal ca
rina continued on lower part of frons; propodeum posteriorly on each side with a
tooth above the apical spine formed by the submarginal carina (Fig. 46); metasomal
petiole dark ferruginous with exception of apical yellow band (Fig. 44), the linear part
of petiole long and slender, and slightly rugosely punctate, total length of petiole 7.7×
basal width and 2.6× apical width, swollen part of metasomal petiole less than (0.47×)
half of the length of the petiole. Distribution. China (new record); Indonesia. Distribution. China (new record); Indonesia. Labus sumatrensis Giordani Soika, 1991 Labus sumatrensis Giordani Soika, 1991: 163; Girish Kumar et al. 2014: 36. Material examined. No specimens examined. Labus vandervechti Giordani Soika, 1958 Labus vandervechti Giordani Soika, 1958: 83; van der Vecht 1963: 9; Gusenleitner
1988: 195, 197, 198; Girish Kumar et al. 2014: 36. Material examined. No specimens examined. Diagnosis. Allied to L. angularis van der Vecht, 1935 mainly distinguished by
the shape of clypeus which is very weakly marginalized at the apex, with apical teeth
replaced by two undeveloped tubercles; punctures on frons thicker than those in L. angularis and on thorax visibly larger, punctuation of metasoma approximately as in L. angularis (Giordani Soika 1991). Distribution. Indonesia. A taxonomic account of the genus Labus de Saussure, 1867... 41 Figures 42–46. Labus spiniger de Saussure, 1867. 42 habitus, ♂ 43 head in frontal view, ♀ 44 metasomal
petiole, ♂ 45 head in frontal view, ♂ 46 propodeum in lateral view, ♂. Figures 42–46. Labus spiniger de Saussure, 1867. 42 habitus, ♂ 43 head in frontal view, ♀ 44 metasomal
petiole, ♂ 45 head in frontal view, ♂ 46 propodeum in lateral view, ♂. Material examined. No specimens examined. Diagnosis. Very closely allied to L. spiniger de Saussure, 1867 (van der Vecht 1935:
165), but the horizontal part of the propodeum more distinctly punctate, the punctures
not much closer, but slightly larger, and better defined; the sides of the propodeum more
shiny between the punctures (rugose and rather dull in L. spiniger), the transverse ridge
at apex of propodeum more pronounced, as seen in profile forming a more distinctly
projecting tooth; swollen part of metasomal petiole relatively longer, nearly half as long
as the petiole; second metasomal segment relatively longer (length: width = 1.2 : 1, in
L. spiniger = 1.1: 1); metasomal petiole red, with narrow yellow apical band; legs ferrugi T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 42 nous, coxae and trochanters dark brown to black, fore femur I with vague yellow spot at
apex, mid and hind femora fuscous at base, hind tibiae fuscous at apex, tarsal segments
2–5 of mid legs and 1–5 of hind legs fuscous (van der Vecht 1963). Distribution. Indonesia. Distribution. Indonesia. Key to the world species of Labus de Saussure In the below key, information on L. clypeatus van der Vecht, L. exiguus (de Saussure),
L. humbertianus de Saussure, L. rufomaculatus van der Vecht, L. sumatrensis Giordani
Soika and L. vandervechti Giordani Soika is extracted from the original descriptions,
and the characters of other species were studied on specimens. 1
Propodeum posteriorly on each side without a tooth above the apical spine
formed by the submarginal carina (Figs 5, 11, 40).......................................2
–
Propodeum posteriorly on each side with a tooth above the apical spine
formed by the submarginal carina (Figs 18, 29, 46).....................................5
2
Swollen part of metasomal petiole as long as or longer than half of the length
of the petiole (Figs 4, 41).............................................................................3
–
Swollen part of metasomal petiole less than half of the length of the petiole
(Fig. 12).......................................................................................................4
3
The median portion of clypeus less produced, its emargination wide medi
ally and lateral teeth slightly blunter (Fig. 2); frontal fovea big and rounded,
much larger than the anterior ocellus (Fig. 3)................... L. edentatus sp. n. –
The median portion of clypeus slightly more produced, its emargination nar
rower medially and lateral teeth sharper (Figs 37, 38); frontal fovea smaller and
not distinctly defined, slightly larger than the anterior ocellus (Fig. 37)............. ........................................................................L. pusillus van der Vecht, 1963
4
Frons sparsely punctate and more shiny between the punctures (Figs 8, 10);
total length of petiole shorter, 7.53×its basal width in female and 8.07×its
basal width in male ...................................................L. sparsipunctus sp. n. –
Frons coarsely and densely punctate, and duller between the punctures; total
length of petiole more than 9× basal width.... L. clypeatus van der Vecht, 1935
5
The linear part of metasomal petiole short and wide, and distinctly rugosely punc
tuate, total length of petiole about or less than 4.51× basal width (Figs 16, 19)....6
–
The linear part of metasomal petiole long and narrow, and just slightly ru
gosely punctuate, total length of petiole at least more than 6.5× basal width
(Figs 23, 30, 32–33, 44)..............................................................................7
6
The tooth above the apical spine of propodeum sharply acute and high, and
far away from apical spine (Fig. 18); swollen part of metasomal petiole more
densely punctate (Fig. 19)................... L. postpetiolatus Gusenleitner, 1988
–
The tooth above the apical spine of propodeum comparatively blunter and
lower, and closer to the apical spine (Fig. 15); swollen part of metasomal petiole
sparsely punctate (Fig. 16)..................................................... L. robustus sp. n. Key to the world species of Labus de Saussure 1
Propodeum posteriorly on each side without a tooth above the apical spine
formed by the submarginal carina (Figs 5, 11, 40).......................................2
–
Propodeum posteriorly on each side with a tooth above the apical spine
formed by the submarginal carina (Figs 18, 29, 46).....................................5
2
Swollen part of metasomal petiole as long as or longer than half of the length
of the petiole (Figs 4, 41).............................................................................3
–
Swollen part of metasomal petiole less than half of the length of the petiole
(Fig. 12).......................................................................................................4
3
The median portion of clypeus less produced, its emargination wide medi
ally and lateral teeth slightly blunter (Fig. 2); frontal fovea big and rounded,
much larger than the anterior ocellus (Fig. 3)................... L. edentatus sp. n. –
The median portion of clypeus slightly more produced, its emargination nar
rower medially and lateral teeth sharper (Figs 37, 38); frontal fovea smaller and
not distinctly defined, slightly larger than the anterior ocellus (Fig. 37)............. ........................................................................L. pusillus van der Vecht, 1963
4
Frons sparsely punctate and more shiny between the punctures (Figs 8, 10);
total length of petiole shorter, 7.53×its basal width in female and 8.07×its
basal width in male ...................................................L. sparsipunctus sp. n. –
Frons coarsely and densely punctate, and duller between the punctures; total
length of petiole more than 9× basal width.... L. clypeatus van der Vecht, 1935
5
The linear part of metasomal petiole short and wide, and distinctly rugosely punc
tuate, total length of petiole about or less than 4.51× basal width (Figs 16, 19)....6
–
The linear part of metasomal petiole long and narrow, and just slightly ru
gosely punctuate, total length of petiole at least more than 6.5× basal width
(Figs 23, 30, 32–33, 44)..............................................................................7
6
The tooth above the apical spine of propodeum sharply acute and high, and
far away from apical spine (Fig. 18); swollen part of metasomal petiole more
densely punctate (Fig. 19)................... L. postpetiolatus Gusenleitner, 1988
–
The tooth above the apical spine of propodeum comparatively blunter and
lower, and closer to the apical spine (Fig. 15); swollen part of metasomal petiole
sparsely punctate (Fig. 16)..................................................... L. robustus sp. n. 6 A taxonomic account of the genus Labus de Saussure, 1867... Key to the world species of Labus de Saussure 43 7
Swollen part of metasomal petiole half of the length of the petiole...............8
–
Swollen part of metasomal petiole less than half of the length of the petiole
(Figs 23, 30, 32–33, 44)..............................................................................9
8
Legs black, with the following parts yellow: fore tibiae on outerside and mid
tibiae with a small mark at base (van der Vecht 1963; Girish Kumar et al. 2014) ...................................................L. humbertianus de Saussure, 1867
–
Legs ferrugineous, only with some parts of femora and midtibia black (van
der Vecht 1935)............................................L. exiguus (de Saussure, 1855)
9
Head in frontal view as long as wide, its sides rounded (Figs. 24, 34–35)... 10
–
Head in frontal view longer than wide, its sides somewhat flattened (Figs
20–21, 27, 31, 43, 45)...............................................................................12
10
Clypeus distinctly punctate, punctures larger and relatively denser (Figs 34–
35)..................................................L. philippinensis Giordani Soika, 1986
–
Clypeus less distinctly punctate, punctures sparser (Fig. 24)......................11
11
Punctures on frons thicker than in the L. angularis and those on mesosoma
visibly larger....................................... L. sumatrensis Giordani Soika, 1991
–
Punctures on frons sparser and those on mesosoma smaller than the above
species....................................................... L. angularis van der Vecht, 1935
12
Frons not carinate; body with red or ferruginous spots and without yellow
marks................................................L. rufomaculatus van der Vecht, 1963
–
Inter-antennal carina continued on lower part of frons; body with distinct
yellow spots or bands.................................................................................13
13
Metasomal petiole at least partly red or reddish brown, with yellow apical
narrow band; frontal fovea relatively bigger (Figs 20, 28)...........................14
–
Metasomal petiole black, with narrow yellow apical band; frontal fovea smaller
(Fig. 43)......................................................................................................... 15
14
The horizontal part of the propodeum punctate, the punctures comparatively
smaller; the sides of the propodeum rugose and rather dull between the punc
tures; the transverse ridge at apex of propodeum less pronounced.................. .......................................................................L. spiniger de Saussure, 1867
–
The horizontal part of the propodeum more distinctly punctate, the punc
tures not much closer, but slightly larger, and better defined; the sides of the
propodeum more shiny between the punctures, the transverse ridge at apex
of propodeum more pronounced, as seen in profile forming a more distinctly
projecting tooth (Giordani Soika 1958; van der Vecht 1963)......................... .........................................................L. vandervechti Giordani Soika, 1958
15
In female, frontal fovea much deeper, distinctly defined, and rounded (Fig. 28); the swollen part of the metasomal petiole with uniform punctures......... ................................................................L. Key to the world species of Labus de Saussure lofuensis Giordani Soika, 1973
–
In female, frontal fovea shallow, longer than wide, not distinctly defined (Fig. 22); the swollen part of the metasomal petiole irregularly punctate................ .................................................................. L. amoenus van der Vecht, 1935 T-J. Li & J.M. Carpenter / Journal of Hymenoptera Research 65: 23–46 (2018) 44 Acknowledgements We are very grateful to Christophe Barthélémy (Hongkong, China) for sending the
specimen and allowing its deposition in AMNH. This study was funded by the Na
tional Natural Science Foundation of China (Nos: 31772490, 31372247, 31000976),
Young Talent Incubation Programme of Chongqing Normal University (14CSDG07). References Bingham CT (1896) A contribution to the knowledge of the hymenopterous fauna
of Ceylon. Proceedings of the Zoological Society, London: 401–459. https://doi. org/10.1111/j.1096-3642.1896.tb03051.x Bingham CT (1897) Fauna of British India, including Ceylon and Burma – Hymenoptera
I – Wasps and Bees. Taylor and Francis, London, 579 pp. https://doi.org/10.5962/bhl. title.100738 Cameron P (1900) Descriptions of new genera and species of Hymenoptera. Annals
and Magazine of Natural History (7)6: 410–419, 495–506, 530–539. https://doi. org/10.1080/00222930008678422 Dalla Torre KW von (1904) Vespidae. Genera Insectorum 19: 1–108. Dover C (1925) Further notes on the Indian Diplopterous wasps. Journal of Asiatic Society of
Bengal, NS 22: 289–305. Giordani Soika A (1960 [1958]) Notulae vespidologicae 4–9. Bollettino del Museo Civico di
Storia Naturale di Venezia 11: 35–102. Giordani Soika A (1973) Notulae vespidologicae 35. Descrizione di nuovi Eumenidi. Bollet
tino del Museo Civico di Storia Naturale di Venezia 24: 97–131. Giordani Soika A (1986) Eumenidi di Okinawa e delle Filippine raccolti da J. Kojima. Bollet
tino del Museo Civico di Storia Naturale di Venezia 35: 67–89. Giordani Soika A (1991) Eumenidi raccolti in Indonesia da G. Osella e J. Klapperich. Bollet
tino del Museo Civico di Storia Naturale di Verona 15: 163–166. Girish Kumar P, Mohammed Shareef KP, Kishore L, Carpenter JM (2014) A taxonomic review
of the Oriental genus Labus de Saussure, 1867 (Hymenoptera: Vespidae: Eumeninae) from
the Indian subcontinent. Biosystematica 7(2): 29–37. Gusenleitner J (1987) Über Eumenidae aus Nepal (Hymenoptera: Vespoidea). Linzer Biologis
che Beiträge 19(1): 255–270. Gusenleitner J (1988) Über Eumenidae aus Thailand, mit einer Bestimmungstabelle fur ori
entalischer Labus-Arten (Hymenoptera: Vespoidea). Linzer biologische Beiträge 20(1):
173–198. Gusenleitner J (2006) Über Aufsammlungen von Faltenwespen in Indien (Hymenoptera: Vesp
idae). Linzer biologische Beiträge 38(1): 677–695. Gusenleitner J (2011) Eine Aufsammlung von Faltenwespen aus Laos im Biologiezentrum Linz
(Hymenoptera: Vespidae: Vespinae, Stenogastrinae, Polistinae, Eumeninae). Linzer biolo
gische Beiträge 43(2): 1351–1368. A taxonomic account of the genus Labus de Saussure, 1867... 45 Kohl FF (1907) Zoologische Ergebnisse der Expedition der K. Akademie dei Wissenschaften
nach Südarabien und Sokotra im Jahre 1898-1899. Denkschriften Akademie der Wissen
schaften in Wien, Mathematisch-Naturwissenschaftliche Klasse 71(1): 170–302. Madl M (1995) Contribution to the Hymenoptera – Fauna of Thale Ban National Park. Linzer
biologische Beiträge 27(1): 397–399. Nguyen LTP, Dang HT, Kojima J, Carpenter JM (2014) An annotated distributional checklist
of solitary wasps of the subfamily Eumeninae (Hymenoptera: Vespidae) of Vietnam. Ento
mologica Americana 120(1/2): 7–17. References https://doi.org/10.1664/13-RA-010.1 Saussure H de (1855) Études sur la Famille des Vespides 2. La Monographie des Masariens et
un supplément la Monographie des Euméniens. V. Masson, Paris & J. Kessmann, Genéve,
49–288 pp. Saussure H de (1867) Reise der Österreichischen Fregatte Novara um die Erde in den jahren
1857, 1858, 1859., Zool. Teil, 2 Band 1. Abteil A. 2. Hymenoptera, Wien, 142 pp. Vecht J van der (1935) Notes on Oriental Labus, with descriptions of three new species from
Java (Hymenoptera: Vespidae). Treubia 15(2): 157–167. Vecht J van der (1963) Studies on Indo-Australian and East Asiatic Eumenidae (Hymenoptera:
Vespoidea). Zoologische Verhandelingen Leiden 60: 1–116.
|
https://openalex.org/W4233357843
|
https://zenodo.org/record/19896/files/Jiao_Biogeosciences.pdf
|
English
| null |
Mechanisms of microbial carbon sequestration in the ocean – future research directions
| null | 2,014
|
cc-by
| 21,360
|
Biogeosciences, 11, 5285–5306, 2014
www.biogeosciences.net/11/5285/2014/
doi:10.5194/bg-11-5285-2014
© Author(s) 2014. CC Attribution 3.0 License. Biogeosciences, 11, 5285–5306, 2014
www.biogeosciences.net/11/5285/2014/
doi:10.5194/bg-11-5285-2014
© Author(s) 2014. CC Attribution 3.0 License. Mechanisms of microbial carbon sequestration in
the ocean – future research directions Thomas (helmuth.thomas@dal.ca) Received: 8 May 2014 – Published in Biogeosciences Discuss.: 3 June 2014 Received: 8 May 2014 – Published in Biogeosciences Discuss.: 3 June 2014
Revised: 27 August 2014 – Accepted: 27 August 2014 – Published: 1 October 2014 y
g
Revised: 27 August 2014 – Accepted: 27 August 2014 – Published: 1 October 2014 Analyses of biomarkers and isotopic records show inten-
sive MCP processes in the Proterozoic oceans when the MCP
could have played a significant role in regulating climate. Un-
derstanding the dynamics of the MCP in conjunction with
the better constrained biological pump (BP) over geological
timescales could help to predict future climate trends. Inte-
gration of the MCP and the BP will require new research
approaches and opportunities. Major goals include under-
standing the interactions between particulate organic carbon
(POC) and RDOC that contribute to sequestration efficiency,
and the concurrent determination of the chemical composi-
tion of organic carbon, microbial community composition
and enzymatic activity. Molecular biomarkers and isotopic
tracers should be employed to link water column processes Abstract. This paper reviews progress on understanding bi-
ological carbon sequestration in the ocean with special refer-
ence to the microbial formation and transformation of recal-
citrant dissolved organic carbon (RDOC), the microbial car-
bon pump (MCP). We propose that RDOC is a concept with
a wide continuum of recalcitrance. Most RDOC compounds
maintain their levels of recalcitrance only in a specific en-
vironmental context (RDOCt). The ocean RDOC pool also
contains compounds that may be inaccessible to microbes
due to their extremely low concentration (RDOCc). This dif-
ferentiation allows us to appreciate the linkage between mi-
crobial source and RDOC composition on a range of tempo-
ral and spatial scales. Mechanisms of microbial carbon sequestration in
the ocean – future research directions N. Jiao1, C. Robinson2, F. Azam3, H. Thomas4, F. Baltar5, H. Dang1, N. J. Hardman-Mountford6, M. Johnson2,
D. L. Kirchman7, B. P. Koch8, L. Legendre9,10, C. Li11, J. Liu1, T. Luo1, Y.-W. Luo1, A. Mitra12, A. Romanou13,
K. Tang1, X. Wang14, C. Zhang15, and R. Zhang1 1State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China
2School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, UK
3Scripps Institution of Oceanography, UCSD, La Jolla, CA 920193, USA 1State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen 361005, China
2School of Environmental Sciences, University of East Anglia, Norwich Research Park, Norwich, UK
3Scripps Institution of Oceanography, UCSD, La Jolla, CA 920193, USA
4 pp
g p y
4Dalhousie University, Halifax, Nova Scotia, Canada 4Dalhousie University, Halifax, Nova Scotia, Canada 5Department of Marine Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
6CSIRO Marine and Atmospheric Research, Floreat, WA 6014, Australia 5Department of Marine Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
6CSIRO M i
d At
h i R
h Fl
t WA 6014 A
t li 5Department of Marine Science, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
6CSIRO Marine and Atmospheric Research Floreat WA 6014 Australia 6CSIRO Marine and Atmospheric Research, Floreat, WA 6014, Australia 7School of Marine Science and Policy, University of Delaware, DE 19958, USA
8 7School of Marine Science and Policy, University of Delaware, DE 19958, USA 8Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung, 27570 Bremerhaven, Germany
9Sorbonne Universités, UPMC Univ. Paris 06, UMR7093, Laboratoire d’Océanographie de Villefranche,
06230 Villefranche-sur-Mer, France 8Alfred-Wegener-Institut Helmholtz-Zentrum für Polar- und Meeresforschung, 27570 Bremerhaven, Germany
9Sorbonne Universités, UPMC Univ. Paris 06, UMR7093, Laboratoire d’Océanographie de Villefranche,
06230 Villefranche-sur-Mer, France 10CNRS, UMR7093, Laboratoire d’Océanographie de Villefranche, 06230 Villefranche-sur-Mer, France
11Chinese University of Geology, Wuhan, China
12 12Centre for Sustainable Aquatic Research, Swansea University, Swansea, UK
13 Dept. of Applied Physics and Applied Math., Columbia University and NASA-Goddard Institute fo
880 Broadway, New York, NY 10025, USA 14South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
15Tongji University, Shanghai, China 14South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, China
15Tongji University, Shanghai, China Correspondence to: N. Jiao (jiao@xmu.edu.cn), C. Robinson (carol.robinson@uea.ac.uk
F. Azam (fazam@ucsd.edu), and H. Thomas (helmuth.thomas@dal.ca) Correspondence to: N. Jiao (jiao@xmu.edu.cn), C. Robinson (carol.robinson@uea.ac.uk),
F. Azam (fazam@ucsd.edu), and H. 1
Introduction The ocean absorbs approximately 30 % of anthropogenic
CO2 (IPCC, 2013), mitigating global warming in a profound
way. However, the biological mechanisms for long-term car-
bon sequestration in the ocean are not fully understood. The
biological pump (BP) is the collective term for a suite of
processes by which carbon dioxide that is fixed by phyto-
plankton photosynthesis in the euphotic zone is exported to
the deep ocean. These processes include the passive flux of
sinking organic particles (dead cells, faecal pellets, etc.), the
active flux of dissolved and particulate organic material me-
diated by vertical migration of zooplankton, and the verti-
cal transport of dissolved organic material by physical pro-
cesses. Around 50 % of the photosynthetically produced par-
ticulate organic carbon (POC) is transformed through mech-
anisms including excretion, zooplankton grazing, viral lysis
and the action of microbial ectohydrolases into dissolved or-
ganic carbon (DOC) (Anderson and Tang, 2010). The pro-
duction rate and chemical composition of this dissolved or-
ganic matter (DOM) is influenced by the nutrient status and
community composition of the microbial food web. Opera-
tionally DOC is defined as all compounds less than 0.2 µm
in size (Carlson et al., 2002), and thus will include micropar-
ticulates (e.g., cell wall fragments, membranes, viruses etc.)
and metabolites leaked/released by photo-autotrophs, defe-
cated by phagoheterotrophs and associated with viral lysis of
host cells. Marine microbes readily utilize most of this DOC,
producing CO2 and in turn transforming the composition of
the DOM. However, an estimated ∼5–7 % of the microbially
produced DOC is recalcitrant (RDOC) and resists rapid rem-
ineralization (Ogawa et al., 2001; Gruber et al., 2006; Koch
et al., 2014), which enables the DOC to be exported below
the seasonal thermocline and sequestered in the oceans’ inte-
rior. The production and transformation of RDOC is intricately
linked with the production and transformation of POC; thus
it is timely to investigate these interactions. It is not known
whether the cycling of POC and DOC would interact to en-
hance or decrease their individual effects, but it is possible
that perturbations such as eutrophication or increasing tem-
perature could cause a shift in the balance of carbon seques-
tration via dissolved versus particulate forms. In addition,
their combined response to environmental conditions may be
regulated differently under different conditions, for example,
in coastal eutrophic waters compared to oceanic oligotrophic
waters. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5286 to sediment records, as well as to link present-day observa-
tions to paleo-evolution. Ecosystem models need to be devel-
oped based on empirical relationships derived from bioassay
experiments and field investigations in order to predict the
dynamics of carbon cycling along the stability continuum
of POC and RDOC under potential global change scenarios. We propose that inorganic nutrient input to coastal waters
may reduce the capacity for carbon sequestration as RDOC. The nutrient regime enabling maximum carbon storage from
combined POC flux and RDOC formation should therefore
be sought. rable to the inventory of atmospheric CO2 (Hansell et al.,
2009), trade off between the two carbon pools would influ-
ence climate change. Hence the relative rates of POC export,
production of RDOC and respiration of POC and DOC regu-
late the timescale over which carbon is stored in the ocean’s
interior, and small changes to these rates would have a major,
potentially detrimental, impact on atmospheric CO2. While numerous experiments have assessed the sensitiv-
ity of POC export to changes in stratification, mixing and
remineralization depth (Kriest et al., 2010; Romanou et al.,
2014), little attention has been paid to the environmental fac-
tors and anthropogenic perturbations, such as ocean acidi-
fication (OA) and eutrophication, which might control the
rates of RDOC production and transformation. Given the
vast abundance and diversity of microbes (ranging from auto-
and heterotrophic prokaryotes through to photoauto-, mixo-
and phagoheterotrophic protists), the complexity of micro-
bial ecosystems and the sensitivity of microbes to environ-
mental change, small shifts in microbial metabolic efficiency
potentially cause large changes to carbon sequestration (Mi-
tra et al., 2014). Without fully understanding the microbial
processes, we risk overlooking a crucial feedback of the over-
all system that is caused by a seemingly minor perturbation
of an individual process. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. www.biogeosciences.net/11/5285/2014/ 2
The nature and controls of DOC in the ocean Up-
per panel: microbial response (in terms of abundance or uptake rate)
to DOC availability as a reference to conceptualize the microbial
uptake threshold for RDOCc, microbial abundance corresponding
to DOC concentrations of 40 µM in the deep and 70 µM in the sur-
face oceans; LDOC – labile DOC, a fraction of DOC, which is im-
mediately accessible to microbial utilization; SLDOC – semi-labile
DOC, a fraction of DOC, which resides mainly in the upper layer
but which becomes labile when transported to deep water. Figure 1. Linking RDOC at multiple dimensions: temporal (age)
and spatial (depth) transformations of RDOC. Lower panel: succes-
sive microbial processing of organic carbon results in the generation
of RDOC of different recalcitrance and different potential residence
time; MCP – microbial carbon pump; RDOCt – RDOC compounds
that are resistant to microbial consumption in certain environments,
but subject to further cleaving and decomposition when the situation
changes; RDOCc – composed of diverse small molecules which are
inaccessible to microbial uptake due to their low concentration. Up-
per panel: microbial response (in terms of abundance or uptake rate)
to DOC availability as a reference to conceptualize the microbial
uptake threshold for RDOCc, microbial abundance corresponding
to DOC concentrations of 40 µM in the deep and 70 µM in the sur-
face oceans; LDOC – labile DOC, a fraction of DOC, which is im-
mediately accessible to microbial utilization; SLDOC – semi-labile
DOC, a fraction of DOC, which resides mainly in the upper layer
but which becomes labile when transported to deep water. While age can be used to identify RDOC, not all old
DOC is RDOC. For example, petroleum components can
be very old but when exposed to microbial action they can
be rapidly decomposed. In addition, different microbes have
different decomposition capabilities under different environ-
mental conditions. Thus, recalcitrance can vary between dif-
ferent species, different functional groups and different en-
vironments (Carlson et al., 2011; Jiao et al., 2011; Kujawin-
ski, 2011). The classification of RDOC as microbial species-
specific, functional group-specific or environmental context-
specific recalcitrant could diminish confusion between bio-
logical and geochemical descriptions. Therefore we propose
the term RDOCcontext (RDOCt) (Fig. 1). the principal components of the DOC pool. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5287 n in the ocean
5287
40μM
70μM
LDOC
RDOCt
LMW
103yr
Uptake Threshold
106
105
Abundance
Uptake Rate
SLDOC
HMW
RDOCc
0yr
Epipelagic
Mesopelagic
Bathypelagic
Figure 1. Linking RDOC at multiple dimensions: temporal (age)
and spatial (depth) transformations of RDOC. Lower panel: succes-
sive microbial processing of organic carbon results in the generation
of RDOC of different recalcitrance and different potential residence
time; MCP – microbial carbon pump; RDOCt – RDOC compounds
that are resistant to microbial consumption in certain environments,
but subject to further cleaving and decomposition when the situation
changes; RDOCc – composed of diverse small molecules which are
inaccessible to microbial uptake due to their low concentration. Up-
per panel: microbial response (in terms of abundance or uptake rate)
to DOC availability as a reference to conceptualize the microbial
uptake threshold for RDOCc, microbial abundance corresponding
to DOC concentrations of 40 µM in the deep and 70 µM in the sur-
face oceans; LDOC – labile DOC, a fraction of DOC, which is im-
mediately accessible to microbial utilization; SLDOC – semi-labile
DOC, a fraction of DOC, which resides mainly in the upper layer
but which becomes labile when transported to deep water. 40μM
70μM
LDOC
RDOCt
LMW
103yr
Uptake Threshold
106
105
Abundance
Uptake Rate
SLDOC
HMW
RDOCc
0yr
Epipelagic
Mesopelagic
Bathypelagic on the composition and processing of DOC, interactions be-
tween POC and DOC cycling, impacts of anthropogenic per-
turbations on the BP and MCP in different environments, and
approaches for ecosystem sustainability and management. 1
Introduction A multidisciplinary effort is required to address these
challenges. To this end, the international IGBP/SCOR pro-
gramme Integrated Marine Biogeochemistry and Ecosystem
Research (IMBER), convened a workshop entitled “The im-
pact of anthropogenic perturbations on open ocean carbon
sequestration via the dissolved and particulate phases of the
biological carbon pump”, at the IMBIZO III conference in
Goa, India, in January 2013. Microbial ecologists, marine
biogeochemists, organic chemists, climatologists, fisheries
scientists and economists presented recent research on the
biological and microbial carbon pumps, discussed future nat-
ural and social science research needs to integrate POC and
DOC research, and brainstormed to better understand the mi-
crobial carbon storage mechanisms of the ocean. The microbial carbon pump (MCP) (Jiao et al., 2010a) de-
scribes the ecological processes and chemical mechanisms
that produce RDOC throughout the water column. The re-
silience of RDOC to degradation by marine microbes is an
important mediator of the global carbon cycle and the marine
carbon pool. Since the current reservoir of RDOC is compa- The objective of this paper is to identify the challenges of
and devise strategies for the integration of observations and
models of ocean POC and DOC cycling and sequestration,
with reference to the chemical composition of marine DOC,
the microbial processing of DOC, the environmental controls Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ 2
The nature and controls of DOC in the ocean One promising
approach is ultrahigh-resolution mass spectrometry (Fourier
transform ion cyclotron resonance mass spectrometry; FT-
ICR-MS), which is based on the analysis of exact molecular
masses from which the molecular elemental composition of
marine DOM can be deduced (Koch et al., 2005; Hertkorn et
al., 2006). To date several thousand molecular formulae have
been identified and many of them may act as potential indica-
tors for microbial sources and transformation processes (e.g.,
Kujawinski et al., 2004; Gonsior et al., 2009). Solid-phase
extracted DOM is composed of highly oxygenated molecules
(average oxygen to carbon ratio ∼0.45), which implies that For a better understanding of the nature and behavior of
RDOC, effort needs to be directed to isolation of differ-
ent DOC molecules and subsequent chemical analyses of 2
The nature and controls of DOC in the ocean Most of the DOC produced by photosynthesis is labile and
can be remineralized or assimilated by microbes within min-
utes to a few days (Fuhrman, 1987). The remaining DOC
can be gradually degraded and transformed by microbes and
abiotic processes to a huge variety of new compounds with
residence times from days to months, decades, hundreds and
even thousands of years (Sherr, 1988; Marchant and Scott,
1993). The oceanic DOC pool has been classified into two
major classes: labile DOC (LDOC) which does not accumu-
late in the ocean due to rapid microbial turnover and recal-
citrant DOC (RDOC) which serves as a reservoir until its
eventual mineralization or removal. In some studies, RDOC
is subdivided into fractions defined by their lifetimes: semi-
labile (∼1.5 years), semi-refractory (∼20 years), refrac-
tory (∼16 000 years) and ultra-refractory (∼40 000 years)
(Hansell et al., 2012). Obviously there are major gaps be-
tween the timescales describing the recalcitrance of RDOC,
to say nothing of the difference in definitions between sci-
entific disciplines. While geochemists can define RDOC ac-
cording to the 14C age on timescales of thousands of years
and modelers use the turnover rates as the basis of their defi-
nition for the different types of RDOC, microbiologists may
identify RDOC according to the absence of the genes that en-
code the enzymes required to metabolize the specific RDOC
compound with no link to the timescale required. Such dif-
ferences must be identified and addressed to ensure the inter-
disciplinary collaborations needed for a comprehensive un-
derstanding of the interactions between microbes and their
geochemical environment and the consequences of micro-
bial processing of carbon on outgassing of CO2 and carbon
sequestration. Figure 1. Linking RDOC at multiple dimensions: temporal (age)
and spatial (depth) transformations of RDOC. Lower panel: succes-
sive microbial processing of organic carbon results in the generation
of RDOC of different recalcitrance and different potential residence
time; MCP – microbial carbon pump; RDOCt – RDOC compounds
that are resistant to microbial consumption in certain environments,
but subject to further cleaving and decomposition when the situation
changes; RDOCc – composed of diverse small molecules which are
inaccessible to microbial uptake due to their low concentration. www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Although D-amino acids can be transformed into L-
amino acids by racemases inside the cell (Jørgensen and
Middelboe, 2006), the transformation can not be carried out
extracellularly, and if no membrane transporter is available
then this leads to the accumulation of D-amino acids in
the water column as RDOC. Structural RDOC molecules,
such as many D-amino acids (D-cysteine, D-tryptophan, D-
tyrosine), are intrinsically very resistant to microbial utiliza-
tion. Theoretically, an organic molecule/compound can be
intrinsically recalcitrant to a specific microbial species or
functional group if the microbes do not have the gene that
encodes the corresponding enzyme to take up or decom-
pose the molecule/compound. However, in the natural en-
vironment of diverse microbes and variable conditions, all
RDOC molecules/compounds are in a transitional stage, sub-
ject to further cleaving or decomposition, and their recalci-
trance is a continuum dependent upon microbial community
structure and environmental conditions. Throughout the wa-
ter column, microbial processing alters the nature of DOC
through decomposition, assimilation and regeneration. Mi-
crobes can produce complex structures, such as biofilms, and
low molecular weight (LMW) molecules such as antibiotics,
toxins and virulence factors. In addition, in the surface wa-
ter, photochemical reactions alter the composition of DOC,
produce LMW organic compounds (Kieber et al., 1990) and
influence the availability of DOC to microbes. The succes-
sive and repetitive processing of DOC compounds by the
diverse prokaryote community could generate smaller and
smaller fragments forming a LMW DOC pool. Although
the total concentration of this LMW DOC pool is not low
(∼40 µM, about half of the surface ocean DOC concentra-
tion), since it is composed of billions of different molec-
ular species (Baldock et al., 2004; Koch et al., 2005), the
concentration of most individual LMW DOC constituents
would be extremely low. This, rather than their recalcitrance,
could prevent energy-efficient microbial uptake (Kattner et Besides describing the molecular composition of RDOC,
understanding the microbial inaccessibility of RDOC is es-
sential to determining why the ocean holds such a huge DOC
pool in the presence of such an abundance of microbes. Ap-
propriate (meta-) genomic and (meta-) transcriptomic meth-
ods are now available to examine the microbial genetic and
enzymatic repertoire for cleaving and decomposing as well
as taking up and transforming DOC compounds (Kujaw-
inski, 2011). N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5288 they should be utilizable by prokaryotes. This high oxygen
content, which primarily exists in carboxylic functions, re-
flects a high degree of polarity and may therefore need a
highly specific and energy-efficient uptake system by distinct
microbes (Kattner et al., 2011). A low average proportion of
hydrogen (average hydrogen to carbon ratio ∼1.25) reflects
a substantial proportion of stable aromatic backbones, struc-
tures known to be difficult for prokaryotes to degrade. Ap-
proximately one-third of the detectable formulae are present
in all marine samples and most likely represent a common
refractory background in DOM (Koch et al., 2005; Kattner
et al., 2011). Within limits (on a molecular formula level),
FT-ICR-MS allows us to distinguish between labile and re-
fractory marine DOM generated within the MCP. D-Glucose
incubations show that microbially derived marine DOM re-
sembles labile material and that longer incubations are re-
quired to reach refractory element compositions (Koch et al.,
2014). Recently, the chemical composition of DOM has been
related to the degradation state and age of DOM (Flerus et al.,
2012; Lechtenfeld et al., 2014). These studies reveal that the
most persistent compounds encompass a very narrow range
of average molecular elemental ratios H / C and O / C and
show a continuum of residence times of refractory DOM in
the ocean; the longest of which substantially exceeded the
average age of marine DOC of ∼5000 years (Bauer et al.,
1992). ganisms can respond to microscale DOC gradients and ac-
cess nutrient-enriched patches (Stocker, 2012). High-molecular-weight (HMW) compounds must be
cleaved into smaller chemical units by extracellular enzymes
before microbial uptake (Arnosti, 2011). It is hard for cell
wall materials such as peptidoglycan (accounting for 2 %
of the cell biomass) (Park and Uehara, 2008) to be de-
composed completely when they are released as fragments
into the environment during viral lysis or grazing processes. Usually heterotrophic prokaryotes need at least seven com-
bined enzymatic transformations to cleave and decompose
peptidoglycan for reutilization (Jiang et al., 2010). Even if
peptidoglycan is cleaved, the fragments containing certain
components such as N-acetylglucosamine–N-acetylmuramic
acid and anhydro-N-acetylmuramic acid can remain inacces-
sible to the uptake and assimilation systems of some mi-
crobes; however, many heterotrophic prokaryotes can take up
and metabolize N-acetylglucosamine (Riemann and Azam,
2002). www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 4
Interactions between POC and DOC sequestration Differentiation of carbon sequestration to either the particu-
late or dissolved phase depends on the size threshold used
to divide POC from DOC, and the lifetime of the various
size fractions. Organic matter produced by the marine food
web covers a size range of almost 10 orders of magnitude,
from the smallest organic molecules (e.g., glucose, 0.7 nm)
to baleen whales (up to 30 m). For convenience, researchers
divide this size range into DOC and POC (the threshold de-
pending on the filter used to retain particles), but actually the
size distribution is almost continuous. The lifetime of any
substance is defined, assuming exponential decay, as the time
over which its concentration decreases to 1/e of its initial
value, where e is the Napierian constant 2.71828; this corre-
sponds to the “e-folding lifetime”, which is different from the
related concept of “half-life” where 1/2 is used instead of 1/e
(Hansell, 2013). Organic matter has lifetimes that range from
less than a day to tens of thousands of years. The rates of
production of the DOC fractions defined by Hansell (2013)
are inversely related to their average lifetimes, i.e., organic
compounds with a long lifetime are produced at small rates
according to the following equation (based on values given
in Table 1 of Hansell, 2013): In addition to its present-day role, the MCP may have been
crucial in the formation of a huge RDOC reservoir in the
Precambrian Ocean. A MCP mediated by sulfate-reducing
or iron-reducing microbes under hypoxic or anoxic condi-
tions may have facilitated the accumulation of authigenic car-
bonate (i.e., derived from DOC) in sediments or bottom wa-
ter, which may have played an important role in the global
carbon budget through Earth’s history (Canfield and Kump,
2013; Schrag et al., 2013). Geochemical records indicate an intensive prokaryote
driven MCP, and production of a large RDOC reservoir in
ancient oceans. Logan et al. (1995) found that the n-alkyl
lipids preserved in Proterozoic rocks are generally isotopi-
cally heavier than coexisting isoprenoidal lipids, while the
opposite is observed for most modern and Phanerozoic sed-
iments. This suggests that in the Proterozoic ocean, the n-
alkyl lipids received stronger heterotrophic reworking than
recalcitrant isoprenoidal lipids and that the MCP was there-
fore stronger in the Proterozoic oceans relative to that in
modern and Phanerozoic oceans. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5289 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean indicate the presence of an unusually large RDOC reservoir
at this time (Rothman et al., 2003). exploit substrates at very low concentrations, a low threshold
exists (Jannasch, 1995) for energetically profitable substrate
utilization (Barber, 1968; Kattner et al., 2011). These LMW
DOC molecules would stay inaccessible to microbial uptake
until they accumulate to a threshold level. The development of this large RDOC reservoir coin-
cided with a series of extreme “snowball Earth” glaciations
(Swanson-Hysell et al., 2010), culminating in the birth of the
earliest animals on Earth (Fike et al., 2006; McFadden et al.,
2008). It is likely that the great glaciations set up a lateral gra-
dient of oxidants in the postglacial oceans (Li et al., 2010),
which not only favored an intensive anaerobic MCP in shal-
low subsurface waters, but also created an extremely reduced
deep ocean for storage of the resulting RDOC; these favor-
able geochemical conditions allowed the accumulation of the
largest ocean RDOC reservoir known in the Earth’s history
(at least 102–103 times larger than the modern RDOC reser-
voir in size and 104 years longer in turnover time, Rothman
et al., 2003). Further work is required on the biogeochemi-
cal mechanisms and effects of the unusual accumulation of
RDOC in the Neoproterozoic deep ocean, as understanding
the processes involved in the MCP in the deep past is impor-
tant to improve our predictive capability for a future ocean,
where anoxia is likely to increase (and thus potentially in-
crease the ocean carbon storage capacity via the RDOC reser-
voir). y
Based on the above considerations, RDOC can be clas-
sified into two categories, environmental context-dependent
RDOCt and concentration-constrained RDOC (RDOCc). RDOCt would be recalcitrant in a given biogeochemical con-
text but could become accessible to microbial degradation in
a different context, RDOCc would be composed of molecules
at extremely low individual concentrations, which are below
the corresponding microbial uptake thresholds. Organic car-
bon ages as it is transformed gradually and successively from
labile to recalcitrant, from young HMW compounds in the
upper ocean to old LMW compounds in the deep ocean, thus
creating the continuum between microbial and geochemical
processing of RDOC in the ocean (Fig. 1). 3
RDOC processing in current and ancient oceans The activity of marine microorganisms leaves fingerprints
in the geological records that are traceable using organic
biomarkers and stable carbon isotopes. Thus integration of
microbial identity and function with stable carbon isotopes
on a geological timescale can improve our understanding of
the mechanisms and processes of DOC production, accumu-
lation and transformation in the modern ocean as well as their
relationships with climate variability. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean For example, the average genome of marine
bacteria contains 3000–5000 proteins, which according to
comparative genomics analysis share similarities for primary
metabolic pathways but differences for specific substrate as-
similation. Genes associated with cross-membrane transport, extra-
cellular hydrolysis, motility and chemotaxis are critical for
accessing the breadth of DOC molecules available for mi-
crobial assimilation. Roseobacter strains tend to assimi-
late carbohydrate-rich DOC while SAR11 bacteria prefer
nitrogen-containing DOM because they have suites of high-
affinity carbohydrate and amino acid ABC transporter sys-
tems, respectively (Jiao and Zheng, 2011). Bacteroides and
Gammaproteobacteria are able to consume a diverse array of
DOC because they have TonB-dependent transporter genes
(Tang et al., 2012). Bacteroides can take up and assimilate
N-acetyl glucosamine while SAR11 cannot, due to a lack of
N-acetyl glucosamine transporter and its deacetylase. Genes
associated with motility and chemotaxis vary from ∼0.5 to
∼1.2 % in the metagenomes of common marine environ-
ments. These genes provide a mechanism by which microor- Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ 4
Interactions between POC and DOC sequestration Economists are interested in the timescale of carbon se-
questration because companies or countries can earn carbon
credits by artificially capturing and securing the storage of
carbon that would otherwise be emitted to or remain in the at-
mosphere. Within the context of ocean fertilization, Lampitt
et al. (2008) proposed that sequestration requires carbon
which persists at least 100 years. According to this 100-year
timescale (e.g., Legendre and Le Fèvre, 1991, 1995; Legen-
dre and Rassoulzadegan, 1996; Passow and Carlson, 2012),
POC that reaches deep-ocean waters is sequestered, as are
the most refractory fractions of RDOC. Carbon sequestration
is also achieved by the solubility and the carbonate pumps
(Volk and Hoffert, 1985). Figure 2. Transformation of DOC and POC through decomposition
and scavenging processes that could influence carbon sequestration
and 14C dating (see text for details). MCP – microbial carbon pump,
BP – biological pump, POC – particulate organic carbon, DOC –
dissolved organic carbon, LDOC – labile DOC, SLDOC – semi-
labile DOC, RDOC – refractory DOC, RDOC coating – the process
of RDOC attaching to the exterior of or being incorporated into
a particle, RDOC aggregation – the process of RDOC molecules
accumulating and combining. (
)
The rate of POC sequestration can be estimated from the
POC sinking flux measured in sediment traps at 2000 m. Esti-
mates of this flux range from 0.43 Pg C year−1 (Honjo et al.,
2008) to 0.66 Pg C year−1 (Henson et al., 2012). The POC
flux to the sediment is estimated to be 0.1–0.16 Pg C year−1
(Hedges and Keil, 1995; Prentice et al., 2001). In con-
trast, there are few estimates of the rate of DOC seques-
tration. Using the 100-year sequestration criterion, a min-
imum estimate would be the combined production rates
of refractory and ultra-refractory DOC (average lifetimes
of 16 000 and 40 000 years and production of 0.043 and
0.000012 Pg C year−1, respectively) (Hansell, 2013). In ad-
dition, at least part of the production of semi-refractory DOC
should be included, since its average lifetime is 20 years (pro-
duction of 0.34 Pg C year−1) (Hansell, 2013) and that of the
next fraction, refractory DOC, is 16 000 years. Hence the
combined production rates of these three fractions of RDOC
would be 0.38 Pg C year−1, roughly consistent to earlier es-
timates of 0.5–0.6 Pg C year−1 (Brophy and Carlson, 1989). 4
Interactions between POC and DOC sequestration A strong negative shift
(down to −15 ‰) in the C-isotopic composition of sedimen-
tary carbonates alongside a generally unchanged C-isotopic
composition of coexisting organic matter (Fike et al., 2006;
Swanson-Hysell et al., 2010; Grotzinger et al., 2011) during
the Neoproterozoic (∼0.85 to 0.54 Ga) has been proposed to (1) log10(production within a DOC fraction)
(1) log10(production within a DOC fraction)
(1) = 0.29 −0.40log10(average lifetime of the fraction), with r2 = 0.96. In addition, the intrinsic lifetimes of organic
compounds may be significantly lengthened by their storage
in geochemical reservoirs. For example, organic matter that www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5290 sinks into deep water may be either remineralized (respired)
to CO2 or buried in sediments. In the first case, the CO2
that is dissolved in deep waters will return to the atmosphere
on average about 1000 years later. In the latter case, the or-
ganic carbon may be incorporated in sediments, carried by
the ocean floor until it is subducted in trenches (the age of
the ocean floor is generally < 125 million years), and released
to the atmosphere by volcanoes, or incorporated into conti-
nental rocks until ultimately released to the atmosphere by
weathering. POC
BP
Epipelagic
Mesopelagic
Bathypelagic
MCP
RDOC Coating
RDOC aggregation
LDOC
RDOC
CO2
CO2
14C dating error
RDOC
SLDOC
Figure 2. Transformation of DOC and POC through decomposition
and scavenging processes that could influence carbon sequestration
and 14C dating (see text for details). MCP – microbial carbon pump,
BP – biological pump, POC – particulate organic carbon, DOC –
dissolved organic carbon, LDOC – labile DOC, SLDOC – semi-
labile DOC, RDOC – refractory DOC, RDOC coating – the process
of RDOC attaching to the exterior of or being incorporated into
a particle, RDOC aggregation – the process of RDOC molecules
accumulating and combining. POC
BP
Epipelagic
Mesopelagic
Bathypelagic
MCP
RDOC Coating
RDOC aggregation
LDOC
RDOC
CO2
CO2
14C dating error
RDOC
SLDOC The Intergovernmental Panel on Climate Change (IPCC,
2013) defines carbon sequestration as the addition of car-
bon containing substances to a reservoir, e.g., the ocean,
which has the capacity to store, accumulate or release car-
bon. www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean hypothesized that the strategy of heterotrophic prokaryotes
in oligotrophic environments is to grow in biofilms on sur-
faces where nutrients are locally available, and to persist in
nutrient-deprived zones such as floating biofilms with the ca-
pacity to return to optimum growth when nutrients again be-
come available (Costerton et al., 1995). This is supported by
evidence suggesting that carbon-limited deep-ocean prokary-
otes show a preferential particle-related life strategy (De-
Long et al., 2006; Arístegui et al., 2009; Baltar et al., 2009,
2010b). with diameter of order 0.01 mm, a very low scavenge proba-
bility p = 4×10−6 will roughly give r ≈1, i.e., about half of
the carbon in the particle reaching the seabed is from RDOC. For a larger particle with diameter of ∼1 mm, the same scav-
enge probability would predict that ∼1 % of the carbon of
the particle reaching the seabed is from RDOC. This calculation is a simplification as it does not limit the
maximum number of RDOC molecules that can be scav-
enged by a particle and does not consider eddies and currents
generated by the sinking particles. In addition, if a particle
does not sink directly to the sea floor but also moves hori-
zontally or even upwards, its path can be even longer and so
it would encounter more RDOC molecules. Aggregating and scavenging processes are common in
the water column. Sinking POC particles could be nuclei
for RDOC molecules to attach to or aggregate with (Druf-
fel and Williams, 1990; Hwang and Druffel, 2003; Roland
et al., 2008). If the POC particles scavenge enough RDOC
molecules, they could become coated with RDOC (Fig. 2). As POC is relatively labile, it is considered to be an im-
portant food source for deep ocean microbes. In the case of
“RDOC-coated POC”, since RDOC is resistant to microbial
utilization, if the POC particle is coated with enough RDOC,
molecules, it is theoretically no longer subject to microbial
attack and can safely reach the seabed where it can be buried
for millions of years. In a simplified scenario that a spherical
particle with diameter of d is sinking directly from the bot-
tom of the euphotic zone to the sea floor, the carbon content
of this particle is Another effect of the “RDOC coating process” is bias in
14C dating (Fig. 2). N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean As indicated in the above calculation,
assuming the average age of RDOC is 5000 year, a 1 year old
POC particle landing at the seabed could be falsely dated as
50–2500 year old depending on its size (0.01–1 mm) using
our assumed scavenge probability. In fact, one of the loss
processes of RDOC in the water column is aggregation that
ultimately leads to transfer of aged organic carbon as POC to
the sediment (Engel et al., 2004b; Jiao et al., 2010a). Physical processes such as stratification, mixing and ocean
currents influence carbon sequestration in the ocean. Increas-
ing stratification restricts nutrient supply from deep water to
the euphotic zone, and therefore primary production which
will in turn impact the export of POC (Doney, 2006; Capo-
tondi et al., 2012; Passow and Carlson, 2012). Episodic ver-
tical water movement such as solitary waves could enhance
POC flux and these are thought to be responsible for the
unexpected presence of Prochlorococcus in aphotic waters
(300–1000 m) in the western Pacific marginal seas (Jiao et
al., 2014). Mesoscale eddies are ubiquitous features in the
ocean (Cheney and Richardson, 1976; Arístegui et al., 1997;
van Haren et al., 2006), and could play a major role in the
generation, accumulation and downward transport of bio-
genic production in the ocean. Cyclonic eddies enhance nu-
trient inputs to the surface ocean increasing new produc-
tion (Falkowski et al., 1991; Harris et al., 1997; Moran et
al., 2001) and chlorophyll concentrations (Arístegui et al.,
1997; McGillicuddy Jr. et al., 1998; Tarran et al., 2001). The presence of eddies has also been related to increased
bacterial abundance (Arístegui and Montero, 2005) and pro-
duction (Bode et al., 2001; Baltar et al., 2007), even in the
mesopelagic zone (Baltar et al., 2010a). However, the con-
tribution of eddies to particle flux is still poorly constrained. Mesoscale eddies were shown to enhance POC export by a
factor of 2–4 (Alonso-González et al., 2010) in the Canary
Island region, whereas eddies in Hawaii did not increase the
efficiency of POC export to mesopelagic waters as most of
the particle production was rapidly remineralized in the up-
per 150 m (Maiti et al., 2008). POC = 4
3π ·
d
2
3
· ρ,
(2) (2) where ρ is the particle’s carbon density. 4
Interactions between POC and DOC sequestration It seems that sequestration from the RDOC-based MCP and
the POC-based BP are of the same order of magnitude. particles large enough to sink (Fig. 2). About 10 % of marine
DOC exists in the form of gels which harbor heterotrophic
prokaryotes (Azam and Malfatti, 2007) and can accelerate
carbon transformation (Ziervogel et al., 2011), while POC or
aggregates attract copiotrophs such as Bacteroides (Arnosti
et al., 2012) that have motility and chemotaxis genes and
can potentially follow DOC gradients (Stocker, 2012). Many
marine heterotrophic prokaryotes produce polysaccharides,
which help them attach to biotic and abiotic surfaces to form
aggregates. The matrix of the aggregate, known as extracel-
lular polymeric substances (EPS) or transparent exopolymer
particles (TEP), is composed of polysaccharides, proteins,
nucleic acids and lipids. These cohesive, three-dimensional
polymers interconnect cells, forming aggregates which then
contribute to POC flux. Microbes colonize sinking aggre-
gates, and can grow by means of exoenzymatic decomposi-
tion of the aggregated organic particles, which in turn could
lead to a DOC plume following the sinking aggregate. In
fact, this plume may account for a significant fraction of
the microbial production and remineralization (Kiorboe and
Jackson, 2001). Thus the balance between the rate at which
aggregates form and sink on the one hand and the rate at
which they are remineralized and secrete DOC on the other
hand has a major impact on ocean carbon flux. It has been Sequestration would be high when there is rapid down-
ward transport of POC or substantial transformation of or-
ganic matter to RDOC. The interactions between POC flux
and RDOC production are numerous. For example, the at-
tenuation of POC flux is accompanied by DOC generation
throughout the water column, while the microbial transfor-
mation of DOC can also be accompanied by the formation of www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 5291 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Assuming a weight
density of the particle of 3 mg mm−3 (Carder et al., 1982)
and the molar ratio of 106 : 16 : 1 : 106 for C : N : P : O of the
particle, we can estimate ρ = 0.1 mmol C mm−3. If the depth
from the bottom of the euphotic zone to the seabed is z, the
path of the sinking particle forms a water column with di-
ameter d and length z, in which all the RDOC molecules
(RDOCect) are encountered by the particle: RDOCect = π ·
d
2
2
· z · [RDOC]. (3) (3) We can estimate [RDOC] to be 40 µM and z to be 4000 m
for the typical open ocean. If the probability that a RDOC
molecule is scavenged by the particle is p, the carbon ratio of
the RDOC and the original POC in this particle after reaching
the sea floor is r =
POC
RDOCect · p = 2
3 ·
ρ
z · [RDOC] · d
p. (4) (4) By using the parameter values we estimated above, Eq. (4)
simplifies to By using the parameter values we estimated above, Eq. (4)
simplifies to r = 2400 · p
d mm−1. (5) (5) A recent study of cyclonic eddies in the western South
China Sea (Jiao et al., 2014) suggested that the inten-
sity, timing and duration of nutrient input influenced the This equation indicates that the ratio r depends on the par-
ticle size and the scavenge probability. For a small particle This equation indicates that the ratio r depends on the par-
ticle size and the scavenge probability. For a small particle www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5292 plankton community structure which affected whether eddy
induced upwelling was associated with an increase (di-
atom dominated) or decrease (dominance of cyanobacte-
ria) in POC flux. Legendre and Le Fèvre (1995) previ-
ously stressed the significant role of the microbial food web
in carbon export. Along a nutrient gradient from eutrophy
to oligotrophy, POC export decreases as there is a transi-
tion in the structure of the microbial food web from phyto-
plankton prey-microzooplankton predators to picoplanktonic
cyanobacteria, heterotrophic bacteria, and Archaea prey and
mixotrophic protist predators (Zubkov and Tarran, 2008;
Hartmann et al., 2012). The ratio of DOC production to to-
tal primary production increases with increasing oligotrophy
(Teira et al., 2001), with some of this DOC likely converted
to RDOC. Thus, the contribution of the MCP to carbon stor-
age could be expected to be relatively high in the oligotrophic
ocean. A similar transition from dominance of the BP to
dominance of the MCP might be expected along a latitudinal
gradient from polar regions to the tropics and from surface
waters to the mesopelagic (Fig. 3). Figure 3. A demonstration of trends in the relative dominance of
the BP and the MCP along environmental gradients. Figure 3. A demonstration of trends in the relative dominance of
the BP and the MCP along environmental gradients. nificant additional annual carbon sequestration. Conversely,
a decrease of the input term or increase to the output could
lead to significant additional emissions. Thus understanding
the interactions between our actions (and their subsequent
effects) and the efficiency of the BP and MCP are of particu-
lar importance, both for understanding the likely response to
future global change and in informing whether or how ma-
rine management options might be employed to enhance (or
reduce degradation of) pump efficiency. 5
Impact of anthropogenic perturbations on carbon
sequestration Many of the interactions over which we may be able to
exercise management options take place in shelf seas, which
are active areas for DOM cycling (Prowe et al., 2009; John-
son et al., 2013) and carbon export (Tsunogai and Noriki,
1991; Thomas et al., 2004). Although covering only 8 % of
the ocean’s surface area, they account for 20 % of the ocean’s
capacity to absorb CO2 (Thomas et al., 2004). Shelf seas are
also the regions subject to strongest human pressures (Emeis
et al., 2014), thus they represent a strong “pressure point” for
controlling BP and MCP efficacy. These human pressures in-
clude nutrient input, hypoxia and trawling amongst others,
and we do not yet know how these pressures or combina-
tions of pressures will affect carbon storage. In the follow-
ing section, we consider the potential effects of two impor-
tant anthropogenic forcings: (i) nutrient input to the oceans
(a largely shelf sea pressure) and (ii) ocean acidification (a
global pressure). 5.1
Relevance to society The BP and MCP operate in concert to keep a large reser-
voir of carbon out of the atmosphere by storing POC, DOC
and dissolved inorganic carbon (DIC) in the ocean. With-
out marine biological carbon sequestration, it has been es-
timated that the atmospheric CO2 concentration would be
50 % (200 ppmv) higher than the current value (Parekh et
al., 2006). This storage of carbon thus has intrinsic value as
an “ecosystem service” (e.g., Luisetti et al., 2011). The term
“blue carbon” has previously been applied to coastal ecosys-
tems which have the capacity to store carbon year-on-year,
with the intention of valuation and possible subsequent car-
bon trading (Ullman et al., 2013) but that definition has not
previously been extended to continental shelf sediments or
the open ocean (Grimsditch et al., 2013). The deep-ocean natural carbon store is relatively secure on
short (decadal to centennial) timescales due to the long resi-
dence times of DIC (103 years) and DOC (104 years) in the
deep ocean. More relevant to society and how our activities
may impact this system is the balance between input and out-
put terms of the ocean carbon inventory. Any action we can
take to increase the efficiency of the BP and MCP, or reduce
the rate of the return pathway(s), will lead to net accumula-
tion of carbon in the deep ocean. Assuming the net biolog-
ical pump (BP+MCP) is ∼1–10 Pg C year−1 (IPCC, 2013),
this represents roughly 1–10 % of net global primary pro-
duction and between 10 and 100 % of global anthropogenic
CO2 emissions. Thus, for example, a 10 % increase in the
annual input term to the ocean carbon store could lead to sig- N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5293 DOC
POC
High N Input
Phytoplankton
BP
CO2
Ambient SLDOC
CO2
ATP
RDOC
MCP
Bacteria
Low N Input
BP
CO2
ATP
RDOC
MCP
Ambient SLDOC
MP
MP
DOC
POC
C
N
C
N
C
N
C
N
Primary Productivity
Bacterial Productivity
Bacterial Respiration
Bacterial Growth
Efficiency
NO3-N
Figure 4. The impact of nutrient supply on the sequestration of carbon via the BP and the MCP. Left panel: primary production, bacterial
respiration and bacterial growth efficiency as functions of nutrients. The arrow in the top graph shows a tipping point of nutrient concentration
beyond which primary production could drop down due to the constrains of limiting factors other than nutrients, such as light availability
and environmental carrying capacity. The red lines after the tipping point emphasize the differences between phytoplankton and bacteria in
their response to high nutrients. The appropriate nutrient concentrations for a healthy ecosystem would range between the vertical dashed
lines where the ecosystem could remain sustainable while running at a high level of biological efficiency. Right panel: responses of the BP
and the MCP to nutrient inputs. (MP – membrane potential; ATP-adenosine triphosphate). With high nutrient input, although the BP could
be enhanced, the MCP could be reduced, because microbial respiration can also be stimulated by nutrients. Meanwhile ambient semi-labile
DOC could be remobilized for microbial utilization, especially with the priming effects of the labile DOC generated by enhanced primary
production. In contrast, if nutrient input is appropriate, the BP could remain moderate, semi-labile DOC could remain persistent, the MCP
could be enhanced, and the storage capacity of the combined BP and MCP could be maximized. DOC
POC
High N Input
Phytoplankton
BP
CO2
Ambient SLDOC
CO2
ATP
RDOC
MCP
Bacteria
Low N Input
BP
CO2
ATP
RDOC
MCP
Ambient SLDOC
MP
MP
DOC
POC
C
N
C
N
C
N
C
N
Primary Productivity
Bacterial Productivity
Bacterial Respiration
Bacterial Growth
Efficiency Figure 4. The impact of nutrient supply on the sequestration of carbon via the BP and the MCP. Left panel: primary production, bacterial
respiration and bacterial growth efficiency as functions of nutrients. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean The phytoplank-
ton bloom would provide a steady supply of labile DOC for
bacterial growth, together with riverine-derived semi-labile
DOC (SL-DOC) (Fig. 4). Oxygen consumption due to this
bacterial respiration can eventually lead to hypoxia. Under
hypoxic and anoxic conditions, anaerobic bacteria would de-
grade the remaining organic matter, generating gases such
as methane and H2S. The former is a potent greenhouse gas
and the latter is a potential source of acidic rain (Fig. 5). Such
scenarios could have occurred during geological events in the
history of the Earth. In terms of carbon preservation, even
if more carbon is fixed, it does not necessarily lead to in-
creased carbon storage. This is verified by a systematic field
survey which indicated an inverse correlation between nitrate
and organic carbon in all terrestrial and marine environments
(Taylor and Townsend, 2010). Thus excess nutrients can lead
to lower organic carbon storage. Figure 5. Hypothesized consequences of excess nutrients in coastal
waters. Excess nutrients from river discharge cause eutrophication,
harmful algal blooms and hypoxia, which in turn influence overall
carbon sequestration efficiency. On the other hand, if nutrient input were reduced, although
the BP is apparently decreased, the organic matter that is pro-
duced would have relatively high C / N or C / P ratios, and
thus be of poor food quality for zooplankton resulting in high
ingestion and gut transit rates and low digestion. Such or-
ganic matter would also be relatively resistant to microbial
utilization, enhancing the MCP. Microbial carbon accumu-
lation is known to occur where/when nutrients are limiting
(Carlson et al., 2002; Gasol et al., 2009; Lauro et al., 2009; Thomas et al., 1999; Jiao et al., 2010b). For example, in post-
bloom, nutrient depleted conditions in temperate systems, net
CO2 fixation continues with the carbon likely being stored in
high C / N or high C / P DOM, while the POC / PON still ap-
proximates Redfield values (Craig et al., 2013). In support
of this, oceanic DOC concentrations tend to be highest in the
low nutrient oligotrophic gyres (Hansell et al., 2009). Even in Thomas et al., 1999; Jiao et al., 2010b). N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean The arrow in the top graph shows a tipping point of nutrient concentration
beyond which primary production could drop down due to the constrains of limiting factors other than nutrients, such as light availability
and environmental carrying capacity. The red lines after the tipping point emphasize the differences between phytoplankton and bacteria in
their response to high nutrients. The appropriate nutrient concentrations for a healthy ecosystem would range between the vertical dashed
lines where the ecosystem could remain sustainable while running at a high level of biological efficiency. Right panel: responses of the BP
and the MCP to nutrient inputs. (MP – membrane potential; ATP-adenosine triphosphate). With high nutrient input, although the BP could
be enhanced, the MCP could be reduced, because microbial respiration can also be stimulated by nutrients. Meanwhile ambient semi-labile
DOC could be remobilized for microbial utilization, especially with the priming effects of the labile DOC generated by enhanced primary
production. In contrast, if nutrient input is appropriate, the BP could remain moderate, semi-labile DOC could remain persistent, the MCP
could be enhanced, and the storage capacity of the combined BP and MCP could be maximized. Figure 5. Hypothesized consequences of excess nutrients in coastal
waters. Excess nutrients from river discharge cause eutrophication,
harmful algal blooms and hypoxia, which in turn influence overall
carbon sequestration efficiency. would be less influenced by the light field. The phytoplank-
ton bloom would provide a steady supply of labile DOC for
bacterial growth, together with riverine-derived semi-labile
DOC (SL-DOC) (Fig. 4). Oxygen consumption due to this
bacterial respiration can eventually lead to hypoxia. Under
hypoxic and anoxic conditions, anaerobic bacteria would de-
grade the remaining organic matter, generating gases such
as methane and H2S. The former is a potent greenhouse gas
and the latter is a potential source of acidic rain (Fig. 5). Such
scenarios could have occurred during geological events in the
history of the Earth. In terms of carbon preservation, even
if more carbon is fixed, it does not necessarily lead to in-
creased carbon storage. This is verified by a systematic field
survey which indicated an inverse correlation between nitrate
and organic carbon in all terrestrial and marine environments
(Taylor and Townsend, 2010). Thus excess nutrients can lead
to lower organic carbon storage. would be less influenced by the light field. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean For example, in post-
bloom, nutrient depleted conditions in temperate systems, net
CO2 fixation continues with the carbon likely being stored in
high C / N or high C / P DOM, while the POC / PON still ap-
proximates Redfield values (Craig et al., 2013). In support
of this, oceanic DOC concentrations tend to be highest in the
low nutrient oligotrophic gyres (Hansell et al., 2009). Even in 5.2
Nutrient supply Generally, an increase in nutrient supply to coastal waters is
expected to lead to an increase in primary production, POC,
and consequently an increase of the BP. However, high nu-
trient concentrations could have a negative impact on the
MCP (Jiao et al., 2010b). Primary production, bacterial res-
piration and bacterial growth efficiency would respond dif-
ferently to increasing nutrients (Fig. 4). As phytoplankton
populations increase with increasing nutrients, a maximum
will be reached when light shading becomes important and
primary production starts to decrease. In contrast, providing
DOC was in adequate supply, heterotrophic bacterial growth www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 5.3
Ocean acidification The absorption of CO2 by the ocean results in an increase in
the partial pressure of CO2 (pCO2), bicarbonate ion [HCO−
3 ]
and hydrogen ion [H+] concentration, and a decrease in car-
bonate ion concentration [CO2−
3 ] – so-called ocean acidifi-
cation (OA) (Doney et al., 2009). Enhanced photosynthesis
and nitrogen fixation have been shown to occur under higher
pCO2 conditions in laboratory and field experiments (Fu et
al., 2007; Riebesell and Tortell, 2011). Phytoplankton pro-
duction of TEP is stimulated by higher pCO2 treatments at
both species and community levels (Engel, 2002; Engel et
al., 2004a; Mari, 2008; Pedrotti et al., 2012), which con-
tributes to the formation of aggregates and so the vertical
flux of POC and DOC. OA inhibits phytoplankton calcifi-
cation which will decrease the ballast effect of calcium car-
bonate, so decreasing the vertical transport of POC (Barker
et al., 2003). In addition, the efficient degradation of TEP
by marine microbes (Koch et al., 2014) may be enhanced at
lower pH (Piontek et al., 2010). Shifts in phytoplankton com-
munity structure have occurred in high CO2 treatments that
could impact the composition and bioavailability of the DOC
produced (Tortell et al., 2002; Paulino et al., 2008; Brussaard
et al., 2013). Although there is no clear impact of OA on
bacterial abundance (Rochelle-Newall et al., 2004; Grossart
et al., 2006; Allgaier et al., 2008; Paulino et al., 2008; New-
bold et al., 2012; Brussaard et al., 2013), gross- and cell-
specific bacterial production are usually stimulated by high
pCO2 treatments (Grossart et al., 2006; Allgaier et al., 2008;
Motegi et al., 2013; Piontek et al., 2013). The activity of
bacterial protease, glucosidase and leucine-aminopeptidase
is also stimulated by higher pCO2 (Grossart et al., 2006; Pio-
ntek et al., 2010, 2013). Changes in the community structure
of bacterioplankton were observed when pCO2 was artifi-
cially adjusted (Allgaier et al., 2008; Newbold et al., 2012;
Zhang et al., 2013). However, it is possible that the observed
responses of bacterioplankton to OA are due to tight cou-
pling of phytoplankton and heterotrophs in experiments with
whole water samples. Nevertheless, higher bacterial activi-
ties in high pCO2 conditions may reduce carbon sequestra-
tion by POC flux but enhance the efficiency of the MCP by
producing more RDOC (Piontek et al., 2010, 2013). Indeed,
Kim et al. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean (such as viral lysis and grazing) involved in both POC and
DOC cycling under high CO2 conditions. eutrophic waters, as long as the C / N ratio reaches a thresh-
old value, microbial cells will start to store carbon and pro-
duce more recalcitrant compounds or polymers (Bhaskar and
Bhosle, 2005; Kadouri et al., 2005; Jiao et al, 2010b). In a
14-day in situ nutrient enrichment experiment undertaken in
the western Pacific oligotrophic gyre, more than 30 % of the
ambient organic carbon was respired in the incubation with
addition of inorganic nutrients compared to the control (Liu
et al., 2014). 6.1
Monitoring Satellite remote sensing of the surface ocean’s optical prop-
erties (ocean color) has been fundamental in developing the
prevailing view of global ocean phytoplankton production
and the BP. However, standard band-ratio chlorophyll prod-
ucts, designed for “Class I” open ocean waters, have limi-
tations when dealing with other optically active constituents
related to DOC transformation. The premise underpinning
the “Class I” water classification is that all optical proper-
ties co-vary with phytoplankton. However, increasing evi-
dence that aged DOC occurs at significant concentrations in
the open ocean (Hansell et al., 2009), independent of phy-
toplankton dynamics, may require this assumption to be re-
evaluated. A range of products have been developed that give
an indication of the surface distributions of pools of organic
carbon constituents in the ocean, including colored (or chro-
mophoric) dissolved organic material (CDOM; Siegel et al.,
2002, 2005; Maritorena and Siegel, 2005; Morel and Gen-
tili, 2009), DOC (Mannino et al., 2008), POC (Stramski et
al., 1999; Loisel et al., 2002; Gardner et al., 2006; Sathyen-
dranath et al., 2009; Stramska, 2009), phytoplankton size
classes (PSC; Ciotti and Bricaud, 2006; Hirata et al., 2008a,
2011; Brewin et al., 2010a, b; Uitz et al., 2010; Devred et
al., 2011) and particle size distribution (PSD; Hirata et al.,
2008b; Kostadinov et al., 2009, 2010). Key uncertainties re-
late to the relationship between the absorption of light by
CDOM and concentrations of DOC and to the contribution
of very small particles (e.g., viruses) to particle backscatter-
ing signals used in the derivation of POC and PSD products
(Stramski et al., 2008; Dall’Olmo et al., 2009). www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 5294 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean q
Increasing concentration
Inorganic e-
acceptors
Microbial respiration processes
Increasing water depth
Oxic zone
Oxygen
Suboxic zone
Anoxic zone
Sulfate
Nitrate,
Metal oxides
Efficiencies
Decreasing respiratory energy metabolic efficiency
Decreasing MCP efficiency and carbon sequestration
Aerobic respiration:
(CH2O)x(NH3)y(H3PO4)z + XO2 xCO2 + xH2O + yNH3 + zH3PO4
Denitrification:
5"CH2O" + 4NO3- 4HCO3- + CO2 + 2N2 + 3H2O
Manganese oxide reduction:
"CH2O" + 2MnO2 + 3CO2 + H2O 2Mn2+ + 4HCO3-
Nitrate reduction:
2"CH2O" + NO3- + 2H+ 2CO2 + NH4+ + H2O
Iron oxide reduction:
"CH2O" + 4Fe(OH)3 + 7CO2 8HCO3- + 3H2O + 4Fe2+
Sulfate reduction:
2CH3CHOCOOH + SO42- 2CH3COOH + 2HCO3- + H2S
NO3-
NO2-
O2
H2S
NH4+
Mn(II)
Figure 6. The influence of microbial respiration processes on the efficiency of the MCP. Left panel after Moore et al. (2009). Inorganic e-
acceptors Anoxic zone Figure 6. The influence of microbial respiration processes on the efficiency of the MCP. Left panel after Moore et al. (2009). to describe prokaryote diversity throughout the water column
alongside a better characterization of DOM. karyotic microbes, such as cooperation or competition for
nutrients, exist in the marine environment. For example, Mi-
tra et al. (2014) have proposed a new paradigm where, in
oligotrophic waters, the mixotrophic protists through pro-
duction of DOM effectively engage in “bacterial farming”
to ensure ample provision of food. Thus, in different biogeo-
chemical environments, the MCP could be expected to oper-
ate with different efficiencies for carbon storage. Marine biogeochemical time series data sets, such as the
Hawaii Ocean Time-Series (HOT), the Bermuda Atlantic
Time-Series Study (BATS) and the Porcupine Abyssal Plain
(PAP) Observatory are vital to the study of inter-annual vari-
ability in the linkage between phyto- and bacterioplankton
community structure and the BP and MCP (Carlson et al.,
2004; Bidigare et al., 2009). These single point data sets
are complemented with transect time series data sets such
as the Atlantic Meridional Transect (Robinson et al., 2006)
and multi-decadal biological data sets such as those col-
lected with the Continuous Plankton Recorder (CPR; Hin-
der et al., 2012). 6.2
Environmental context The sequestration of carbon in the ocean is indispensably
linked to the cycling of nitrogen, phosphorus, sulfur and
iron. Bacterial and Archaeal activities contribute to the re-
generation of N and P by consuming DOC (White et al.,
2012). Complex interactions between prokaryotes and eu- N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Data from the CPR survey show the link
between climate variability and the dominant phytoplank-
ton functional group (PFG) (Hays et al., 2005), and a global
collation of sediment trap data demonstrate the relation-
ship between dominant PFG and POC export efficiency and
transfer efficiency (Henson et al., 2012). Inclusion of mea-
surements of DOC quantity, quality and reactivity along-
side microbial community structure in these monitoring pro-
grammes would improve our understanding of linkages be-
tween climate-derived changes in plankton community struc-
ture and oceanic storage of organic carbon. g
An example of the changing efficiency of DOC-derived
carbon sequestration is that which occurs along an estuar-
ine gradient. Due to high terrigenous input of nutrients and
organic matter, estuarine ecosystems usually experience in-
tense heterotrophic respiration processes that rapidly con-
sume dissolved oxygen, potentially producing extensive hy-
poxic and anoxic zones in the water column. The lowered
availability of dissolved oxygen and the increased load of
nutrients such as nitrate from river input prompt enhanced
anaerobic respiration processes. Thus, most of the nutrients
may be consumed by anaerobically respiring heterotrophic
microorganisms instead of being utilized by phytoplankton
for POC and DOC production (Fig. 6). Anthropogenic eu-
trophication in estuarine and coastal areas may thus reduce
the efficiency of the MCP (Dang and Jiao, 2014). This re-
duced efficiency may be exacerbated by the potential “prim-
ing” effect of labile organic matter addition stimulating the
respiration of RDOC, as recently seen in soil environments
(Wieder et al., 2013). 5.3
Ocean acidification (2011) found an enhanced DOC : POC production
ratio in higher pCO2 treatments in a mesocosm study. Still,
we have a very limited understanding of ecological processes The major limitation of using satellite observations for in-
vestigation of the MCP is that they are restricted to surface
layers of the ocean. Nonetheless, there may be some regions
and seasons where this limitation can be partially overcome,
for example, in upwelling regions or regions of deep seasonal
overturning, where deeper DOC-rich waters are mixed to the
surface. Recent advances in sub-surface remote sensing, by
the addition of biogeochemical and optical sensors to profil-
ing floats (such as those deployed in the Argo array), provide
a new opportunity to investigate distributions of CDOM and
POC throughout the water column, extending our knowledge
of surface bio-optical distributions into the interior ocean and
connecting them with environmental gradients in nutrients,
oxygen and pH (Johnson et al., 2009). Future incorporation
on the floats of novel sensors for rapid characterization of ge-
netic material in situ would significantly advance our ability www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 5295 6.3
Bioassay and perturbation experiments In order to investigate mechanistic relationships between
changing environmental parameters such as temperature, OA www.biogeosciences.net/11/5285/2014/ 6.4
Improved chemical analytical and
genomic approaches Lipid biomarkers and their carbon isotopes can be powerful
tools for identification of the microorganisms participating
in POM and DOM cycling (White et al., 1979; Zhang et al.,
2002), which may also help link biogeochemical processes
in the water column and sediments. The concentrations of
ester-linked phospholipid fatty acids (PLFA) and intact po-
lar Archaeal lipids (IPAL) indicate the biomasses of extant
bacteria and Archaea, respectively, in complex ecosystems
(White et al., 1979; Zhang et al., 2002; Sturt et al., 2004; Lipp
et al., 2008; Liu et al., 2011). Furthermore, certain lipids can
be used as biomarkers because they are characteristic of, or
unique to, certain microbes. Such lipid biomarkers or their
combinations can reflect community structure, physiologi-
cal and nutritional status, and the dynamic biogeochemical
processes carried out by the microbes (White et al., 1998;
Suzumura, 2005). In addition, the carbon isotopes of lipid
biomarkers can be used as tracers for molecular level flow of
carbon and thus serve to evaluate the efficiency of the MCP
and quantify its relationship with the BP because the prod-
ucts of BP-based organisms may serve as the substrates of
MCP-based organisms. Mesocosm experiments have been instrumental in observ-
ing the influence of OA on DOC concentration (Schulz et al.,
2008), TEP production (Engel et al., 2004a) and community
respiration (Egge et al., 2009), the impact of nutrient sup-
ply on production, partitioning and the elemental composi-
tion of dissolved and particulate material (DOM, POM), and
the impact of increasing temperatures on the accumulation
and stoichiometry of DOM and POM (Wohlers-Zöllner et al.,
2012), the coupling of phytoplankton and bacterial processes
(Hoppe et al., 2008) and the balance between autotrophic and
heterotrophic metabolism (Müren et al., 2005). p
Most data on the effect of climate change on organic
matter dynamics were obtained in perturbation experiments
studying the response to a single factor. Recent data highlight
the need to study the interactions between multiple drivers
(e.g., temperature, nutrients, light and OA). For example, the
contradictory responses of phytoplankton TEP production to
OA (Engel et al., 2004a; Schulz et al., 2008; Egge et al.,
2009) indicates that additional factors, such as total alkalinity
(Mari, 2008; Passow, 2012) or nutrient stoichiometry (Corzo
et al., 2000; Staats et al., 2000; Passow, 2002; Beauvais et al.,
2006), should be considered in future experiments that inves-
tigate TEP aggregation (Passow and Carlson, 2012). 6.4
Improved chemical analytical and
genomic approaches Syner-
gistic effects of increased temperature and OA on microbial
community composition (Lindh et al., 2012), and OA and
increased inorganic nutrients on bacterial production (Bal-
tar et al., 2013) have also been found. It is therefore crucial
to move towards a multiple-factor approach in the design of
mesocosm experiments to better constrain the effect of mul-
tiple environmental drivers on the MCP and the BP. How-
ever, studying the impact of multiple factors demands a more
complex experimental design and statistical approach to dis-
tinguish between subtle and interacting effects (Breitburg et
al., 1998). Oceanographers would benefit from the experi-
ence of multiple stressor studies undertaken in freshwater
and terrestrial environments by ecotoxicologists to develop
hypotheses and concepts linking global, regional and local
anthropogenic drivers and their combined effects on ocean
biota (Calow, 1989; Boyd and Hutchins, 2012). g
Radiocarbon is another powerful approach for quantifica-
tion of MCP or BP activities in the ocean. From POC to DIC
to DOC, the 14C values decrease sequentially. If the BP plays
a dominant role in the ocean, a more 14C positive signature
would be seen in the water column and sediment organic car-
bon; on the other hand, if MCP dominates, the reworking of
POC in the water column may shift organic carbon toward
older DOC with depleted 14C (McNichol and Aluwihare,
2007). The 14C of lipid biomarkers can help to evaluate path-
ways of carbon metabolism by deep-ocean microbes. For ex-
ample, the 14C values of glycerol dialkyl glycerol tetraethers
from deep sea ammonia-oxidizing Archaea are closer to the
14C value of DIC, indicating that these organisms fix CO2
in the deep ocean (Ingalls et al., 2006). Such an approach
would help evaluate the autotrophic versus heterotrophic ca-
pabilities of meso- and bathypelagic prokaryotes. Studying
the 14C signature of DNA collected from mesopelagic Pa-
cific waters, Hansman et al. (2009) concluded that both DIC
and fresh DOC (presumably released from sinking POC) are
utilized, while ambient DOC is not a major substrate. What is more challenging is linking the taxonomic com-
position of microbial communities with their possible func-
tions in the carbon cycle. In particular, it is not clear
if the biological formation of RDOC is carried out by
all microbes or by a subset of the microbial community. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5296 and nutrients, and microbial organic carbon cycling, bioassay
or manipulation experiments are required. Mesocosm exper-
iments have become the preferred approach for these ma-
nipulations due to their ability to (a) study community dy-
namics of three or more trophic levels for a prolonged period
of time, (b) measure the pools and fluxes of bio-active com-
pounds and to perform mass balance estimates, and (c) study
the interactions of ecosystem dynamics and biogeochemical
processes under defined experimental conditions (Riebesell
et al., 2013). study the effect of changing DOC concentration and compo-
sition associated with for example whale or zooplankton ex-
cretion, melting sea-ice, increased river flow or the offshore
movement of upwelled water (Loucaides et al., 2012). www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5297 Newly-Produced DOC
Ventilation
Recalcitrancy
Distribution
Bacterial
modification
Photo-
degradation
RDOC:
Slow, Constant Degradation
With Thermohaline Circulation
RDOC
LDOC
Used locally
Epipelagic box
Mesopelagic box
Bathypelagic box
Sea Floor
Surface
200 m
1000 m
Figure 7. A conceptual framework for ecosystem modeling. proliferation of genomic approaches for exploring potential
metabolisms and biogeochemical roles of microbes in the
oceans. These approaches include metagenomic and meta-
transcriptomic methods, which are based on the direct se-
quencing of genomes and transcripts from all organisms in
a particular size class (usually the one dominated by bac-
teria and Archaea) without isolation or separation of indi-
vidual taxa (Moran, 2008; Kirchman, 2012). These methods
give insights into the potential function of microbes and have
suggested new pathways, such as light-harvesting by prote-
orhodopsin (Béjà et al., 2000) in the open oceans. Data from
genomic sequencing of single cells isolated by flow cytom-
etry have suggested metabolisms, such as chemolithotrophy
based on sulfur oxidation in mesopelagic waters (Swan et al.,
2011). Photo-
degradation Epipelagic box These “omic” approaches have provided insights into pro-
cesses involving labile DOC (Poretsky et al., 2010), but work
is needed to get a complete picture of how microbes interact
with all DOC components in the oceans. Microbial oceanog-
raphers face several challenges in using omic approaches
to explore DOC use and formation. One challenge is that
analysis of sequence data heavily depends on databases of
sequences from laboratory-grown organisms with known
metabolic functions. These laboratory-grown organisms may
not be representative of uncultivated oceanic microbes. An-
other problem is that not all of the genes in even heavily
studied organisms, such as Escherichia coli, are completely
known, and typically 10–20 % of a prokaryotic genome will
not be similar to genes from any organism (Koonin and Wolf,
2008). Even when identified by its similarity to known genes,
more detailed enzymatic analysis may not show the predicted
function (Cottrell et al., 2005). Consequently, there are prob-
lems in using genomic information to examine even known
functions. It is even more difficult to use genomic data to
gain insights into processes such as RDOC formation when
the basic biogeochemical mechanisms are unknown. Figure 7. A conceptual framework for ecosystem modeling. to quantify the relative importance of the MCP and BP, iden-
tify processes which contribute the greatest uncertainties in
their quantification, and guide the priorities for future field
and laboratory work. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean The key processes to be represented in an MCP model are
those that describe the production and removal of RDOC in
different vertical regimes. An initial step could be to set up a
conceptual model representing the ocean as simply three ver-
tical boxes: the epipelagic (surface 200 m), mesopelagic (200
to 1000 m) and bathypelagic zones (below 1000 m) (Fig. 7). In the epipelagic zone, such a model would focus on quan-
tifying the production of SLDOC. The model of Flynn et
al. (2008) provides a basis for such work, describing the pro-
duction of different types of DOM as a function of the growth
and nutrient status of primary producers. Similar models
are needed for DOM production by zooplankton, and for
the consumption of all fractions of DOM by bacteria and
other microbes. The proposal made by Mitra et al. (2014)
places an additional reason to develop these simulations, with
mixotrophs simultaneously producing DOM through pho-
totrophy and phagotrophy, while consuming bacteria whose
growth is supported by the same DOM. It is unlikely that a single gene or gene cluster will pro-
vide the answer to RDOC formation, just as few diseases can
be traced to defects in one or two genes. However, genomes
represent the evolutionary record of how microbes have in-
teracted with DOC over millennia. If we can read that record,
we are likely to learn much about DOC–microbe interac-
tions. Coupling omic studies with geochemical studies on all
DOC components is likely to give new insights into the MCP. In the mesopelagic zone, the model would need to ad-
dress the lability continuum of RDOC. A mechanism would
be needed to determine a flexible lability continuum accord-
ing to the nutrient conditions in the overlying epipelagic
layer: the continuum would move toward recalcitrance in
oligotrophic oceans while being more labile in eutrophic re-
gions (Jiao and Zheng, 2011). Such a model could also in-
clude a description of processes in the mesopelagic layer
allowing the modification of the accessibility of RDOC to
heterotrophic bacteria. This requires the development of a
model which describes the ability of bacteria to discriminate
between different types of DOM with changes in stoichiome-
try (C : N : P), and which can reflect changes in growth rates, 6.4
Improved chemical analytical and
genomic approaches New sequencing technology has been instrumental in the In situ mesoscale addition experiments where a single wa-
ter mass tagged with the inert tracer sulfur hexafluoride along
with a potentially limiting nutrient (e.g., iron) or nutrients
(e.g., iron and phosphate) (Boyd et al., 2007) could also be
adapted for the study of the BP and MCP. The unequivocal
lagrangian sampling mode which this allows could be used to www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 6.5
Ecosystem modeling Biogeochemical models have been used to study DOC cy-
cling and its interactions with heterotrophic prokaryotes in
the surface ocean, indicating that oligotrophic conditions can
lead to the accumulation and export of semi-labile DOC
(SLDOC, a fraction of DOC, which resides mainly in the up-
per layer but becomes labile when transported to deep water)
(Polimene et al., 2006; Luo et al., 2010). To better understand
carbon sequestration, further modeling studies are required N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5298 growth efficiency and respiration. While such models exist
(e.g., Flynn, 2005; Luo et al., 2010), other than for use in con-
ceptualized scenarios, all these models remain largely empir-
ical because of the practical challenges surrounding the char-
acterization and quantification of different DOM fractions. 2005) or taxonomy (e.g., diatoms, dinoflagellates, Blackford
et al., 2004). While understanding of microbial heterotrophic
communities in the ocean is advancing rapidly, there is no
clear differentiation of a set of bacterial and Archaeal func-
tional types. As with other marine ecosystem modeling ap-
proaches, the resolution and descriptions used will vary ac-
cording to the question posed. For example, taxonomically it
may be desirable to separate Archaea from bacteria, whereas
in the context of understanding biogeochemical transforma-
tions of POC and RDOC, it may be more opportune to model
the affinities of organisms to different substrates (particles,
gels or DOC) or their evolutionary strategies for responding
to different resource environments. In this regard, partition-
ing into copiotrophs that use chemotaxis, motility and fast
uptake kinetics to exploit microscale gradients of high nu-
trient concentration, versus non-motile oligotrophs that are
adapted to life in nutrient poor environments (Stocker, 2012)
may provide a good starting point. Accepting that limitation , assuming that the DOC concen-
tration in the bathypelagic zone does not change substantially
with depth (Hansell, 2013), the model can address the sug-
gestion that RDOC is not directly transported to local bathy-
pelagic zones, but moves with the thermohaline circulation
and is degraded slowly within all three vertical layers. This
initial model simulates CO2 production as the result of de-
grading RDOC in different vertical layers. Together with an
estimation of deep water ventilation and exchange rates of
water masses between the three vertical boxes, it is expected
to quantify the role of the MCP in carbon sequestration on
various timescales (Fig. 7). Models of these processes within coastal ocean scenarios
will help in evaluating anthropogenic impacts on carbon se-
questration by both the BP and the MCP. In silico experi-
ments could be set up with different rates and stoichiome-
tries of riverine nutrient inputs. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Carbon sequestration result-
ing from the BP in coastal oceans also needs to consider
the remineralization of POC in subsurface layers, as well
as the resuspension of benthic POC; however, while mod-
els may clearly differentiate between POC and DOC, in real
world sampling the so-called DOC fraction is contaminated
by micro-particulates (i.e., POC) that compounds the already
challenging definition of labile, semi-labile and refractory
fractions. Here, perhaps more than for ocean systems, it is
necessary to describe the variable stoichiometry of the DOM
fractions. To quantify carbon sequestration by the MCP, pa-
rameters which control both DOC lability and its interactions
with bacteria should be described in the model. To further
quantify the fate of DOC after use by bacteria, variable stoi-
chiometry for bacterial cellular composition is required. The
model ultimately needs to calculate the net rate of supply of
RDOC to the adjacent open ocean and thus give estimates for
the net carbon sequestration rate by the MCP. Modeling studies would eventually need to extend to the
global scale, aiming to reproduce the distribution of DOC
and predict how the MCP and BP will change with chang-
ing anthropogenic influences. Overall, two-way interaction
should be maintained between the MCP–BP modeling and
observational scientific communities: the results from exper-
iments will help parameterize the model, and model results
will then guide further experimentation. First and foremost,
however, is the need to rationalize the chemical, biological
and modeling descriptions of different types of DOM. www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ References This synthesis of current research and gaps in our knowledge
leads to the following conclusions and suggestions for future
research: Allgaier, M., Riebesell, U., Vogt, M., Thyrhaug, R., and Grossart,
H.-P.: Coupling of heterotrophic bacteria to phytoplankton
bloom development at different pCO2 levels: a mesocosm
study, Biogeosciences, 5, 1007–1022, doi:10.5194/bg-5-1007-
2008, 2008. RDOC can be classified as RDOCt and RDOCc depend-
ing on the composition and concentration of the RDOC
molecules as well as the prevailing environmental conditions. State-of-the-art analytical chemical and genomic methods
should be used to determine the microbial source and com-
position of RDOC and assess the environmental conditions
which influence the recalcitrance of RDOC. State-of-the-art analytical chemical and genomic methods
should be used to determine the microbial source and com-
position of RDOC and assess the environmental conditions
which influence the recalcitrance of RDOC. Alonso-González, I. J., Arístegui, J., Lee, C., and Calafat, A.:
Regional and temporal variability of sinking organic matter
in the subtropical northeast Atlantic Ocean: a biomarker diag-
nosis, Biogeosciences, 7, 2101–2115, doi:10.5194/bg-7-2101-
2010, 2010. Analyses of biomarkers and isotopic records show inten-
sive MCP processes in the Proterozoic oceans when the MCP
could have played a more significant role in regulating cli-
mate. Understanding MCP dynamics in the past will aid in
predicting how carbon storage could change with changing
climatic conditions in the future. Anderson, T. R. and Tang, K. W.: Carbon cycling and POC turnover
in the mesopelagic zone of the ocean: Insights from a simple
model, Deep-Sea Res. Pt. II., 57, 1581–1592, 2010. Arístegui, J. and Montero, M. F.: Temporal and spatial changes in
plankton respiration and biomass in the Canary Islands region:
the effect of mesoscale variability, J. Marine. Syst., 54, 65–82,
2005. Future research programs should integrate the study of
POC flux and DOC transformation in order to elucidate the
interactions between POC and DOC cycling and the environ-
mental controls on these interactions. This includes an inves-
tigation of the occurrence and lability of RDOC-coated POC. Arístegui, J., Tett, P., Hernández-Guerra, A., Basterretxea, G., Mon-
tero, M. F., Wild, K., Sangrá, P., Hernández-León, S., Cantón,
M., García-Braun, J. A., Pacheco, M., and Barton, E. D.: The
influence of island-generated eddies on chlorophyll distribution:
a study of mesoscale variation around Gran Canaria, Deep-Sea
Res., 44, 71–96, 1997. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Microbiology Initiative. A. Mitra was supported in part by project
EURO-BASIN (ref. 264933, 7FP, European Union) and also by a
Leverhulme International Networking Grant. Microbiology Initiative. A. Mitra was supported in part by project
EURO-BASIN (ref. 264933, 7FP, European Union) and also by a
Leverhulme International Networking Grant. nutrient concentration/stoichiometry” could be obtained for
a given ecosystem through theoretical calculation and field
experimentation. Edited by: G. Herndl 6.6
Strategies to enhance carbon sequestration Based on the discussion above (Sect. 5.2, Fig. 4) and a statis-
tical study which shows that concentrations of organic car-
bon and nitrate in natural environments ranging from soils
and rivers, to coastal and oceanic waters are inversely corre-
lated (Taylor and Townsend, 2010), it seems that if we want
to store more organic carbon in the environment we must
avoid high concentrations of nutrients. Therefore, reduced
nutrient levels in otherwise eutrophic coastal waters could
result in a greater proportion of the fixed carbon becoming
RDOC (or RDOCt) (Jiao et al., 2010b). Then we may be
able to enhance carbon sequestration in the coastal zone by
controlling the discharge of nutrients from land. This can be
achieved through the integrative management of the land–
ocean system, for example by using methods of fertilization
which avoid loss of nutrients to rivers and reducing sewage
discharge to coastal waters. Such an integrative management
approach would have the additional advantage of reducing
eutrophication. Furthermore, the carbon storage capacity of
coastal regions could be enhanced by optimizing both the BP
and the MCP (Fig. 4). If the key to the efficiency of carbon
storage in an ecosystem is the nutrient status, then it is critical
to perform field-based research to fully understand the tip-
ping point of the nutrient concentration or stoichiometry that
leads to different types of carbon metabolism. An “optimal These model experiments could be used to determine a re-
lationship between carbon sequestration (from the BP and
the MCP) and the nutrient input from rivers, in order to es-
timate an optimal rate and stoichiometry of riverine nutrient
input for maximum carbon sequestration. Reverse modeling
methods (Friedrichs et al., 2007; Luo et al., 2010), may be
applied to improve these estimates, and to unveil the uncer-
tainties associated with model processes and parameters. Modeling of ocean ecosystems has relied heavily on the
concept of functional groups, with more complex mod-
els having multiple functional types within each modeled
group. For example, the singular phytoplankton box of early
nutrient–phytoplankton–zooplankton–detritus (NPZD) mod-
els has been extended to take account of size (e.g., pico, nano
and micro size classes), biogeochemical function (e.g., sili-
cifiers, calcifiers, dimethylsulfide producers, Le Quere et al., www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 5299 Acknowledgements. We thank the organisers, sponsors and par-
ticipants of the IMBIZO III workshop. Particular thanks go to our
hosts at the National Institute of Oceanography, Goa, India, and the
staff of the IMBER International Project Office, Lisa Maddison,
Bernard Avril and Liuming Hu. We acknowledge financial support
from the MOST 973 program 2013CB955700, the NSFC projects
91028001, 91328209, 41376132 and 91028011, and the SOA
projects 201105021 and GASI-03–01-02–05. FA was supported
by a grant from the Gordon and Betty Moore Foundation Marine References Bioassay and field experiments should be undertaken
to assess the combined effects of multiple environmental
drivers (temperature, OA and nutrient supply) on marine car-
bon storage. Ecosystem models need to be developed and
tested with mechanistic relationships derived from these ex-
periments in order to predict the dynamics of and interactions
between the BP and the MCP under global change scenarios
including changing mixing and circulation, changing nutri-
ent and oxygen distributions, and increasing temperature and
decreasing pH. Arístegui, J., Gasol, J. M., Duarte, C. M., and Herndl, G. J.: Micro-
bial Oceanography of the dark ocean’s pelagic realm, Limnol. Oceanogr., 54, 1501–1529, 2009. Arnosti, C.: Microbial Extracellular Enzymes and the Marine Car-
bon Cycle, Ann. Rev. Mar. Sci., 3, 401–425, 2011. Arnosti, C., Fuchs, B. M., Amann, R., and Passow, U.: Contrast-
ing extracellular enzyme activities of particle-associated bacteria
from distinct provinces of the North Atlantic Ocean, Front. Mi-
crobiol., 3, 425, doi:10.3389/fmicb.2012.00425, 2012. Azam, F. and Malfatti, F.: Microbial structuring of marine ecosys-
tems, Nat. Rev. Microbiol., 5, 782–791, 2007. We hypothesize that increased nutrient supply to an
ecosystem above a tipping point will decrease the efficiency
of carbon storage by the MCP and in the long term by the
BP as well. If supported, this could have important impli-
cations for management strategies for carbon sequestration
and coastal ecosystem health including reduced eutrophica-
tion and hypoxia. Baldock, J. A., Masiello, C. A., Gelinas, Y., and Hedges, J. I.: Cy-
cling and composition of organic matter in terrestrial and marine
ecosystems, Mar. Chem., 92, 39–64, 2004. Baltar, F., Arístegui, J., Gasol, J. M., Hernández-León, S., and
Herndl, G. J.: Strong coast – ocean and surface – depthgradi-
ents in prokaryotic assemblage structure and activity in a coastal
transition zone region, Aquat. Microb. Ecol., 50, 63–74, 2007. Baltar, F., Arístegui, J., Gasol, J. M., Sintes, E., and Herndl, G. J.:
Evidence of prokaryotic metabolism on suspended particulate or-
ganic matter in the dark waters of the subtropical North Atlantic,
Limnol. Oceanogr., 54, 182–193, 2009. Baltar, F., Arístegui, J., Gasol, J. M., Lekunberri, I., and Herndl, G. J.: Mesoscale eddies: Hotspots of prokaryotic activity and differ-
ential community structure in the ocean, ISME J., 4, 975–988,
2010a. Baltar, F., Arístegui, J., Gasol, J. M., Sintes, E., van Aken, H. M.,
and Herndl, G. J.: High dissolved extracellular enzymatic activ- Biogeosciences, 11, 5285–5306, 2014 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean N., and DeLong, E. F.: Bacterial
rhodopsin: Evidence for a new type of phototrophy in the sea,
Science, 289, 1902–1906, 2000. Carlson, C. A., Giovannoni, S. J., Hansell, D. A., Goldberg, S. J.,
Parsons, R., Otero, M. P., Vergin, K., and Wheeler, B. R.: Effect
of nutrient amendments on bacterioplankton production, com-
munity structure, and DOC utilization in the northwestern Sar-
gasso Sea, Aquat. Microb. Ecol., 30, 19–36, 2002. Bhaskar, P. V. and Bhosle, N. B.: Microbial extracellular polymeric
substances in marine biogeochemical processes, Curr. Sci. India,
88, 45–53, 2005. Carlson, C. A., Giovannoni, S. J., Hansell, D. A., Goldberg, S. J.,
Parsons, R., and Vergin, K.: Interactions among dissolved or-
ganic carbon, microbial processes, and community structure in
the mesopelagic zone of the northwestern Sargasso Sea, Limnol. Oceanogr., 49, 1073–1083, 2004. Bidigare, R. R., Chai, F., Landry, M. R., Lukas, R., Hannides, C. C. S., Christensen, S. J., Karl, D. M., Shi, L., and Chao, Y.: Subtrop-
ical ocean ecosystem structure changes forced by North Pacific
climate variations, J. Plankton. Res., 31, 1131–1139, 2009. Blackford, J. C., Allen, J. I., and Gilbert, F. J.: Ecosystem dynam-
ics at six contrasting sites: a generic modelling study, J. Marine. Syst., 52, 191–215, 2004. Carlson, C. A., Hansell, D. A., and Tamburini, C.: DOC Persistence
and its Fate After Export Within the Ocean Interior, in: Microbial
Carbon Pump in the Ocean, edited by: Jiao, N., Azam, F., and
Sanders, S., Science/AAAS Business Office, Washington, DC,
57–59, 2011. Bode, A., Barquero, S., Varela, M., Braun, J. A., and de Armas,
D.: Pelagic bacteria and phytoplankton in oceanic waters near
the Canary Islands in summer, Mar. Ecol.-Prog. Ser., 209, 1–17,
2001. Cheney, R. E. and Richardson, P. L.: Observed decay of a cyclonic
Gulf Stream ring, Deep-Sea Res., 23, 143–155, 1976. Boyd, P. W. and Hutchins, D. A.: Understanding the responses of
ocean biota to a complex matrix of cumulative anthropogenic
change, Mar. Ecol.-Prog. Ser., 470, 125–135, 2012. Ciotti, A. M. and Bricaud, A.: Retrievals of a size parameter for
phytoplankton and spectral light absorption by colored detrital
matter from water-leaving radiances at SeaWiFS channels in a
continental shelf region off Brazil, Limnol. Oceanogr.-Meth., 4,
237–253, 2006. Boyd, P. W., Jickells, T., Law, C. S., Blain, S., Boyle, E. A., Bues-
seler, K. O., Coale, K. H., Cullen, J. J., de Baar, H. J. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5300 ity in the deep central Atlantic Ocean, Aquat. Microb. Ecol., 58,
287–302, 2010b. Brewin, R. J. W., Sathyendranath, S., Hirata, T., Lavender, S. J., Barciela, R. M., and Hardman-Mountford, N. J.: A three-
component model of phytoplankton size class for the Atlantic
Ocean, Ecol. Model., 221, 1472–1483, 2010b. Baltar, F., Palovaara, J., Vila-Costa, M., Calvo, E., Pelejero, C., Mar-
rase, C., Salazar, G., Gasol, J. M., and Pinhassi, J.: Response
of rare versus abundant bacterioplankton to disturbances in a
Mediterranean coastal site, in 13th Symposium on Aquatic mi-
crobial Ecology, Stresa, Italy, 8–13 September 2013, 2013. Brophy, J. E. and Carlson, D. J.: Production of biologically refrac-
tory dissolved organic carbon by natural seawater microbial pop-
ulations, Deep-Sea. Res. Pt. I., 36, 497–507, 1989. Brussaard, C. P. D., Noordeloos, A. A. M., Witte, H., Collenteur,
M. C. J., Schulz, K., Ludwig, A., and Riebesell, U.: Arctic mi-
crobial community dynamics influenced by elevated CO2 lev-
els, Biogeosciences, 10, 719–731, doi:10.5194/bg-10-719-2013,
2013. Barber, R. T.: Dissolved organic carbon from deep waters resists
microbial oxidation, Nature, 220, 274–275, 1968. Barker, S., Higgins, J. A., and Elderfield, H.: The future of the car-
bon cycle: review, calcification response, ballast and feedback on
atmospheric CO2, Philos. Trans. A Math. Phys. Eng. Sci., 361,
1977–1999, 2003. Calow, P.: Proximate and ultimate responses to stress in biological
systems, Biol. J. Linn. Soc., 37, 173–181, 1989. Bauer, J. E., Williams, P. M., and Druffel, E. R. M.: 14C activity
of dissolved organic carbon fractions in the north-central Pacific
and Sargasso Sea, Nature, 357, 667–670, 1992. Canfield, D. E. and Kump, L. R.: Geochemistry, Carbon cycle
makeover, Science, 339, 533–534, 2013. Beauvais, S., Pedrotti, M. L., Egge, J., Iversen, K., and Marrasé,
C.: Effects of turbulence on TEP dynamics under contrasting nu-
trient conditions: implications for aggregation and sedimentation
processes, Mar. Ecol.-Prog. Ser., 323, 47–57, 2006. Capotondi, A., Alexander, M. A., Bond, N. A., Curchitser, E. N.,
and Scott, J. D.: Enhanced upper ocean stratification with climate
change in the CMIP3 models, J. Geophys. Res., 117, C04031,
doi:10.1029/2011jc007409, 2012. Carder, K. L., Steward, R. G., and Betzer, P. R.: In situ holographic
measurements of the sizes and settling rates of oceanic particu-
lates, J. Geophys. Res., 87, 5681–5685, 1982. Béjà, O., Aravind, L., Koonin, E. V., Suzuki, M. T., Hadd, A.,
Nguyen, L. P., Jovanovich, S., Gates, C. M., Feldman, R. A.,
Spudich, J. L., Spudich, E. www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean J.: Global POC
concentrations from in-situ and satellite data, Deep-Sea Res. Pt. II., 53, 718–740, 2006. Druffel, E. R. M. and Williams, P. M.: Identification of a deep ma-
rine source of particulate organic-carbon using bomb C-14, Na-
ture, 347, 172–174, 1990. Gasol, J. M., Vázquez-Domínguez, E., Vaqué, D., Agustí, S., and
Duarte, C. M.: Bacterial activity and diffusive nutrient supply in
the oligotrophic Central Atlantic Ocean, Aquat. Microb. Ecol.,
56, 1–12, 2009. Egge, J. K., Thingstad, T. F., Larsen, A., Engel, A., Wohlers, J.,
Bellerby, R. G. J., and Riebesell, U.: Primary production during
nutrient-induced blooms at elevated CO2 concentrations, Bio-
geosciences, 6, 877–885, doi:10.5194/bg-6-877-2009, 2009. Gonsior, M., Peake, B. M., Cooper, W. T., Podgorski, D.,
D’Andrilli, J., and Cooper, W. J.: Photochemically Induced
Changes in Dissolved Organic Matter Identified by Ultrahigh
Resolution Fourier Transform Ion Cyclotron Resonance Mass
Spectrometry, Environ. Sci. Technol., 43, 698–703, 2009. Emeis, K.-C., Beusekom, J. V., Callies, U., Ebinghaus, R., Kan-
nen, A., Kraus, G., Kröncke, I., Lenhart, H., Lorkowski, I.,
Matthias, V., Möllmann, C., Pätsch, J., Scharfe, M., Thomas,
H., Weisse, R., and Zorita, E.: The North Sea – a shelf sea
in the anthropocene, J. Marine Syst., IMBIZO special issue,
doi:10.1016/j.jmarsys.2014.03.012, in press, 2014. Grimsditch, G., Alder, J., Nakamura, T., Kenchington, R., and
Tamelander, J.: The blue carbon special edition – Introduction
and overview, Ocean. Coast. Manage., 83, 1–4, 2013. Grossart, H. P., Allgaier, M., Passow, U., and Riebesell, U.: Testing
the effect of CO2 concentration on the dynamics of marine het-
erotrophic bacterioplankton, Limnol. Oceanogr., 51, 1–11, 2006. Engel, A.: Direct relationship between CO2 uptake and transpar-
ent exopolymer particles production in natural phytoplankton, J. Plankton. Res., 24, 49–53, 2002. Grotzinger, J. P., Fike, D. A., and Fischer, W. W.: Enigmatic origin
of the largest-known carbon isotope excursion in Earth’s history,
Nat. Geosci., 4, 285–292, 2011. Engel, A., Delille, B., Jacquet, S., Riebesell, U., Rochelle-Newall,
E., Terbrüggen, A., and Zondervan, I.: Transparent exopolymer
particles and dissolved organic carbon production by Emiliania
huxleyi exposed to different CO2 concentrations: a mesocosm
experiment, Aquat. Microb. Ecol., 34, 93–104, 2004a. Gruber, D. F., Simjouw, J. P., Seitzinger, S. P., and Taghon, G. L.:
Dynamics and characterization of refractory dissolved organic
matter produced by a pure bacterial culture in an experimental
predator-prey system, Appl. Environ. Microb., 72, 4184–4191,
2006. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5301 Flynn, K. J.: Incorporating plankton respiration in models of aquatic
ecosystem function, in: Respiration in Aquatic Ecosystems,
edited by: Giorgio, P. A. D. and Williams, P. J. L. B., Oxford
University Press, 2005. consumption in the ocean, Biogeosciences Discuss., 10, 11255–
11282, doi:10.5194/bgd-10-11255-2013, 2013. consumption in the ocean, Biogeosciences Discuss., 10, 11255–
11282, doi:10.5194/bgd-10-11255-2013, 2013. Dall’Olmo, G., Westberry, T. K., Behrenfeld, M. J., Boss, E., and
Slade, W. H.: Significant contribution of large particles to optical
backscattering in the open ocean, Biogeosciences, 6, 947–967,
doi:10.5194/bg-6-947-2009, 2009. Flynn, K. J., Clark, D. R., and Xue, Y.: Modeling the release of
dissolved organic matter by phytoplankton, J. Phycol., 44, 1171–
1187, 2008. Dang, H. and Jiao, N.: Perspectives on the microbial carbon pump
with special reference to microbial respiration and ecosystem ef-
ficiency in large estuarine systems, Biogeosciences, 11, 3887–
3898, doi:10.5194/bg-11-3887-2014, 2014. Friedrichs, M. A. M., Dusenberry, J. A., Anderson, L. A., Arm-
strong, R. A., Chai, F., Christian, J. R., Doney, S. C., Dunne, J.,
Fujii, M., Hood, R., McGillicuddy, D. J., Moore, J. K., Schar-
tau, M., Spitz, Y. H., and Wiggert, J. D.: Assessment of skill
and portability in regional marine biogeochemical models: Role
of multiple planktonic groups, J. Geophys. Res., 112, C08001,
doi:10.1029/2006jc003852, 2007. DeLong, E. F., Preston, C. M., Mincer, T., Rich, V., Hallam, S. J., Frigaard, N. U., Martinez, A., Sullivan, M. B., Edwards, R.,
Brito, B. R., Chisholm, S. W., and Karl, D. M.: Community ge-
nomics among stratified microbial assemblages in the ocean’s in-
terior, Science, 311, 496–503, 2006. Fu, F. X., Warner, M. E., Zhang, Y., Feng, Y., and Hutchins, D. A.: Effects of increased temperature and CO2 on photosynthe-
sis, growth, and elemental ratios in marine Synechococcus and
Prochlorococcus (Cyanobacteria), J. Phycol., 43, 485–496, 2007. Devred, E., Sathyendranath, S., Stuart, V., and Platt, T.: A three
component classification of phytoplankton absorption spectra:
Application to ocean-color data, Remote Sens. Environ., 115,
2255–2266, 2011. Fuhrman, J. A.: Close Coupling between Release and Uptake of
Dissolved Free Amino-Acids in Seawater Studied by an Isotope-
Dilution Approach, Mar. Ecol.-Prog. Ser., 37, 45–52, 1987. Doney, S. C.: Oceanography: Plankton in a warmer world, Nature,
444, 695–696, 2006. Doney, S. C., Fabry, V. J., Feely, R. A., and Kleypas, J. A.: Ocean
acidification: the other CO2 problem, Annu. Rev. Mar. Sci., 1,
169–192, 2009. Gardner, W. D., Mishonov, A., and Richardson, M. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean W., Fol-
lows, M., Harvey, M., Lancelot, C., Levasseur, M., Owens, N. P. J., Pollard, R., Rivkin, R. B., Sarmiento, J., Schoemann, V.,
Smetacek, V., Takeda, S., Tsuda, A., Turner, S., and Watson, A. J.: Mesoscale iron enrichment experiments 1993–2005: Synthe-
sis and future directions, Science, 315, 612–617, 2007. Corzo, A., Morillo, J. A., and Rodríguez, S.: Production of transpar-
ent exopolymer particles (TEP) in cultures of Chaetoceros calci-
trans under nitrogen limitation, Aquat. Microb. Ecol., 23, 63–72,
2000. Costerton, J. W., Lewandowski, Z., Caldwell, D. E., Korber, D. R.,
and Lappinscott, H. M.: Microbial Biofilms, Annu. Rev. Micro-
biol., 49, 711–745, 1995. Breitburg, D. L., Baxter, J. W., Hatfield, C. A., Howarth, R. W.,
Jones, C. G., Lovett, G. M., and Wigand, C.: Understanding ef-
fects of multiple stressors: ideas and challenges, in: Successes,
Limitations, and Frontiers in ecosystem science, edited by: Pace,
M. L., and Groffman, P. M., Springer-Verlag New York, Inc.,
New York, 416–431, 1998. Cottrell, M. T., Yu, L., and Kirchman, D. L.: Sequence and expres-
sion analyses of Cytophaga-like hydrolases in a western Arctic
metagenomic library and the Sargasso Sea, Appl. Environ. Mi-
crobiol., 71, 8506–8513, 2005. Brewin, R. J. W., Lavender, S. J., Hardman-Mountford, N. J., and
Hirata, T.: A spectral response approach for detecting domi-
nant phytoplankton size class from satellite remote sensing, Acta
Oceanol. Sin., 29, 14–32, 2010a. Craig, S. E., Thomas, H., Jones, C. T., Li, W. K. W., Greenan,
B. J. W., Shadwick, E. H., and Burt, W. J.: Temperature and
phytoplankton cell size regulate carbon uptake and carbon over- Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean A., Benner, R., Kattner, G., Wil-
helm, S. W., Kirchman, D. L., Weinbauer, M. G., Luo, T., Chen,
F., and Azam, F.: Microbial production of recalcitrant dissolved
organic matter: long-term carbon storage in the global ocean,
Nat. Rev. Microbiol., 8, 593–599, 2010a. Hinder, S. L., Manning, J. E., Gravenor, M. B., Edwards, M., Walne,
A. W., Burkill, P. H., and Hays, G. C.: Long-term changes in
abundance and distribution of microzooplankton in the NE At-
lantic and North Sea, J. Plankton. Res., 34, 83–91, 2012. Jiao, N., Tang, K., Cai, H., and Mao, Y.: Increasing the microbial
carbon sink in the sea by reducing chemical fertilization on the
land, Nat. Rev. Microbiol., 9, 75–75, doi:10.1038/nrmicro2386-
c2, 2010b. Hirata, T., Aiken, J., Hardman-Mountford, N., Smyth, T. J., and Bar-
low, R. G.: An absorption model to determine phytoplankton size
classes from satellite ocean colour, Remote Sens. Environ., 112,
3153–3159, 2008a. Jiao, N., Herndl, G. J., Hansell, D. A., Benner, R., Kattner, G., Wil-
helm, S. W., Kirchman, D. L., Weinbauer, M. G., Luo, T., Chen,
F., and Azam, F.: The microbial carbon pump and the oceanic
recalcitrant dissolved organic matter pool, Nat. Rev. Microbiol.,
9, 555–555, doi:10.1038/nrmicro2386-c5, 2011. Hirata, T., Hardman-Mountford, N. J., Aiken, J., Martinez-Vicente,
V., Fishwick, J., and Bernard, S.: Particle size distribution de-
termined from ocean colour as a descriptor of nano/micro phyto-
plankton community in the oceans, in Proceedings of the Remote
Sensing and Photogrammetry Society Conference 2008, Univer-
sity of Exeter, Falmouth, UK, 15–17 September 2008, 155–156,
2008b. Jiao, N., Luo, T., Zhang, R., Yan, W., Lin, Y., Johnson, Z. I., Tian,
J., Yuan, D., Yang, Q., Zheng, Q., Sun, J., Hu, D., and Wang, P.:
Presence of Prochlorococcus in the aphotic waters of the western
Pacific Ocean, Biogeosciences, 11, 2391–2400, doi:10.5194/bg-
11-2391-2014, 2014. Johnson, K. S., Berelson, W. M., Boss, E. S., Chase, Z., Claustre,
H., Emerson, S. R., Gruber, N., Kortzinger, A., Perry, M. J., and
Riser, S. C.: Observing Biogeochemical Cycles at Global Scales
with Profiling Floats and Gliders Prospects for a Global Array,
Oceanography, 22, 216–225, 2009. Hirata, T., Hardman-Mountford, N. J., Brewin, R. J. W., Aiken,
J., Barlow, R., Suzuki, K., Isada, T., Howell, E., Hashioka, T.,
Noguchi-Aita, M., and Yamanaka, Y.: Synoptic relationships be-
tween surface Chlorophyll-a and diagnostic pigments specific
to phytoplankton functional types, Biogeosciences, 8, 311–327,
doi:10.5194/bg-8-311-2011, 2011. Johnson, M. T., Greenwood, N., Sivyer, D. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5302 mesopelagic ocean, P. Natl. Acad. Sci. USA, 106, 6513–6518,
2009. community autotrophy in the mesopelagic ocean using natural
radiocarbon, P. Natl. Acad. Sci. USA, 103, 6442–6447, 2006. community autotrophy in the mesopelagic ocean using natural
radiocarbon, P. Natl. Acad. Sci. USA, 103, 6442–6447, 2006. Harris, R. P., Boyd, P., Harbour, D. S., Head, R. N., Pingree, R. D.,
and Pomroy, A. J.: Physical, chemical and biological features of
a cyclonic eddy in the region of 61◦10′N 19◦50′W in the North
Atlantic., Deep-Sea Res. Pt. I, 11, 1815–1839, 1997. IPCC: Climate Change 2013: The Physical Science Basis. Working
Group I Contribution to the Fifth Assessment Report of the In-
tergovernmental Panel on Climate Change, edited by: Stocker, T. F., Qin, D., Plattner, G., Tignor, M., Allen, S. K., Boschung, J.,
Nauel, A., Xia, Y., Bex, V., and Midgley, P. M., Cambridge Uni-
versity Press, Cambridge, United Kingdom and New York, NY,
USA, 1535 pp., 2013. Hartmann, M., Grob, C., Tarran, G. A., Martin, A. P., Burkill, P. H.,
Scanlan, D. J., and Zubkov, M. V.: Mixotrophic basis of Atlantic
oligotrophic ecosystems, P. Natl. Acad. Sci. USA, 109, 5756–
5760, 2012. Jannasch, H. W.: The Microbial Turnover of Carbon in the Deep-
Sea Environment, in: Direct Ocean Disposal of Carbon Dioxide,
edited by: Handa, N. and Ohsumi, T., Terra Scientific Publishing
Company (TERRAPUB), Tokyo, 1–11, 1995. Hays, G. C., Richardson A. J., and Robinson C. Climate change and
marine plankton, Trend. Ecol. Evol., 20, 337–244, 2005 Hedges, I. J. and Keil, R. G.: Sedimentary organic matter preserva-
tion: an assessment and speculative synthesis, Mar. Chem., 49,
81–115, 1995. Company (TERRAPUB), Tokyo, 1–11, 1995. Jiang, H. B., Kong, R. Q., and Xu, X. D.: The N-Acetylmuramic
Acid 6-Phosphate Etherase Gene Promotes Growth and Cell Dif-
ferentiation of Cyanobacteria under Light-Limiting Conditions,
J. Bacteriol., 192, 2239–2245, 2010. Henson, S. A., Sanders, R., and Madsen, E.: Global patterns
in efficiency of particulate organic carbon export and trans-
fer to the deep ocean, Global. Biogeochem. Cy., 26, GB1028,
doi:10.1029/2011gb004099, 2012. Jiao, N. and Zheng, Q.: The microbial carbon pump: from genes to
ecosystems, Appl. Environ. Microbiol., 77, 7439–7444, 2011. Hertkorn, N., Benner, R., Frommberger, M., Schmitt-Kopplin, P.,
Witt, M., Kaiser, K., Kettrup, A., and Hedges, J. I.: Characteriza-
tion of a major refractory component of marine dissolved organic
matter, Geochim. Cosmochim. Ac., 70, 2990–3010, 2006. Jiao, N., Herndl, G. J., Hansell, D. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Engel, A., Thoms, S., Riebesell, U., Rochelle-Newall, E., and Zon-
dervan, I.: Polysaccharide aggregation as a potential sink of ma-
rine dissolved organic carbon, Nature, 428, 929–932, 2004b. Hansell, D. A.: Recalcitrant Dissolved Organic Carbon Fractions,
Annu. Rev. Mar. Sci., 5, 421–445, 2013. Falkowski, P. G., Ziemann, D. A., Kolber, D. A., and Bienfang, P. K.: Role of eddy pumping in enhancing primary production in
the ocean, Nature, 352, 55–58, 1991. Hansell, D. A., Carlson, C. A., Repeta, D. J., and Schlitzer, R.: Dis-
solved organic matter in the ocean – A controversy stimulates
new insights, Oceanography, 22, 202–211, 2009. Fike, D. A., Grotzinger, J. P., Pratt, L. M., and Summons, R. E.:
Oxidation of the Ediacaran ocean, Nature, 444, 744–747, 2006. Hansell, D. A., Carlson, C. A., and Schlitzer, R.: Net re-
moval of major marine dissolved organic carbon fractions in
the subsurface ocean, Global. Biogeochem. Cy., 26, GB1016,
doi:10.1029/2011gb004069, 2012. Flerus, R., Lechtenfeld, O. J., Koch, B. P., McCallister, S. L.,
Schmitt-Kopplin, P., Benner, R., Kaiser, K., and Kattner, G.: A
molecular perspective on the ageing of marine dissolved organic
matter, Biogeosciences, 9, 1935–1955, doi:10.5194/bg-9-1935-
2012, 2012. Hansman, R. L., Griffin, S., Watson, J. T., Druffel, E. R. M., and In-
galls, A. E.: The radiocarbon signature of microorganisms in the www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Koonin, E. V. and Wolf, Y. I.: Genomics of bacteria and archaea:
the emerging dynamic view of the prokaryotic world, Nucleic. Acids. Res., 36, 6688–6719, 2008. Lindh, M. V., Riemann, L., Baltar, F., Romero-Oliva, C., Salomon,
P. S., Granéli, E., and Pinhassi, J.: Consequences of increased
temperature and acidification on bacterioplankton community
composition during a mesocosm spring bloom in the Baltic Sea,
Environ. Microbiol. Rep., 5, 252–262, 2012. Kostadinov, T. S., Siegel, D. A., and Maritorena, S.: Retrieval of the
particle size distribution from satellite ocean color observations,
J. Geophys. Res., 114, C09015, doi:10.1029/2009jc005303,
2009. Lipp, J. S., Morono, Y., Inagaki, F., and Hinrichs, K. U.: Significant
contribution of Archaea to extant biomass in marine subsurface
sediments, Nature, 454, 991–994, 2008. Kostadinov, T. S., Siegel, D. A., and Maritorena, S.: Global vari-
ability of phytoplankton functional types from space: assessment
via the particle size distribution, Biogeosciences, 7, 3239–3257,
doi:10.5194/bg-7-3239-2010, 2010. Liu, J., Jiao, N., and Tang, K.: An experimental study on the effects
of nutrient enrichment on organic carbon persistence in the west-
ern Pacific oligotrophic gyre, Biogeosciences, 11, 5115–5122,
doi:10.5194/bg-11-5115-2014, 2014. Kriest, I., Khatiwala, S., and Oschlies, A.: Towards an assessment of
simple global marine biogeochemical models of different com-
plexity, Prog. Oceanogr., 86, 337–360, 2010. Liu, Y., Yao, T., Jiao, N., Tian, L., Hu, A., Yu, W., and Li, S.: Mi-
crobial diversity in the snow, a moraine lake and a stream in Hi-
malayan glacier, Extremophiles, 15, 411–421, 2011. Kujawinski, E. B.: The Impact of Microbial Metabolism on Ma-
rine Dissolved Organic Matter, Annu. Rev. Mar. Sci., 3, 567–599,
2011. Kujawinski, E. B., Del Vecchio, R., Blough, N. V., Klein, G. C.,
and Marshall, A. G.: Probing molecular-level transformations of
dissolved organic matter: insights on photochemical degradation
and protozoan modification of DOM from electrospray ioniza-
tion Fourier transform ion cyclotron resonance mass spectrome-
try, Mar. Chem., 92, 23–37, 2004. Logan, G. A., Hayes, J. M., Hieshima, G. B., and Summons, R. E.:
Terminal Proterozoic Reorganization of Biogeochemical Cycles,
Nature, 376, 53–56, 1995. Loisel, H., Nicolas, J. M., Deschamps, P. Y., and Frouin, R.:
Seasonal and inter-annual variability of particulate organic
matter in the global ocean, Geophys. Res. Lett., 29, 2196,
doi:10.1029/2002gl015948, 2002. Lampitt, R. S., Achterberg, E. P., Anderson, T. R., Hughes, J. A.,
Iglesias-Rodriguez, M. D., Kelly-Gerreyn, B. A., Lucas, M.,
Popova, E. E., Sanders, R., Shepherd, J. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5303 Le Quere, C., Harrison, S. P., Prentice, I. C., Buitenhuis, E. T., Au-
mont, O., Bopp, L., Claustre, H., Da Cunha, L. C., Geider, R.,
Giraud, X., Klaas, C., Kohfeld, K. E., Legendre, L., Manizza, M.,
Platt, T., Rivkin, R. B., Sathyendranath, S., Uitz, J., Watson, A. J., and Wolf-Gladrow, D.: Ecosystem dynamics based on plank-
ton functional types for global ocean biogeochemistry models,
Glob. Change. Biol., 11, 2016–2040, 2005. Kieber, R. J., Zhou, X. L., and Mopper, K.: Formation of Carbonyl-
Compounds from UV-Induced Photodegradation of Humic Sub-
stances in Natural-Waters – Fate of Riverine Carbon in the Sea,
Limnol. Oceanogr., 35, 1503–1515, 1990. Kim, J. M., Lee, K., Shin, K., Yang, E. J., Engel, A., Karl,
D. M., and Kim, H. C.: Shifts in biogenic carbon flow from
particulate to dissolved forms under high carbon dioxide and
warm ocean conditions, Geophys. Res. Lett., 38, L08612,
doi:10.1029/2011gl047346, 2011. Lechtenfeld, O. J., Kattner, G., Flerus, R., McCallister, S. L.,
Schmitt-Kopplin, P., and Koch, B. P.: Molecular transformation
and degradation of refractory dissolved organic matter in the
Atlantic and Southern Ocean, Geochim. Cosmochim. Ac., 126,
321–337, 2014. Kiorboe, T. and Jackson, G. A.: Marine snow, organic solute
plumes, and optimal chemosensory behavior of bacteria, Limnol. Oceanogr., 46, 1309–1318, 2001. Legendre, L. and Le Fèvre, J.: From individual plankton cells to
pelagic marine ecosystems and to global biogeochemical cy-
cles, in: Particle analysis in oceanography, edited by: Demers,
S., Springer-Verlag, Berlin, 261–300, 1991. Kirchman, D. L.: Processes in Microbial Ecology, Oxford Univer-
sity Press, Oxford, 312 pp., 2012. Koch, B. P., Witt, M., Engbrodt, R., Dittmar, T., and Kattner, G.:
Molecular formulae of marine and terrigenous dissolved organic
matter detected by electrospray ionization Fourier transform ion
cyclotron resonance mass spectrometry, Geochim. Cosmochim. Ac., 69, 3299–3308, 2005. Legendre, L. and Le Fèvre, J.: Microbial Food Webs and the Export
of Biogenic Carbon in Oceans, Aquat. Microb. Ecol., 9, 69–77,
1995. Koch, B. P., Kattner, G., Witt, M., and Passow, U.: Molecular in-
sights into the microbial formation of marine dissolved organic
matter: recalcitrant or labile?, Biogeosciences, 11, 4173–4190,
doi:10.5194/bg-11-4173-2014, 2014. Legendre, L. and Rassoulzadegan, F.: Food-web mediated export of
biogenic carbon in oceans: Hydrodynamic control, Mar. Ecol.-
Prog. Ser., 145, 179–193, 1996. Li, C., Love, G. D., Lyons, T. W., Fike, D. A., Sessions, A. L., and
Chu, X. L.: A Stratified Redox Model for the Ediacaran Ocean,
Science, 328, 80–83, 2010. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean B., Thomson, M., Reeve,
A., Weston, K., and Jickells, T. D.: Characterising the seasonal
cycle of dissolved organic nitrogen using Cefas SmartBuoy high-
resolution time-series samples from the southern North Sea, Bio-
geochemistry, 113, 23–36, 2013. Honjo, S., Manganini, S. J., Krishfield, R. A., and Francois, R.: Par-
ticulate organic carbon fluxes to the ocean interior and factors
controlling the biological pump: A synthesis of global sediment
trap programs since 1983, Prog. Oceanogr., 76, 217–285, 2008. Jørgensen, N. O. G. and Middelboe, M.: Occurrence and bacterial
cycling of D amino acid isomers in an estuarine environment,
Biogeochemistry, 81, 77–94, 2006. Hoppe, H.-G., Breithaupt, P., Walther, K., Koppe, R., Bleck, S.,
Sommer, U., and Jürgens, K.: Climate warming in winter af-
fects the coupling between phytoplankton and bacteria during the
spring bloom: a mesocosm study, Aquat. Microb. Ecol., 51, 105–
115, 2008. Kadouri, D., Jurkevitch, E., Okon, Y., and Castro-Sowinski, S.:
Ecological and agricultural significance of bacterial polyhydrox-
yalkanoates, Crit. Rev. Microbiol., 31, 55–67, 2005. Hwang, J. and Druffel, E. R.: Lipid-like material as the source of
the uncharacterized organic carbon in the ocean?, Science, 299,
881–884, 2003. Kattner, G., Simon, M., and Koch, B.: Molecular characterization of
dissolved organic matter and constraints for prokaryotic utiliza-
tion, in: Microbial Carbon Pump in the Ocean, edited by: Jiao,
N., Azam, F., and Sanders, S., Science/AAAS, Washington DC,
60–61, 2011. Ingalls, A. E., Shah, S. R., Hansman, R. L., Aluwihare, L. I., Santos,
G. M., Druffel, E. R. M., and Pearson, A.: Quantifying archaeal Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5304 by semilabile DOM as revealed by data assimilative modeling,
Aquat. Microb. Ecol., 60, 273–287, 2010. Müren, U., Berglund, J., Samuelsson, K., and Andersson, A.: Po-
tential effects of elevated sea-water temperature on pelagic food
webs, Hydrobiologia, 545, 153–166, 2005. Maiti, K., Benitez-Nelson, C. R., Rii, Y., and Bidigare, R.: The in-
fluence of a mature cyclonic eddy on particle export in the lee of
Hawaii, Deep-Sea Res. Pt II, 55, 1445–1460, 2008. Newbold, L. K., Oliver, A. E., Booth, T., Tiwari, B., DeSantis, T.,
Maguire, M., Andersen, G., van der Gast, C. J., and Whiteley, A. S.: The response of marine picoplankton to ocean acidification,
Environ. Microbiol., 14, 2293–2307, 2012. Mannino, A., Russ, M. E., and Hooker, S. B.: Algorithm develop-
ment and validation for satellite-derived distributions of DOC Ogawa, H., Amagai, Y., Koike, I., Kaiser, K., and Benner, R.: Pro-
duction of refractory dissolved organic matter by bacteria, Sci-
ence, 292, 917–920, 2001. and CDOM in the US Middle Atlantic Bight, J. Geophys. Res.,
113, C07051, doi:10.1029/2007jc004493, 2008. Marchant, H. J. and Scott, F. J.: Uptake of sub-micrometre particles
and dissolved organic material by Antarctic choanoflagellates,
Mar. Ecol.-Prog. Ser., 92, 59–64, 1993. Parekh, P., Dutkiewicz, S., Follows, M. J., and Ito, T.: Atmospheric
carbon dioxide in a less dusty world, Geophys. Res. Lett., 33,
L03610, doi:10.1029/2005gl025098, 2006. Mari, X.: Does ocean acidification induce an upward flux of marine
aggregates?, Biogeosciences, 5, 1023–1031, doi:10.5194/bg-5-
1023-2008, 2008. Park, J. T. and Uehara, T.: How bacteria consume their own ex-
oskeletons (Turnover and recycling of cell wall peptidoglycan),
Microbiol. Mol. Biol. R, 72, 211–227, 2008. Maritorena, S. and Siegel, D. A.: Consistent merging of satellite
ocean color data sets using a bio-optical model, Remote Sens. Environ., 94, 429–440, 2005. Passow, U.: Production of transparent exopolymer particles (TEP)
by phyto-and bacterioplankton, Mar. Ecol.-Prog. Ser., 236, 1–12,
2002. McFadden, K. A., Huang, J., Chu, X., Jiang, G., Kaufman, A. J.,
Zhou, C., Yuan, X., and Xiao, S.: Pulsed oxidation and biological
evolution in the Ediacaran Doushantuo Formation, P. Natl. Acad. Sci. USA, 105, 3197–3202, 2008. Passow, U.: The abiotic formation of TEP under different ocean
acidification scenarios, Mar. Chem., 128, 72–80, 2012. Passow, U. and Carlson, C. A.: The biological pump in a high CO2
world, Mar. Ecol.-Prog. Ser., 470, 249–271, 2012. McGillicuddy Jr., D. J., Robinson, A. R., Siegel, D. A., Jannasch, H. W., Johnson, R., Dickey, T. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean D., McNeil, J., Michaels, A. F., and
Knap, A. H.: Influence of mesoscale eddies on new production
in the Sargasso Sea, Nature, 394, 263–266, 1998. Paulino, A. I., Egge, J. K., and Larsen, A.: Effects of increased at-
mospheric CO2 on small and intermediate sized osmotrophs dur-
ing a nutrient induced phytoplankton bloom, Biogeosciences, 5,
739–748, doi:10.5194/bg-5-739-2008, 2008. McNichol, A. P. and Aluwihare, L. I.: The power of radiocarbon
in biogeochemical studies of the marine carbon cycle: insights
from studies of dissolved and particulate organic carbon (DOC
and POC), Chem. Rev., 107, 443–466, 2007. Pedrotti, M. L., Fiorini, S., Kerros, M. E., Middelburg, J. J., and
Gattuso, J. P.: Variable production of transparent exopolymeric
particles by haploid and diploid life stages of coccolithophores
grown under different CO2 concentrations, J. Plankton. Res., 34,
388–398, 2012. Mitra, A., Flynn, K. J., Burkholder, J. M., Berge, T., Calbet, A.,
Raven, J. A., Granéli, E., Glibert, P. M., Hansen, P. J., Stoecker,
D. K., Thingstad, F., Tillmann, U., Våge, S., Wilken, S., and
Zubkov, M. V.: The role of mixotrophic protists in the biological
carbon pump, Biogeosciences, 11, 995–1005, doi:10.5194/bg-
11-995-2014, 2014. Piontek, J., Lunau, M., Händel, N., Borchard, C., Wurst, M.,
and Engel, A.: Acidification increases microbial polysaccha-
ride degradation in the ocean, Biogeosciences, 7, 1615–1624,
doi:10.5194/bg-7-1615-2010, 2010. Moore, T. S., Mullaugh, K. M., Holyoke, R. R., Madison, A. S.,
Yucel, M., and Luther, G. W.: Marine Chemical Technology and
Sensors for Marine Waters: Potentials and Limits, Annu. Rev. Mar. Sci., 1, 91–115, 2009. Piontek, J., Borchard, C., Sperling, M., Schulz, K. G., Riebesell, U.,
and Engel, A.: Response of bacterioplankton activity in an Arctic
fjord system to elevated pCO2: results from a mesocosm pertur-
bation study, Biogeosciences, 10, 297–314, doi:10.5194/bg-10-
297-2013, 2013. Moran, M. A.: Genomics and metagenomics of marine prokaryotes,
in: Microbial Ecology of the Oceans, edited by: Kirchman, D. L.,
John Wiley and Sons, Inc., Hoboken, New Jersey, 91–129, 2008. Polimene, L., Allen, J. I., and Zavatarelli, M.: Model of interac-
tions between dissolved organic carbon and bacteria in marine
systems, Aquat. Microb. Ecol., 43, 127–138, 2006. Moran, X. A. G., Taupier-Letage, I., Vazquez-Dominguez, E., Ruiz,
S., Arin, L., Raimbault, P., and Estrada, M.: Physical-biological
coupling in the Algerian Basin (SW Mediterranean): Influence of
mesoscale instabilities on the biomass and production of phyto-
plankton and bacterioplankton, Deep-Sea Res. Pt. I, 48, 405–437,
2001. Poretsky, R. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean G., Smythe-Wright,
D., and Yool, A.: Ocean fertilization: a potential means of geo-
engineering?, Philos. Trans. R. Soc. London A, 366, 3919–3945,
2008. Loucaides, S., Tyrrell, T., Achterberg, E. P., Torres, R., Nightin-
gale, P. D., Kitidis, V., Serret, P., Woodward, M., and Robin-
son, C.: Biological and physical forcing of carbonate chem-
istry in an upwelling filament off northwest Africa: Results
from a Lagrangian study, Global. Biogeochem. Cy., 26, GB3008,
doi:10.1029/2011gb004216, 2012. Lauro, F. M., McDougald, D., Thomas, T., Williams, T. J., Egan,
S., Rice, S., DeMaere, M. Z., Ting, L., Ertan, H., Johnson, J.,
Ferriera, S., Lapidus, A., Anderson, I., Kyrpides, N., Munk, A. C., Detter, C., Han, C. S., Brown, M. V., Robb, F. T., Kjelleberg,
S., and Cavicchioli, R.: The genomic basis of trophic strategy
in marine bacteria, P. Natl. Acad. Sci. USA, 106, 15527–15533,
2009. Luisetti, T., Turner, R. K., Bateman, I. J., Morse-Jones, S., Adams,
C., and Fonseca, L.: Coastal and marine ecosystem services val-
uation for policy and management: Managed realignment case
studies in England, Ocean. Coast. Manage., 54, 212–224, 2011. Luo, Y. W., Friedrichs, M. A. M., Doney, S. C., Church, M. J.,
and Ducklow, H. W.: Oceanic heterotrophic bacterial nutrition www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 Biogeosciences, 11, 5285–5306, 2014 N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5305 controlling the air-sea CO2 flux in the North Sea, Cont. Shelf. Res., 29, 1801–1808, 2009. Staats, N., Stal, L. J., and Mur, L. R.: Exopolysaccharide produc-
tion by the epipelic diatom Cylindrotheca closterium: effects of
nutrient conditions, J. Exp. Mar. Biol. Ecol., 249, 13–27, 2000. controlling the air-sea CO2 flux in the North Sea, Cont. Shelf. Res., 29, 1801–1808, 2009. Riebesell, U. and Tortell, P. D.: Effects of ocean acidification
on pelagic organisms and ecosystems, in: Ocean Acidification,
edited by: Gattuso, J. and Hanson, L., Oxford University Press,
Oxford, 99–121, 2011. Stocker, R.: Marine microbes see a sea of gradients, Science, 338,
628–633, 2012. Stramska, M.: Particulate organic carbon in the global ocean derived
from SeaWiFS ocean color, Deep-Sea Res. Pt. I, 56, 1459–1470,
2009. Riebesell, U., Czerny, J., von Bröckel, K., Boxhammer, T., Büden-
bender, J., Deckelnick, M., Fischer, M., Hoffmann, D., Krug, S. A., Lentz, U., Ludwig, A., Muche, R., and Schulz, K. G.: Tech-
nical Note: A mobile sea-going mesocosm system – new oppor-
tunities for ocean change research, Biogeosciences, 10, 1835–
1847, doi:10.5194/bg-10-1835-2013, 2013. Stramski, D., Reynolds, R. A., Kahru, M., and Mitchell, B. G.: Es-
timation of particulate organic carbon in the ocean from satellite
remote sensing, Science, 285, 239–242, 1999. Stramski, D., Reynolds, R. A., Babin, M., Kaczmarek, S., Lewis, M. R., Röttgers, R., Sciandra, A., Stramska, M., Twardowski, M. S.,
Franz, B. A., and Claustre, H.: Relationships between the surface
concentration of particulate organic carbon and optical properties
in the eastern South Pacific and eastern Atlantic Oceans, Biogeo-
sciences, 5, 171–201, doi:10.5194/bg-5-171-2008, 2008. Riemann, L. and Azam, F.: Widespread N-acetyl-D-glucosamine
uptake among pelagic marine bacteria and its ecological impli-
cations, Appl. Environ. Microb., 68, 5554–5562, 2002. Robinson, C., Poulton, A. J., Holligan, P. M., Baker, A. R., Forster,
G., Gist, N., Jickells, T. D., Malin, G., Upstill-Goddard, R.,
Williams, R. G., Woodward, E. M. S., and Zubkov, M. V.: The
Atlantic Meridional Transect (AMT) Programme: A contextual
view 1995–2005, Deep-Sea Res. Pt. II, 53, 1485–1515, 2006. Sturt, H. F., Summons, R. E., Smith, K., Elvert, M., and Hin-
richs, K. U.: Intact polar membrane lipids in prokaryotes and
sediments deciphered by high-performance liquid chromatogra-
phy/electrospray ionization multistage mass spectrometry–new
biomarkers for biogeochemistry and microbial ecology, Rapid
Commun. Mass Sp., 18, 617–628, 2004. Rochelle-Newall, E., Delille, B., Frankignoulle, M., Gattuso, J. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean P.,
Jacquet, S., Riebesell, U., Terbrüggen, A., and Zondervan, I.:
Chromophoric dissolved organic matter in experimental meso-
cosms maintained under different pCO2 levels, Mar. Ecol.-Prog. Ser., 272, 25–31, 2004. Suzumura, M.: Phospholipids in marine environments: a review, Ta-
lanta, 66, 422–434, 2005. Roland, L. A., McCarthy, M. D., and Guilderson, T.: Sources of
molecularly uncharacterized organic carbon in sinking particles
from three ocean basins: A coupled 114C and δ13C approach,
Mar. Chem., 111, 199–213, 2008. Swan, B. K., Martinez-Garcia, M., Preston, C. M., Sczyrba, A.,
Woyke, T., Lamy, D., Reinthaler, T., Poulton, N. J., Masland, E. D. P., Gomez, M. L., Sieracki, M. E., DeLong, E. F., Herndl,
G. J., and Stepanauskas, R.: Potential for Chemolithoautotrophy
Among Ubiquitous Bacteria Lineages in the Dark Ocean, Sci-
ence, 333, 1296–1300, 2011. Romanou, A., Romanski, J., and Gregg, W. W.: Natural ocean car-
bon cycle sensitivity to parameterizations of the recycling in a
climate model, Biogeosciences, 11, 1137–1154, doi:10.5194/bg-
11-1137-2014, 2014. Swanson-Hysell, N. L., Rose, C. V., Calmet, C. C., Halverson, G. P., Hurtgen, M. T., and Maloof, A. C.: Cryogenian glaciation and
the onset of carbon-isotope decoupling, Science, 328, 608–611,
2010. Rothman, D. H., Hayes, J. M., and Summons, R. E.: Dynamics of
the Neoproterozoic carbon cycle, P. Natl. Acad. Sci. USA, 100,
8124–8129, 2003. Tang, K., Jiao, N., Liu, K., Zhang, Y., and Li, S.: Distribution and
functions of TonB-dependent transporters in marine bacteria and
environments: implications for dissolved organic matter utiliza-
tion, Plos One, 7, e41204, doi:10.1371/journal.pone.0041204,
2012. Sathyendranath, S., Stuart, V., Nair, A., Oka, K., Nakane, T.,
Bouman, H., Forget, M. H., Maass, H., and Platt, T.: Carbon-
to-chlorophyll ratio and growth rate of phytoplankton in the sea,
Mar. Ecol.-Prog. Ser., 383, 73–84, 2009. Tarran, G. A., Zubkov, M. V., Sleigh, M. A., Burkill, P. H., and
Yallop, M.: Microbial community structure and standing stocks
in the NE Atlantic in June and July of 1996, Deep-Sea Res. Pt. II, 48, 963–985, 2001. Schrag, D. P., Higgins, J. A., Macdonald, F. A., and Johnston, D. T.:
Authigenic carbonate and the history of the global carbon cycle,
Science, 339, 540–543, 2013. Schulz, K. G., Riebesell, U., Bellerby, R. G. J., Biswas, H., Meyer-
höfer, M., Müller, M. N., Egge, J. K., Nejstgaard, J. C., Neill,
C., Wohlers, J., and Zöllner, E.: Build-up and decline of or-
ganic matter during PeECE III, Biogeosciences, 5, 707–718,
doi:10.5194/bg-5-707-2008, 2008. Taylor, P. G. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean S., Sun, S., Mou, X., and Moran, M. A.: Transporter
genes expressed by coastal bacterioplankton in response to dis-
solved organic carbon, Environ. Microbiol., 12, 616–627, 2010. Prentice, I. C., Farquhar, G. D., Fasham, M. J. R., Goulden, M. L.,
Heimann, M., Jaramillo, V. J., Kheshgi, H. S., LeQuéré, C., Sc-
holes, R. J., and Wallace, D. W. R.: The Carbon Cycle and Atmo-
spheric Carbon Dioxide, in: Climate Change 2001: the Scientific
Basis. Contributions of Working Group I to the Third Assess-
ment Report of the Intergovernmental Panel on Climate Change,
edited by: Houghton, J. T., Ding, Y., Griggs, D. J., Noguer, M.,
van der Linden, P. J., Dai, X., Maskell, K., and Johnson, C. A.,
Cambridge University Press, Cambridge, UK, 185–237, 2001. Morel, A. and Gentili, B.: A simple band ration technique to quan-
tify the colored dissolved and detrital organic material from
ocean color remotely sensed data, Remote Sens. Environ., 113,
998–1011, 2009. Motegi, C., Tanaka, T., Piontek, J., Brussaard, C. P. D., Gattuso, J.-
P., and Weinbauer, M. G.: Effect of CO2 enrichment on bacterial
metabolism in an Arctic fjord, Biogeosciences, 10, 3285–3296,
doi:10.5194/bg-10-3285-2013, 2013. Prowe, A. E. F., Thomas, H., Patsch, J., Kuhn, W., Bozec, Y., Schiet-
tecatte, L. S., Borges, A. V., and de Baar, H. J. W.: Mechanisms Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean 5306 in an Equatorial Pacific phytoplankton assemblage, Mar. Ecol.-
Prog. Ser., 236, 37–43, 2002. in an Equatorial Pacific phytoplankton assemblage, Mar. Ecol.-
Prog. Ser., 236, 37–43, 2002. White, D. C., Flemming, C. A., Leung, K. T., and Macnaughton,
S. J.: In situ microbial ecology for quantitative appraisal, moni-
toring, and risk assessment of pollution remediation in soils, the
subsurface, the rhizosphere and in biofilms, J. Microbiol. Meth.,
32, 93–105, 1998. Tsunogai, S. and Noriki, S.: Particulate Fluxes of Carbonate and
Organic-Carbon in the Ocean – Is the Marine Biological-Activity
Working as a Sink of the Atmospheric Carbon, Tellus B, 43, 256–
266, 1991. Wieder, W. R., Bonan, G. B., and Allison, S. D.: Global soil carbon
projections are improved by modelling microbial processes, Nat. Clim. Chang., 3, 909–912, 2013. Uitz, J., Claustre, H., Gentili, B., and Stramski, D.: Phytoplankton
class-specific primary production in the world’s oceans: Seasonal
and interannual variability from satellite observations, Global. Biogeochem. Cy., 24, GB3016, doi:10.1029/2009gb003680,
2010. Wohlers-Zöllner,
J.,
Biermann,
A.,
Engel,
A.,
Dörge,
P.,
Lewandowska, A. M., von Scheibner, M., and Riebesell, U.: Ef-
fects of rising temperature on pelagic biogeochemistry in meso-
cosm systems: a comparative analysis of the AQUASHIFT Kiel
experiments, Mar. Biol., 159, 2503–2518, 2012. Ullman, R., Bilbao-Bastida, V., and Grimsditch, G.: Including Blue
Carbon in climate market mechanisms, Ocean. Coast. Manage.,
83, 15–18, 2013. Zhang, C. L., Li, Y., Wall, J. D., Larsen, L., Sassen, R., Huang, Y.,
Wang, Y., Peacock, A., White, D. C., Horita, J., and Cole, D. R.: Lipid and carbon isotopic evidence of methane-oxidizing and
sulfate-reducing bacteria in association with gas hydrates from
the Gulf of Mexico, Geology, 30, 239–242, 2002. van Haren, H., Millot, C., and Taupier-Letage, I.: Fast deep sink-
ing in Mediterranean eddies, Geophys. Res. Lett., 33, L04606,
doi:10.1029/2005gl025367, 2006. Volk, T. and Hoffert, M. I.: Ocean carbon pumps: analysis of rel-
ative strength and efficiencies of in ocean-driven circulation at-
mospheric CO2 changes, in: The carbon cycle and atmospheric
CO2: Natural variation Archean to Present, edited by: Sundquist,
E. T. and Broecker, W. S., AGU Monograph 32, American Geo-
physical Union, Washington, DC, 99–110, 1985. Zhang, R., Xia, X., Lau, S. C. K., Motegi, C., Weinbauer, M. G.,
and Jiao, N.: Response of bacterioplankton community structure
to an artificial gradient of pCO2 in the Arctic Ocean, Biogeo-
sciences, 10, 3679–3689, doi:10.5194/bg-10-3679-2013, 2013. N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean and Townsend, A. R.: Stoichiometric control of organic
carbon-nitrate relationships from soils to the sea, Nature, 464,
1178–1181, 2010. Teira, E., Pazo, M. J., Serret, P., and Fernandez, E.: Dissolved or-
ganic carbon production by microbial populations in the Atlantic
Ocean, Limnol. Oceanogr., 46, 1370–1377, 2001. Sherr, E. B.: Direct Use of High Molecular-Weight Polysaccharide
by Heterotrophic Flagellates, Nature, 335, 348–351, 1988. Thomas, H., Ittekkot, V., Osterroht, C., and Schneider, B.: Prefer-
ential recycling of nutrients – the ocean’s way to increase new
production and to pass nutrient limitation?, Limnol. Oceanogr.,
44, 1999–2004, 1999. Siegel, D. A., Maritorena, S., Nelson, N. B., Hansell, D. A., and
Lorenzi-Kayser, M.: Global distribution and dynamics of colored
dissolved and detrital organic materials, J. Geophys. Res., 107,
3228, doi:10.1029/2001jc000965, 2002. Thomas, H., Bozec, Y., Elkalay, K., and de Baar, H. J. W.: Enhanced
open ocean storage of CO2 from shelf sea pumping, Science,
304, 1005–1008, 2004. Siegel, D. A., Maritorena, S., Nelson, N. B., Behrenfeld, M. J., and McClain, C. R.: Colored dissolved organic mat-
ter and its influence on the satellite-based characterization
of the ocean biosphere, Geophys. Res. Lett., 32, L20605,
doi:10.1029/2005gl024310, 2005. Tortell, P. D., DiTullio, G. R., Sigman, D. M., and Morel, F. M. M.:
CO2 effects on taxonomic composition and nutrient utilization www.biogeosciences.net/11/5285/2014/ Biogeosciences, 11, 5285–5306, 2014 Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ N. Jiao et al.: Mechanisms of microbial carbon sequestration in the ocean Ziervogel, B., Dhaksnamoorthy, B., Blachowicz, L., and Roux, B.:
Antibiotic Binding and Dynamics within the OmpF Channel Al-
low Transfer Across the Bacterial Outer Membrane, Biophys. J.,
100, 244–244, 2011. White, A. E., Watkins-Brandt, K. S., Engle, M. A., Burkhardt, B.,
and Paytan, A.: Characterization of the rate and temperature sen-
sitivities of bacterial remineralization of dissolved organic phos-
phorus compounds by natural populations, Front Microbiol., 3,
276, doi:10.3389/fmicb.2012.00276, 2012. Zubkov, M. V. and Tarran, G. A.: High bacterivory by the smallest
phytoplankton in the North Atlantic Ocean, Nature, 455, 224–
226, 2008. White, D. C., Davis, W. M., Nickels, J. S., King, J. D., and Bobbie,
R. J.: Determination of the sedimentary microbial biomass by
extractible lipid phosphate, Oecologia, 40, 51–62, 1979. Biogeosciences, 11, 5285–5306, 2014 Biogeosciences, 11, 5285–5306, 2014 Biogeosciences, 11, 5285–5306, 2014 www.biogeosciences.net/11/5285/2014/ www.biogeosciences.net/11/5285/2014/
|
https://openalex.org/W2081894215
|
https://bmchealthservres.biomedcentral.com/counter/pdf/10.1186/1472-6963-8-152
|
English
| null |
Needs-oriented discharge planning and monitoring for high utilisers of psychiatric services (NODPAM): Design and methods
|
BMC health services research
| 2,008
|
cc-by
| 6,985
|
BioMed Central BioMed Central BioMed Central Published: 21 July 2008 BMC Health Services Research 2008, 8:152
doi:10.1186/1472-6963-8-152 BMC Health Services Research 2008, 8:152
doi:10.1186/1472-6963-8-152 alth Services Research 2008, 8:152
doi:10.1186/1472-6963-8-1 This article is available from: http://www.biomedcentral.com/1472-6963/8/152 is article is available from: http://www.biomedcentral.com/147 © 2008 Puschner 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. Open Acc
Study protocol
Needs-oriented discharge planning and monitoring for high utilisers
of psychiatric services (NODPAM): Design and methods
Bernd Puschner*1, Sabine Steffen1, Wolfgang Gaebel2, Harald Freyberger3,
Helmfried E Klein4, Tilman Steinert5, Rainer Muche6 and Thomas Becker1 Address: 1Department of Psychiatry II, Ulm University, BKH Günzburg, Ludwig-Heilmeyer-Str. 2, 89312 Günzburg, Germany, 2Department of
Psychiatry and Psychotherapy, Heinrich-Heine-University Düsseldorf, Bergische Landstr. 2, 40629 Düsseldorf, Germany, 3Department of
Psychiatry and Psychotherapy, Greifswald University, Rostocker Chaussee 70, 18437 Stralsund, Germany, 4Department of Psychiatry and
Psychotherapy, Regensburg University, Universitätsstr. 84, 93053 Regensburg, Germany, 5Department of Psychiatry I, Ulm University,
Ravensburg, Weingartshofer Str. 2, 88214 Ravensburg, Germany and 6Institute for Biometrics, Ulm University, Schwabstr. 13, 89075 Ulm,
Germany mail: Bernd Puschner* - bernd.puschner@bkh-guenzburg.de; Sabine Steffen - Sabine.Steffen@bkh-guenzburg.de;
Wolfgang Gaebel
Wolfgang Gaebel@uni duesseldorf de; Harald Freyberger
Freyberg@uni greifswald de; Email: Bernd Puschner* - bernd.puschner@bkh-guenzburg.de; Sabine Steffen - Sabine.Steffen@bkh-guenzburg.de;
Wolfgang Gaebel - Wolfgang.Gaebel@uni-duesseldorf.de; Harald Freyberger - Freyberg@uni-greifswald.de;
Helmfried E Klein - Helmfried.Klein@medbo.de; Tilman Steinert - Tilman.Steinert@zfp-weissenau.de; Rainer Muche - Rainer.Muche@uni-
ulm.de; Thomas Becker - t.becker@bkh-guenzburg.de mail: Bernd Puschner* - bernd.puschner@bkh-guenzburg.de; Sabine Steffen - Sabine.Steffen@bkh-guenzburg.de;
Wolfgang Gaebel - Wolfgang.Gaebel@uni-duesseldorf.de; Harald Freyberger - Freyberg@uni-greifswald.de; p
@
g
g
;
@
g
g
gang Gaebel - Wolfgang.Gaebel@uni-duesseldorf.de; Harald Freyberger - Freyberg@uni-greifswald.de; Wolfgang Gaebel - Wolfgang.Gaebel@uni-duesseldorf.de; Harald Freyberger - Freyberg@uni-greifswald.de;
Helmfried E Klein - Helmfried.Klein@medbo.de; Tilman Steinert - Tilman.Steinert@zfp-weissenau.de; Rainer Muche - Rainer.Muche@uni-
ulm.de; Thomas Becker - t.becker@bkh-guenzburg.de Helmfried E Klein - Helmfried.Klein@medbo.de; Tilman Steinert - Tilman.Steinert@zfp-weissenau.de; Rainer Muche - Rainer.Muche@uni-
ulm.de; Thomas Becker - t.becker@bkh-guenzburg.de * Corresponding author Received: 1 July 2008
Accepted: 21 July 2008 Received: 1 July 2008
Accepted: 21 July 2008 Study protocol Open Access Background regarding the arrangement of aftercare occur rather fre-
quently (in two thirds of the cases), patient involvement
with outpatient programmes while they are still in hospi-
tal is less common. g
Large numbers of psychiatric patients do not receive after-
care in the community during the period immediately fol-
lowing hospital discharge. The average rate of utilisation
of aftercare is about 50%, with a wide range between 22
and 90% depending on the definition of aftercare [1-3]. More specifically, one third to one half of patients with
schizophrenia and related disorders miss their first sched-
uled outpatient appointment after discharge [4]. In addi-
tion, for those who receive follow-up, the delay between
discharge and receipt of aftercare (operationalised as first
outpatient visit) has been found to be substantial [1,5]. Thus, limited continuity of service provision is pervasive,
and time lags arising in this process have been found to
increase the probability of relapse and to negatively affect
patients' quality of life [1]. Since the 1980s considerable research effort has been
devoted to high utilisation (HU) of psychiatric services. According to Hadley et al. [8], "the concept of 'heavy use'
typically applies to those persons whose frequency of
admission or duration of inpatient service is substantially
beyond that of the majority of persons receiving similar
treatment" (p. 280). Two extensive literature reviews on
the topic (Kent et al. [9]: 200 publications identified, 72
described; Roick et al. [10]: 250 publications identified,
105 described) showed that overall, 10 – 30% of psychi-
atric patients are identified as high utilisers who consume
between 50 and 80% of service resources. There have been some research efforts aimed at identify-
ing the causes for this failure to utilise psychiatric services
(for an overview see [2]). Results based on patient varia-
bles have been mostly negative, while service system vari-
ables, e.g. availability of discharge planning, have been
found to facilitate access to aftercare. There is some evi-
dence suggesting that patients who receive discharge plan-
ning are more likely to utilise outpatient mental health
services and are less prone to become socially isolated and
require rehospitalisation. Thus, in a sample of N = 104
inpatients with schizophrenia or schizoaffective disorder,
Olfson et al. [6] found that those who had received dis-
charge planning showed significantly better aftercare
compliance (98.1 vs. 62.7%), fewer rehospitalisations
(40% reduction), and better clinical outcome (34.5 vs. Background 41.0 BPRS-points, ES = .57) at three-month follow-up. Furthermore, in a sample of 229 inpatients with a primary
psychiatric diagnosis, Boyer et al. [1] found that patients
were significantly more likely to keep their initial outpa-
tient appointment if they were involved in the outpatient
programme before discharge (OR = 2.14 – 3.89) or if the
discharge plan was discussed between inpatient staff and
outpatient clinicians (OR = 3.17). Probably due to large differences between psychiatric serv-
ice systems, there is no consensus on what constitutes HU. In operationalising HU, most authors solely relied on an
arbitrary number of readmissions during a given time
period – usually study duration. An analysis of nine stud-
ies pertaining to psychiatric inpatient treatment (cf. table
1 in Kent et al. [9]) showed that a mean number of 3.25
(SD = 1.75) admissions during 2.38 (SD = 1.51) years, i.e. 1.37 admissions per year, was used as the criterion for HU. This corresponds to Roick et al. [10] who found that over-
all, patients with 1 – 3 inpatient stays during one year are
identified as a high utilisers. This measure is easily acces-
sible, but has also been criticised (e.g. [11]) since patients
with multiple short hospital stays might be wrongly
included (since they do not use excessive resources), and
patients with few very long hospital stays might be
wrongly excluded, especially when observation time is
short. Thus, some authors used cumulated length of stay Table 1: Study instruments by raters and measurement points. T0
T1
T2
T3
Research worker
CAN-EU
CAN-EU
CAN-EU
CAN-EU
CSSRI-EU
CSSRI-EU
CSSRI-EU
CSSRI-EU
BPRS
BPRS
BPRS
BPRS
HAM-D
HAM-D
HAM-D
HAM-D
MANSA
MANSA
MANSA
MANSA
EQ-5D
EQ-5D
EQ-5D
EQ-5D
Clinician
GAF
GAF
CGI
CGI
STAR-C
STAR-C
ZUF-THERA
DP satisfaction
Patient
SCL-90-R
SCL-90-R
SCL-90-R
SCL-90-R
STAR-P
STAR-P
STAR-P
STAR-P
ZUF-8
DP satisfaction
Notes: DP = Discharge Planning. See text for other abbreviations. Table 1: Study instruments by raters and measurement points. It appears that rather simple measures – such as timely
face-to-face contact of the inpatient with the follow-up
outpatient therapist(s) and/or smooth transition between
in- and outpatient treatment – are likely to bring about an
increase in successful community tenure. Also a time-lim-
ited critical time intervention [6,7] has been shown to
yield clinical improvement. http://www.biomedcentral.com/1472-6963/8/152 http://www.biomedcentral.com/1472-6963/8/152 http://www.biomedcentral.com/1472-6963/8/152 Abstract Background: Attempts to reduce high utilisation of psychiatric inpatient care by targeting the critical time of
hospital discharge have been rare. Methods: This paper presents design and methods of the study "Effectiveness and Cost-Effectiveness of Needs-
Oriented Discharge Planning and Monitoring for High Utilisers of Psychiatric Services" (NODPAM), a multicentre
RCT conducted in five psychiatric hospitals in Germany. Inclusion criteria are receipt of inpatient psychiatric care,
adult age, diagnosis of schizophrenia or affective disorder, defined high utilisation of psychiatric care during two
years prior to the current admission, and given informed consent. Consecutive recruitment started in April 2006. Since then, during a period of 18 months, comprehensive outcome data of 490 participants is being collected at
baseline and during three follow-up measurement points. The manualised intervention applies principles of needs-led care and focuses on the inpatient-outpatient
transition. A trained intervention worker provides two intervention sessions: (a) Discharge planning: Just before
discharge with the patient and responsible clinician at the inpatient service; (b) Monitoring: Three months after
discharge with the patient and outpatient clinician. A written treatment plan is signed by all participants after each
session. Primary endpoints are whether participants in the intervention group will show fewer hospital days and
readmissions to hospital. Secondary endpoints are better compliance with aftercare, better clinical outcome and
quality of life, as well as cost-effectiveness and cost-utility. Discussion: If a needs-oriented discharge planning and monitoring proves to be successful in this RCT, a tool
will be at hand to improve patient outcome and reduce costs via harmonising fragmented mental health service
provision. Trial Registration: ISRCTN59603527 Trial Registration: ISRCTN59603527 Page 1 of 8
(page number not for citation purposes) (page number not for citation purposes) BMC Health Services Research 2008, 8:152 http://www.biomedcentral.com/1472-6963/8/152 Secondary Subjects receiving the intervention will show better com-
pliance with aftercare as well as better clinical outcome
and quality of life. Furthermore, the intervention will
show cost-effectiveness and cost-utility, and community-
based psychiatrists whose patients receive the new dis-
charge protocol will show better compliance with treat-
ment recommendations. Objectives and purpose of the study Objective of the study is to test the following hypotheses. Objective of the study is to test the following hypotheses. Trial desig
interventio
Figure 1 Trial design (timing and projected N for data collection and
intervention)
Figure 1
Trial design (timing and projected N for data collection and
intervention). High utilisers of psychiatric services who receive a needs-
oriented discharge planning and monitoring programme
will show fewer hospital days and readmissions to hospi-
tal. Trial design (timing and projected N for data collection and
intervention)
Figure 1
Trial design (timing and projected N for data collection and
intervention). to examine the short-term (three months after baseline),
mid-term (six months) and long-term (18 months) effects
of the intervention, and on the other hand, the burden on
participants (patients and clinicians) is not too high
which will help to keep low study attrition. In the event of
rehospitalisation, data assessment will proceed as origi-
nally scheduled, allowing some delay in case the patient
should be in an acute crisis making assessment impossi-
ble. Assessment will be carried out by a research worker
(different in person from the intervention worker). Methods and design g
A clinical trial entitled "Effectiveness and Cost-Effective-
ness of Needs-Oriented Discharge Planning and Monitor-
ing for High Utilisers of Psychiatric Services" (NODPAM)
will be conducted. Coordinating centre (CC) is Ulm Uni-
versity's Department of Psychiatry II (Günzburg). Partici-
pating centres are the Departments of Psychiatry and
Psychotherapy at the Universities of Düsseldorf, Greif-
swald, and Regensburg, and Ulm University's Department
of Psychiatry I (Ravensburg). This paper describes design
and methods of the NODPAM trial as outlined in the trial
protocol which has been submitted to the funding body
(German Research Foundation) on July 15, 2005 after
having attended to the funders' and independent review-
ers' comments on the original proposal and its revision. Before the start of recruitment in April 2006, minor
changes have been applied1. Randomisation A central randomisation procedure will be conducted by
an independent unit (Ulm University's Institute for Bio-
metrics). Stratification will be applied since we will con-
duct a multicentre trial, and, furthermore, since the
intervention might have different effects as to diagnostic
criteria, gender, and chronicity. In order to ensure an ade-
quate distribution of patients with regard to these aspects,
strata will be centre (five centres), primary diagnosis
(ICD-10 Chapter V codes F20 – F29 vs. F30 – F39), gender
(male vs. female) and chronicity (shorter vs. equal to or
longer than three years). If during recruitment a patient
fulfils inclusion criteria and has given informed consent,
a fax will be sent to the randomisation centre which will
perform randomisation, generate a patient code, and send
it back to the study centre. Background The impact of extensive strat-
egies such as case management or the introduction of dis-
charge coordinators, on the other hand, has been limited
or controversial. Nonetheless, the use of bridging strategies in routine prac-
tice is idiosyncratic at best. Boyer et al. [1] showed that
while discussions between in- and outpatient teams http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 Trial design (timing and projected N for data collection and
intervention)
Figure 1
Trial design (timing and projected N for data collection and
intervention). 490
441
397
357
months
T0
T1
T2
T3
Data collection
Intervention
pre-
discharge
session
discharge
0
3
6
18
post-
discharge
session
N total
N site
98
88
79
71
N total
N site
245
49
208
42 (LOS) during a given period (in addition to or instead of
number of readmissions) to identify HU. E.g. Lucas et al. [12] developed a "heaviness of use" score by combining
the frequency of admissions with the total number of bed
days. Exceptionally, direct treatment costs beyond cumu-
lated inpatient costs including costs for non-inpatient care
(day hospital treatment, outpatient and complementary
care) was applied [13], but data collection is laborious
and might not be worthwhile since costs for inpatient care
make up the largest share (75–92% [9,14]) of total treat-
ment costs. Primary Trial desig
interventio
Figure 1 Rater perspective (T0 – T3) Cumulated LOS and number of readmissions during the
study period (primary outcomes) will be assessed by a
research worker at T1 – T3 via the German version of the
"Client Sociodemographic and Service Receipt Inventory"
(CSSRI-EU). This is a standardised instrument for the
comprehensive assessment of mental health service use
and costs [16] of which a German version is available
[17]. It provides detailed information on direct (e.g. hos-
pital in-patient days, out-patient/day care attendances,
community-based service contacts, medication profile)
and indirect (e.g. days of work loss, state benefits, source/
level of income) costs and has been used in a number of
German studies2. Overview Cluster randomisation (at the clinician level) will not be
employed since it would severely disrupt clinical routine
and might result in undesired between-centre effects. Fur-
thermore, in many wards of the participating services,
only one clinician is present. Moreover, cross-contamina-
tion among inpatient clinicians is not to be expected since NODPAM is a randomised controlled prospective trial
with four measurement points during an 18-month study
period (see Figure 1, upper part). Assessments will take
place at baseline, three, six, and 18 months after dis-
charge. We believe that this design is appropriate to
answer the research questions since on one hand it allows Page 3 of 8
(page number not for citation purposes) Page 3 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 http://www.biomedcentral.com/1472-6963/8/152 http://www.biomedcentral.com/1472-6963/8/152 Outcome measures the intervention is low profile, restricted to one pre-dis-
charge session, and will be carried out by a research
worker with limited clinician involvement. Assessment will take place at baseline and three (3, 6 and
18 months) follow-up measurement points in each partic-
ipating centre (see Figure 1). Patients will receive a remu-
neration of 30 € for each assessment. Secondary outcomes
will be provided by independent raters (research workers
trained in study instrument use), clinicians, and patients. See Table 1 for an overview of the instruments used. Blinding Due to the nature of the intervention, patients, clinicians,
and intervention workers cannot be blinded to patient
allocation. The natural flow of the study will also prevent
the research worker from being blinded. Furthermore,
blinding to patient allocation and centre affiliation of
principal and responsible investigators as well as of the
person performing randomisation is considered neither
feasible nor necessary because no related bias is expected,
and a blinded intermediate risk assessment, e.g. in order
to identify excess mortality of participants as could be the
case with invasive interventions or acutely critical condi-
tions, is not required. Inclusion and exclusion criteria
Inclusion criteria Psychiatric inpatients between 18–65 years with a primary
diagnosis of schizophrenia, bipolar affective disorder, or
major depression (ICD-10 Chapter V codes F20 – F29 or
F30 – F39). Furthermore, subjects have to be identified as
high utilisers of psychiatric inpatient care. In order to
avoid misclassification, a two-fold criterion consisting of
number of hospitalisations and cumulative LOS prior to
the index admission will be applied; a subject will be
included for participation if he or she, during a 24-month-
period prior to admission: a) has been psychiatrically hos-
pitalised at least twice with a cumulative LOS exceeding
30 days or b) has been psychiatric hospitalised at least
once with a cumulative psychiatric LOS of more than 50
days. This criterion is in accordance with existing evidence
and is also more comprehensive than the one suggested
for psychiatric inpatient care in Germany by Roick et al. [15] who, regardless of LOS, categorised patients as high
utilisers who, during a 30-month period, had had more
than three admissions. Assessment of needs will be carried out with the German
version of the "Camberwell Assessment of Need – Euro-
pean Version" (CAN-EU [18]), an interviewer-adminis-
tered instrument comprising 22 individual domains of
need. Staff and user ratings can be obtained; we adminis-
tered only the user rating. Psychopathology will be assessed
using the "Brief Psychiatric Rating Scale" (BPRS [19])
which is a standardised instrument for the assessment of
psychopathological symptoms. Depression will be tapped
into using the "Hamilton Depression Scale" (HAM-D
[20]). Quality of life will be measured via a) the "Manches-
ter Short Assessment of Quality of Life" (MANSA [21])
which is a disease specific instrument for the multidimen-
sional assessment of objective and subjective quality of
life in persons with severe mental illness and b) the Euro-
Qol group's EQ-5D, a generic quality of life assessment
instrument which has been developed for the computa-
tion of quality adjusted life years (QALYs) and whose psy-
chometric properties and applicability in persons with
severe mental illness have been tested [22]. Patient perspective (T0 – T3) Psychological impairment will be measured using the Ger-
man version [30] of Derogatis' [31] "Symptom-Check-
List" (SCL-90-R), a widely used and validated self-report
scale. Therapeutic relationship (patients' point of view) will
be assessed using the "Scale to assess the therapeutic rela-
tionship in community mental health care" patient scale
(STAR-P [26]). Satisfaction with outpatient treatment (T1
only) will be covered for the outpatient setting with the
German version (ZUF-8 [28]) of the "Client Satisfaction
Questionnaire" (CSQ [29]). Satisfaction with discharge
planning (T1 and intervention group only) will be assessed
with a self-developed eight item rating scale. Sample size
l Power calculation for a panel study with four points of
assessment was based on the approach suggested by
Hedeker et al. [32]. For a high utiliser population, previ-
ous research [33] found mean number of inpatient days
during 12 months after discharge to be 47 (SD = 83) days
(projected mean number for 18 months = 71 days). Based
on existing studies, the mean reduction of inpatient days
due to the planned intervention was assumed to be 40%. A small effect size (0.2 SD) should be detected with a
power of 0.80 at a two-tailed significance level of 0.05. Panel attrition was estimated 10% at each measurement
point. With regard to data analysis, a constant group effect
over time with a random-effect structure and auto-corre-
lated results was expected. A baseline sample size of 242
participants in each group was calculated to be sufficient. Post discharge intervention session
Three months after discharge, the intervention worker
arranges a second (post-discharge) intervention session
including all stakeholders involved in the outpatient treat-
ment phase: outpatient clinician, patient and relatives if
the patient wants them to be present. Initially, a written
protocol is drafted to summarise critical domains of need
from the first session, their development since discharge,
and the current CAN ratings (at 3-month follow-up). Par-
ticipants are asked if they can name reasons for change
(improvement or deterioration) or stability of ratings in
the related domains. Accordingly, after rounding to no decimals, a total sample
size of 490 participants (N = 98 at each site) will be
included in the study. Participants will be randomly
assigned to the intervention (N = 245, N = 49 per site) or
control group (N = 245, N = 49 per site). Subsequently, a revised version of the pre-discharge treat-
ment plan is developed on the basis of current met and
unmet needs and signed by all participants. A typed ver-
sion of the plan will be sent to the outpatient clinician and
the patient with the advice that the plan should be contin-
uously integrated in outpatient treatment, i. e. discussed
and monitored during every contact between the outpa-
tient clinician and patient for a period of at least three
months. The outpatient clinician in private practice is Pre-discharge intervention session g
The intervention worker obtains the relevant information
of patients' met and unmet needs in a basic plan (results
of the CAN rating) from the research worker. The first
joint (pre-discharge) intervention session in general takes
place seven days prior to hospital discharge with the fol-
lowing people participating: in-patient clinician, patient
and carers if the patient wants to. There is a guided discus-
sion of the critical domains of need identified on the basis
of CAN ratings which require both current and post-dis-
charge measures and/or interventions. The handwritten
draft of the post-discharge treatment plan contains every
addressed need with a precise problem definition, objec-
tives, time-frame regarding goal attainment and persons
responsible for the implementation. The treatment plan is
signed by all participants. A typed version of the pre-dis-
charge treatment plan will be sent to the outpatient clini-
cian and the patient, and the accompanying letter will
include the advice to discuss the plan and monitor its
progress during every contact between them. The inpa-
tient clinician is remunerated with CME credit points for
each NODAPM intervention session and with a book
voucher worth 50 € (once). http://www.biomedcentral.com/1472-6963/8/152 http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 of the "Scale to assess the therapeutic relationship in com-
munity mental health care" clinician scale (STAR-C [26]). led care (e.g. [34]) and focus on the inpatient-outpatient
transition, with the intervention worker emphasising con-
tinuity of the care process vis-à-vis both patient and clini-
cians (e.g. [7]). The intervention worker will provide (and
patients will be actively involved in) two manualised
intervention sessions (each of about 45 minutes duration,
see Figure 1, lower part): Satisfaction with therapeutic work in the ambulatory setting
(T1 only) will be covered with a self-developed scale
(ZUF-THERA [27]) consisting of six rephrased items of the
ZUF-8 [28], the German version of the "Client Satisfaction
Questionnaire" (CSQ [29]). The items were rephrased in
that way that outpatient clinicians could give an evalua-
tion of the quality of the given treatment. Satisfaction with
discharge planning (T1 and intervention group only) will
be assessed with a self-developed nine item rating scale. Exclusion criteria (a) Primary diagnosis of substance abuse; (b) Presence of
moderate or severe mental handicap (learning disability)
or organic mental disorder; (c) Current treatment by
forensic psychiatric services; (d) Insufficient command of
the German language in order to receive the intervention,
take part in the assessment interviews, and complete
patient questionnaires; (e) Lacking capacity to give valid
consent to participate; (f) Foreseeable inpatient or day
care mental health treatment (including rehabilitation)
extending seven days after discharge from psychiatric
inpatient treatment. Clinician perspective (T0 [inpatient clinician] and T1 [outpatient
clinician]) Clinician perspective (T0 [inpatient clinician] and T1 [outpatient
clinician]) Psychosocial functioning will be rated via the "Global
Assessment of Functioning Scale" (GAF [23]) which has
been shown to be a practical and valid instrument in psy-
chiatric research [24]. Overall psychological impairment will
be tapped into via the "Clinical Global Impression Scale"
(CGI [25]). Therapeutic relationship (clinicians' point of
view) will be assessed using a retranslated German version Page 4 of 8
(page number not for citation purposes) Page 4 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 Monitoring of flow and quality of data Analyses will start once baseline data have been collected
and cleaned. Descriptives of all outcome measures will be
produced, and outcome trajectories from T0 – T3 will be
examined via exploratory analyses. The effect of the inter-
vention on reduction of number of inpatient days as well
as on clinical outcomes, quality of life, and compliance
will be tested by means of random-effect regression mod-
els including a constant group effect across time [35-37]. Due to the application of a generalised least square (GLS)
estimation weighted for the lengths of the time series for
each case, these models allow the inclusion of cases with
incomplete (unbalanced) data across panels. Due to
expected skewness of the primary dependent variable
(LOS), bootstrapping methods will be applied for the esti-
mation of standard errors and confidence intervals. Cost-
effectiveness of intervention will be tested by means of the
net-benefit approach [38], and its cost-utility by using
QALYs generated from EQ-5D. Monitoring of flow and quality of data
(a) At each centre, data will be entered via identical tem-
plates programmed by CC staff using EpiData http://
www.epidata.dk/, a reliable software for entering and doc-
umenting data allowing for e.g. the definition of out-of-
range and outlier data as well as logic checks; (b) After
data entry, centres will send copies of data files and com-
pleted questionnaires to the CC which will carry out: (i)
Review: 10% of the data will be entered again by the CC
and subsequently compared with the data in the files
received from the participating centres. In case of major
differences, proportion of the reviewed data will be
increased; (ii) Query: In case of mismatch, the CC will
contact the respective participating centre in order to cor-
rect data problems. Results of these queries will be com-
municated to the group. Furthermore, a trial steering committee (TSC) was estab-
lished in its function as an independent board of mem-
bers to provide overall supervision and protection for the
trial participants and the principal investigators. In view
of the non-invasive intervention, the risk for participating
patients is considered marginal. http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 remunerated with CME credit points and 100 € for each
NODPAM intervention session. which have been signed by patients are complete and
safely stored at the participating centres; (b) Every partici-
pating patient meets inclusion criteria; (c) Both the inter-
vention and assessment of outcome criteria, in each
individual patient, are carried out according to the trial
protocol and intervention manual. In addition, before
start of recruitment, trial staff responsible for data collec-
tion and application of the intervention will be trained in
the use of study instruments at the CC, also by external
experts if applicable. As the study progresses, adherence to
instrument and intervention manuals will be continu-
ously monitored and training refreshed if necessary, espe-
cially in case of staff changes. Monitoring of flow and quality of data Should, for any unfore-
seeable reason, the intervention or data collection place a
burden on a given participant and should at the same time
he or she be unwilling to terminate participation, the
research worker and/or intervention worker are required
to report this to the TSC who will then decide on whether
or not the participant will have to be excluded. Such
instances will be documented thoroughly. Analysis will be carried out according to the principle of
intention-to-treat. In case attrition should exceed the pro-
jected rate of 10%, additional per-protocol analysis will
be performed. Data monitoring and safety All trial data will be stored safely at the participating cen-
tres. The CC is responsible for the merging of the data
from the centres and – after data cleaning – redistributing
it to the centres for analysis. Data Monitoring will be carried out by persons independ-
ent of trial conduct. Before start of data collection, each
participating centre will name a person responsible for
data entry and monitoring to the CC. The following mon-
itoring strategy will be adhered to. The advisory board, the TSC and Ulm University Hospital
will be continuously informed of study progress including
data quality issues. Intervention
h
i For the intervention group, an intervention worker will
provide a coherent package of needs-oriented discharge
planning and monitoring focusing on the care process. The NODPAM intervention will apply principles of needs- Page 5 of 8
(page number not for citation purposes) Page 5 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 Statistical analysis The primary endpoints will be derived from the difference
in LOS and number of hospitalisations between T0 and
T1, T2, and T3. The secondary endpoints will be calculated
from the differences in sum (or – if applicable – subscale)
scores of the secondary outcome measures between T0
and T1, T2, and T3. Multiple measurement points have
been chosen in order to scrutinise the intervention's effect
over time, especially the question of whether short- and
mid-term effects would persist. Authors' contributions TB is principal and coordinating investigator of NOD-
PAM. BP is responsible investigator in charge of trial man-
agement. The study was initiated by TB and BP who
jointly wrote up and revised the proposal and study pro-
tocol in close collaboration with local PIs (WG, HF, HEK,
TS). RM substantially contributed to the methods section,
in particular to details of the randomisation. SS prepared
the final draft for publication. All authors contributed to
the design and continuing management of the study. Literature reviews conclude that specific interventions tar-
geting the needs of high utilisers and subsequently pre-
venting unnecessary high service use should be developed
and evaluated as to their use for patients and clinicians
[9,10,39]. Furthermore, taking into account the finding that health
service costs make up 76% of the total costs, and that 59%
of these are inpatient costs [14], any intervention that is
effective in reducing the heavy inpatient bed use of heavy
users should have a substantial effect on the total service
costs [12,38]. Since service use patterns of high utilisers
appear to depend on service system rather than on indi-
vidual patient variables [8], it has been specifically recom-
mended to identify gaps in current service provision [9],
and – as outlined above – discharge from psychiatric hos-
pital obviously can be considered such a gap, particularly
in fragmented service systems such as the German one. Footnotes
h 1 These were: (a) Randomisation strata: Originally
planned strata were centre, primary diagnosis, and GAF
score at admission. GAF score at admission was replaced
by gender and illness duration. (b) Original exclusion cri-
terion was only that subjects with a primary diagnosis of
substance abuse will be excluded. (c) Originally the Help-
ing Alliance Questionnaire (HAQ) was intended to be
used for the assessment of the quality of the therapeutic
relationship (changed to STAR); (e) Application of instru-
ments to assess treatment satisfaction (ZUF-8 and ZUF-
THERA) and satisfaction with discharge planning; (d)
Specification of details on amount and kind of clinician
remuneration. In the course of recruitment, the research worker will
devote special attention to providing the patients with
detailed information on the trial so they have a sound
basis on which to decide on informed consent. It will be
made clear to the patient that he or she can withdraw con-
sent at any time during the course of the study without
any consequences. In case the patient is under legal cus-
tody, the custodian's consent does not suffice. All partici-
pating centres' ethics committees will have issued positive
votes on NODPAM before the start of the study. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Acknowledgements This study is supported by a grant from the German Research Foundation
(DFG) in the Special Programme "Clinical Studies" (Grant number BE 2502/
3-1). We are grateful to the responsible investigators at the participating
centres – Birgit Janssen (Düsseldorf), Carsten Spitzer (Greifswald), Her-
mann Spießl (Regensburg), and Jan Bergk (Ulm/Ravensburg) – as well as to
the NODPAM advisory board, especially to Mike Slade (Institute of Psychi-
atry, King's College London), for their substantial contributions during the
preparation of NODPAM. We also thank Annette Schmidtmann at German
Research Foundation (DFG) and the anonymous reviewers for their signif-
icant suggestions. If this needs-oriented discharge planning and monitoring
intervention proves to be successful in this RCT, a tool will
be at hand (a) to harmonise fragmented service provision,
i.e. improve collaboration of in- and outpatient services
and continuity of care; (b) to improve community tenure,
clinical impairment, and quality of life; and (c) to reduce
unnecessary inpatient treatment costs. Ethics Monitoring of patient recruitment, data collection and application of
the intervention Patient confidentiality will be strictly maintained, i.e. by
no means will publications contain person-related infor-
mation. However, the conduct of a longitudinal trial
requires patient follow-up. Thus, study participants' per-
sonal data (e.g. names and addresses) will be recorded for Study visits and regular telephone conferences will allow
for a 100% check of essential trial parameters. In particu-
lar, the CC will see to that: (a) All informed consent forms Page 6 of 8
(page number not for citation purposes) Page 6 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 the duration of the trial. Patient ID data will be stored
safely and kept separate from the data set used for analy-
ses. the duration of the trial. Patient ID data will be stored
safely and kept separate from the data set used for analy-
ses. Discussion 2 The CSSRI-EU also serves to assess necessary informa-
tion pertaining to SES (work, financial and living situa-
tion) and service utilisation beyond inpatient care. Furthermore, basic patient data will be available via the
"BADO" which has been implemented at all participating
centres. There is broad consensus that relapse prevention is one of
the major aims of aftercare. However, the success of
attempts to reduce high re-hospitalisation rates in people
suffering from severe mental disorders has been limited so
far. Insufficient discharge planning and follow-up is con-
sidered one of the main reasons for limited community
tenure and unfavourable clinical outcomes. Only a small
number of RCTs have been conducted to test this assump-
tion. Page 7 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 BMC Health Services Research 2008, 8:152 4. Saarento O, Oiesvold T, Sytema S, Gostas G, Kastrup M, Lonnerberg
O, Muus S, Sandlund M, Hansson L: The Nordic Comparative
Study on Sectorized Psychiatry: continuity of care related to
characteristics of the psychiatric services and the patients. Soc Psychiatry Psychiatr Epidemiol 1998, 33:521-527. tioning Scale]. In Diagnostisches und Statistisches Manual psychischer
Störungen Göttingen: Hogrefe; 1996. 24. g
g
g
Startup M, Jackson MC, Bendix S: The concurrent validity of the Global Assessment of Functioning (GAF). Brit J Clin Psychol
2002, 41:422. 25. y
y
y
p
5. Sytema S: Service utilization research: goals and prospects. Epidemiol Psichiatr Soc 1997, 6:127-136. . Haro JM, Kamath SA, Ochoa S, Novick D, Rele K, Fargas A, Rodriguez
MJ, Rele R, Orta J, Kharbeng A, Araya S, Gervin M, Alonso J, Mavreas
V, Lavrentzou E, SOHO Study Group: The Clinical Global
Impression-Schizophrenia scale: a simple instrument to
measure the diversity of symptoms present in schizophre-
nia. Acta Psychiatr Scand 2003, 416:16-23. 6. Olfson M, Mechanic D, Boyer CA, Hansell S: Linking inpatients
with schizophrenia to outpatient care. Psychiatr Serv 1998,
49:911-917. 7. Hermann D, Opler L, Felix A, Valencia E, Wyatt RJ, Susser E: A crit-
ical time intervention with mentally ill homeless men:
Impact on psychiatric symptoms. J Nerv Ment Dis 2000,
188:135-140. y
26. McGuire-Snieckus R, McCabe R, Catty J, Hansson L, Priebe S: A new
scale to assess the therapeutic relationship in community
mental health care: STAR. Psychol Med 2006, 37:85-95. 27. 27. Puschner B, Bauer S, Kraft S, Kordy H: Zufriedenheit von
Patienten und Therapeuten mit ambulanter Psychotherapie
[Patient and therapist satisfaction in outpatient psychother-
apy]. Psychother Psychosom Med Psychol 2005, 55:517-526. 8. Hadley TR, Culhane D, McGurrin M: Identifying and tracking
heavy users of acute psychiatric inpatient services. Adminin Pol
Ment Health 1992, 19:279-290. ,
9. Kent S, Fogarty M, Yellowlees P: A review of studies of heavy
users of psychiatric services. Psychiatr Serv 1995, 46:1247-1253. 28. Schmidt J, Lamprecht F, Wittmann WW: Zufriedenheit mit der
stationären Versorgung: Entwicklung eines Fragebogens und
erste Validitätsuntersuchungen [Satisfaction with inpatient
management. Development of a questionnaire and initial
validity studies]. Psychother Psychosom Med Psychol 1989,
39:248-255. 10. Roick C, Gärtner A, Heider D, Angermeyer MC: Heavy User psy-
chiatrischer Versorgungsdienste: Ein Überblick über den
Stand der Forschung [Heavy users of psychiatric care: A
review of the state of research]. Psychiat Prax 2002, 29:334-342. 11. http://www.biomedcentral.com/1472-6963/8/152 Wooldridge JM: Econometric Analysis of Cross Section and Panel Data
Cambridge: MIT Press; 2002. 17. Roick C, Kilian R, Matschinger H, Mory C, Angermeyer MC: Die
deutsche Version des Client Sociodemographic and Service
Receipt Inventory: Ein Instrument zur Erfassung psychia-
trischer Versorgungskosten [German adaptation of the cli-
ent sociodemographic and service receipt inventory – an
instrument for the cost of mental health care]. Psychiat Prax
2001, 28:84-90. 38. Hoch JS, Briggs AH, Willan AR: Something old, something new,
something borrowed, something blue: A framework for the
marriage of health econometrics and cost-effectiveness anal-
ysis. Health Econ 2002, 11:415-430. 39. Junghan UM: "Heavy Users": Probleme bei der Identifikation
von Patienten mit überdurchschnittlicher Inanspruchnahme
stationärer Akutversorgung [Heavy users: Problems in the
identification of patients with disproportionate use of acute
inpatient care]. In Fortschritte in Psychiatrie und Psychotherapie. Inter-
disziplinäre und integrative Aspekte Edited by: Konrad P. Wien: Springer;
2002:161-175. 18. McCrone P, Leese M, Thornicroft G, Griffiths G, Padfield S, Schene A,
Knudsen HC, Vazquez-Barquero JL, Lasalvia A, White IR: Reliability
of the Camberwell Assessment of Need – European Version:
EPSILON Study 6. Brit J Psychiatr 2000, 177:34-40. 19. Lukoff D, Nuechterlein KH, Ventura J: Manual for expanded Brief
Psychiatric Rating scale (BPRS). Schizophrenia Bull 1986,
12:594-602. http://www.biomedcentral.com/1472-6963/8/152 Junghan UM: Das ungleiche Ausmass der Nutzung psychia-
trischer Akutstationen: Eine wichtige Beobachtung für die
gemeindepsychiatrische Versorgungsplanung [Dispropor-
tionate extent of use of psychiatric acute care: An important
observation for planning community psychiatric services]. Schizophrenie Beiträge zu Forschung, Therapie und psychosozialem Man-
agement 2000, 15:24-34. 29. Attkisson CC, Zwick R: The Client Satisfaction Questionnaire. Eval Program Plann 1982, 5:233-237. g
30. Franke GH: Die Symptom-Checkliste von Derogatis – Deutsche Version
[Derogatis' Symptom Checklist – German version] Göttingen: Beltz Test;
1995. 31. Derogatis LR: SCL-90-R: Self Report Symptom Inventory. In
Internationale Skalen für Psychiatrie Edited by Collegium Internationale
Psychiatriae Scalarum. Weinheim: Beltz; 1986. g
12. Lucas B, Harrison-Read P, Tyrer P, Ray J, Shipley K, Hickman M, Patel
A, Knapp M, Lowin A: Costs and characteristics of heavy inpa-
tient service users in outer London. Int J Soc Psychiatry 2001,
47:63-74. 32. Hedeker D, Gibbons RD, Waternaux C: Sample size estimation
for longitudinal designs with attrition: comparing time-
related contrasts between two groups. J Educ Behav Stat 1999,
24:70-93. 13. Hadley TR, McGurrin MC, Pulice RT, Holohean EJ: Using fiscal data
to identify heavy service users. Psychiatr Q 1990, 61:41-48. 14. Lang FH, Forbes JF, Murray GD, Johnstone EC: Service provision
for people with schizophrenia: I. Clinical and economical
perspective. Br J Psychiatry 1997, 171:159-164. 33. Kilian R, Matschinger H, Becker T, Angermeyer MC: A longitudinal
analysis of the impact of social and clinical characteristics on
the costs of schizophrenia treatment. Acta Psychiatr Scand 2003,
107:351-360. p
p
J
y
y
15. Roick C, Heider D, Angermeyer MC: Entwicklung eines Screen-
ing-Instruments zur Identifikation von Patienten mit starker
Inanspruchnahme
stationär-psychiatrischer
Versorgung
[Development of a screening instrument for the identifica-
tion of high utilisers of psychiatric inpatient care]. Fortschr
Neurol Psychiat 2003, 71:378-386. 34. Slade M, Leese M, Taylor R, Thornicroft G: The association
between needs and quality of life in an epidemiologically rep-
resentative sample of people with psychosis. Acta Psychiatr
Scand 1999, 100:149-157. 35. Baltagi BH: Econometric analysis of panel data Chichester, New York:
John Wiley & Sons; 2001. y
16. Chisholm D, Knapp M, Knudsen HC, Amaddeo F, Gaite L, van Wijn-
gaarden B, the EPSILON study group: Client Sociodemographic
and Service Receipt Inventory – European version. Br J Psychi-
atry 2000, 177:28-33. 36. Raudenbush SW, Bryk AS: Hierarchical linear models: applications and
data analysis methods 2nd edition. Newbury Park, CA: Sage; 2001. y
y
g
37. References 1. Boyer CA, McAlpine DD, Pottick KJ, Olfson M: Identifying risk fac-
tors and key strategies in linkage to outpatient psychiatric
care. Am J Psychiatr 2000, 157:1592-1598. 1. Boyer CA, McAlpine DD, Pottick KJ, Olfson M: Identifying risk fac-
tors and key strategies in linkage to outpatient psychiatric
care. Am J Psychiatr 2000, 157:1592-1598. J
y
2. Klinkenberg WD, Calsyn RJ: Predictors of receipt of aftercare
and recidivism among persons with severe mental illness: A
review. Psychiatr Serv 1996, 47:487-496. y
3. Miner CR, Rosenthal RN, Hellerstein DJ, Muenz LR: Prediction of
compliance with outpatient referral in patients with schizo-
phrenia and psychoactive substance use disorders. Arch Gen
Psychiat 1997, 54:706-712. y
3. Miner CR, Rosenthal RN, Hellerstein DJ, Muenz LR: Prediction of
compliance with outpatient referral in patients with schizo-
phrenia and psychoactive substance use disorders. Arch Gen
Psychiat 1997, 54:706-712. Page 7 of 8
(page number not for citation purposes) Page 7 of 8
(page number not for citation purposes) http://www.biomedcentral.com/1472-6963/8/152 Pre-publication history 20. Hamilton M: Development of a rating scale for primary
depressive illness. Br J Soc Clin Psychol 1967, 6:278-296. The pre-publication history for this paper can be accessed
here: The pre-publication history for this paper can be accessed
here: 21. Priebe S, Huxley P, Knight S, Evans S: Application and results of
the manchester short assessment of quality of life (MANSA). Int J Soc Psychiatr 1999, 45:7-12. http://www.biomedcentral.com/1472-6963/8/152/pre
pub http://www.biomedcentral.com/1472-6963/8/152/pre
pub J
y
22. Prieto L, Novick D, Sacristán JA, Edgell ET, Alonso J, on behalf of the
SOHO Study Group: A rasch model analysis to test the cross-
cultural validity of the EuroQoL-5D in the schizophrenia out-
patient health outcome study. Acta Psychiatr Scand 2003,
107:S24-S29. 23. Saß H, Wittchen H-U, Zaudig M: Skala zur Globalbeurteilung des
Funktionsniveaus für DSM-IV [Global Assessment of Func- Page 8 of 8
(page number not for citation purposes) Page 8 of 8
(page number not for citation purposes) (page number not for citation purposes)
|
https://openalex.org/W4386414794
|
https://ijirms.in/index.php/ijirms/article/download/1734/1282
|
English
| null |
Fetal Umbilical Artery Velocimetry Indices and Pregnancy Outcome Among Preeclamptic Women at the Federal Teaching Hospital, Abakaliki, Southeast Nigeria
|
International journal of innovative research in medical science
| 2,023
|
cc-by
| 5,079
|
Keywords: Fetal, Umbilical Artery, Velocimetry, Indices, Preeclampsia The main principle underlying the use of Doppler indices is
that Preeclampsia which is due to faulty placentation will lead to
increased resistance to blood flow in the maternal-fetal vessels
indicating compromised maternal-placental perfusion sequence [8]. The subsequent reduction of blood flow in the fetal umbilical arteries
signifies compromised placenta-umbilical circulation. The fall in
blood flow through the umbilical arteries triggers fetal compensation
in the form of brain sparing; by this effect, there is preferential
perfusion of the brain through the middle cerebral arteries, the so-
called centralization [8]. Many Doppler velocimetry indices in
current use are from studies done elsewhere, given that Doppler
indices may be influenced by the patient’s race coupled with the
suboptimal performance of locally available fetal surveillance tools
[3,4,10-12], makes a study of this nature imperative. Accepted 23 August 2023; Received 08 August 2023; International Journal of Innovative Research in Medical Science (IJIRMS)
Volume 08, Issue 09, September 2023,
https://doi.org/10.23958/ijirms/vol08-i09/1734 International Journal of Innovative Research in Medical Science (IJIRMS)
Volume 08, Issue 09, September 2023,
https://doi.org/10.23958/ijirms/vol08-i09/1734 1Department of Obstetrics and Gynaecology, Alex Ekwueme Federal University Teaching Hospital, Abakaliki,
Ni
i 1Department of Obstetrics and Gynaecology, Alex Ekwueme Federal University Teaching Hospital, Abakaliki,
Nigeria. g
2Department of Radiology, Alex Ekwueme Federal University Teaching Hospital, Abakaliki, Nigeria. 3Department of Obstetrics and Gynaecology, David Umahi Federal University Teaching Hospital, Uburu, Nigeria. *Corresponding Author: Darlington-Peter C. UGOJI; darlingtonpeter2012@gmail.com *Corresponding Author: Darlington-Peter C. UGOJI; darlingtonpeter2012@gmail.com Published 01 September 2023 Published 01 September 2023 Accepted 23 August 2023; Fetal Umbilical Artery Velocimetry Indices and
Pregnancy Outcome Among Preeclamptic Women at
the Federal Teaching Hospital, Abakaliki, Southeast
Nigeria Johnbosco E. MAMAH 1, Robinson C. Onoh 1, Michael ORJI 2, Darlington-Peter C. UGOJI
*1,3,
Chichetaram R. OTU 1, Odidika U. J. UMEORA 1 Abstract An accurate tool to identify fetuses at risk of in-utero compromise in women with Preeclampsia is unknown. We studied the hemodynamic
changes in the fetal umbilical and middle cerebral arteries and their association with pregnancy outcomes. This was a cross-sectional study among
eligible pregnant women with Preeclampsia. We conducted a Doppler evaluation of the fetal umbilical and middle cerebral artery indices. The
neonatal APGAR scores, birth weight, NICU admission and perinatal deaths were documented. Maternal complications were documented. Data
analysis was undertaken with Statistical Package for Social Sciences (IBM-SPSS, version 22, Chicago II, USA). Means were compared using
the Z-test for continuous variables, while categorical variables were compared with Chi-square. Relationships were assessed using Pearson's
correlation, with significance at P< 0.05. The accuracy of Doppler indices was calculated using contingency tables. There was a statistically
significant association between fetal complications and Doppler indices but not with maternal complications. The Sensitivity of Doppler indices
was higher with fetal umbilical artery Doppler indices, while the middle cerebral artery indices were more specific. Accuracy is better when
Doppler indices are combined with the cerebro-placental ratio. In women with normal Doppler indices, the indices decreased with advancing
gestational age, but values were higher when compared with nomograms. In conclusion, we found an association between fetal Doppler indices
of the umbilical artery and adverse fetal outcomes. Abnormal umbilical artery Doppler indices suggest fetal compromise, while normal middle
cerebral artery Doppler is reassuring. Keywords: Fetal, Umbilical Artery, Velocimetry, Indices, Preeclampsia Keywords: Fetal, Umbilical Artery, Velocimetry, Indices, Preeclampsia Diagnosis of Preeclampsia For those who had a vaginal delivery, their labour was managed
using the departmental protocol for managing Preeclampsia. This
entailed using a partograph and continuous electronic fetal monitor
for monitoring labour events. Those delivered abdominally had the
procedure performed by a senior registrar or consultant in the
Obstetrics and Gynaecology department. The neonatology team
attended all the deliveries. The Acuson® brand mercury sphygmomanometer was used for the
blood pressure measurement with appropriate cuff size covering at
least 2/3rd of the length of the patient's left arm. Blood pressure was
measured with the patient in a reclined position. Before taking
measurements, the patient is allowed 30 minutes of rest. In taking
the measurement, the cuff was inflated while simultaneously
palpating the radial pulse. Inflation was continued for a further
20mmHg beyond the point where the radial pulse became
impalpable. The pressure was slowly released at a pace of 2mmHg
until the radial pulse became palpable again, indicating systolic
blood pressure. The cuff is then re-inflated, a stethoscope is applied
to the cubital fossa, and the pressure is slowly released as in the
previous fashion. The phase 1 Korotkoff sound is recorded as the
systolic blood pressure (SBP), while the phase 5 Korotkoff sound
was recorded as the diastolic blood pressure (DBP) measurement. Severe hypertension was diagnosed if the patient's blood pressure
was recorded as systolic or diastolic blood pressure equal to or more
than 160mmgHg or 110mmHg, respectively. Urinalysis was done by
dipstick testing of clean catch midstream urine. The participants
were given a wide-bore clean universal bottle and asked to collect a
clean catch midstream urine specimen. Before taking the sample, the
patients were asked to wash their perineum using clean water
provided in the toilet. They were educated to stand astride, open the
sample bottle, and collect a mid-stream urine specimen. After
specimen collection, a urinalysis was performed to test for
proteinuria. Proteinuria of 2+ or more was considered significant. Severe hypertension with significant proteinuria confirms a
diagnosis of severe Preeclampsia. Study design This cross-sectional study involved pregnant women with
Preeclampsia who were followed up from admission till delivery. This study lasted a period of seven months (June to December 2018) The technique of MCA Doppler Velocimetry An axial section of the fetal brain, including the thalami and the
sphenoid bone, was located, and magnified to evaluate the MCA. The pulsed-wave Doppler gate was placed at the proximal third of
the MCA, close to its origin on the internal carotid artery [15,16]. This
was because the systolic velocity decreased with increasing distance
from the point of origin of this vessel. Care was taken to avoid any
undue pressure on the fetal head. The PI, RI, CPI and S/D ratio were
noted. The abnormal recording was reported when the RI, PI and
S/D ratio is <5th percentile [12] while a (CPR) of <1 was taken as
abnormal [17]. Protocol for patient Doppler evaluation Doppler interrogation of the umbilical and middle cerebral arteries
was conducted, and the Pulsatility index (PI), resistive index (RI),
systolic/diastolic ratio (SDR) and cerebro-placenta ratio (CPR) were
documented. This investigation was accomplished using the
SamsungMedison, AccuvixA30 ultrasound machine manufactured
in South Korea in 2013. A 3.5-MHz trans-abdominal transducer was
used. The sonologists trained for the study performed the
investigation using the same instrument setting and technique. Each
patient was investigated in the semi-recumbent position at an angle
of about 300 with a 150 left lateral tilt. Primary outcome measure
Number of patients with abnormal Doppler velocimetry indices. Secondary outcome measures: Number of patients with preterm
delivery, birth asphyxia, low birth weight, NICU admission and
women who suffered perinatal deaths and the number of patients
who suffered maternal complications such as eclampsia, primary
postpartum haemorrhage, abruption placentae, disseminated
intravascular coagulation (DIC), intensive care unit (ICU)
admission, maternal death. Data entry and analysis Data were entered into a personal computer using Microsoft Excel
software and analyzed with Statistical Package for Social Sciences
(IBM-SPSS, version 22, Chicago II, USA). This was represented in
tables using means and standard deviation. Means were compared
using the Z-test for continuous variables, while categorical variables
were compared with Chi-square. Relationships were assessed using
Pearson's correlation, with significance at P< 0.05. Doppler
performance in terms of Sensitivity, specificity, positive predictive Introduction Worldwide, Preeclampsia complicates about 5-10% of all
pregnancies. It is one of the leading causes of maternal and perinatal
mortality, fetal growth restriction and prematurity [1-9]. According to
World Health Organization (WHO), Preeclampsia is about seven
times higher in developing countries [10]. A combination of poor
health-seeking attitudes, lack of uniform protocols for case
management, poverty and ignorance are the main culprits [3,4]. Identifying at-risk fetuses in developing countries where the
perinatal mortality rate is about 14 times higher than in developed
countries is vital to improving perinatal outcomes [5]. Some studies
have found that haemodynamic indices of the fetal umbilical artery
(UmA) and Middle cerebral artery (MCA) may detect fetal
compromise much before any other antepartum test [6,7]. Despite the dangers of Preeclampsia, reliable tool(s) for fetal
surveillance that can guarantee optimal outcomes remain an area of 375 www.ijirms.in International Journal of Innovative Research in Medical Science (IJIRMS) research. This dearth of evidence is rampant in sub-Saharan Africa,
where the burden of Preeclampsia is high, and a sizable portion of
the population is of low socio-economic status with poor health-
seeking
behaviour. The
hemodynamic
derangements
in
Preeclampsia, which may lead to adverse effects, occur in or near
term; some studies have shown the promise of Doppler investigation
in identifying at-risk fetuses before damage is done [6,12-14]. reading was obtained from a free-floating portion of the umbilical
cord in the absence of uterine contraction, breathing or fetal
movement using an insonation angle of 600 at a point 1cm from cord
insertion on the placenta. The pulsed Doppler frequency was
adjusted to suit the flow condition, and the Doppler indices were
electronically read off. The PI, RI, and S/D ratio of the four
waveforms were measured and averaged automatically by an in-built
computer in the ultrasonography machine to ensure validity. Values
of indices greater than the 95th percentile for the gestational age are
abnormal. Doppler indices are affected by race; Caucasian parameters,
when superimposed on Africans, may be misleading. The ability to
accurately identify parturients that will develop Preeclampsia and
the successful development of effective prevention strategies will
significantly reduce the morbidity and mortality associated with
Preeclampsia. Though some studies in Nigeria have been reviewed
on this practice, a local study will help to situate the practice at the
centre and make the results more accessible to inform local policy
changes. Data collection On recruitment of each participant, a structured proforma was used
to collect data on the sociodemographic characteristics (age, marital
status, education level, husband occupation, residence, parity, and
gestational age, which was calculated from first and early second-
trimester ultrasound scan or her last menstrual period if sure). Blood
pressure, proteinuria, and Doppler velocimetry indices of umbilical
and middle cerebral arteries (RI, PI, S/D ratio and CPR) were
documented. The gestational age at delivery, significant intrapartum
events, delivery route, APGAR scores, birth weight, newborn
intensive care unit (NICU) admission, perinatal deaths, and maternal
complications, if any, were also recorded. Recruited participants had
severe Preeclampsia and planned for delivery. The adverse fetal
outcome was low birth weight, birth asphyxia, NICU admission and
perinatal death. The technique of Umbilical Doppler velocimetry Power was set within the fetal study limit, and the pulsed wave
Doppler cursor was positioned on the vessel of interest. The Doppler 376 www.ijirms.in www.ijirms.in International Journal of Innovative Research in Medical Science (IJIRMS) value (PPV) and negative predictive value (NPV) was calculated
using contingency tables. value (PPV) and negative predictive value (NPV) was calculated
using contingency tables. fetuses with adverse outcomes and abnormal fetal middle cerebral
artery Doppler, p<0.05. Abnormal PI of the fetal umbilical and
middle cerebral arteries had a statistically significant association
(p<0.05) with adverse fetal outcomes. Even though more babies with
adverse outcomes had abnormal RI, this was not statistically
significant, p>0.05 (Table 3). fetuses with adverse outcomes and abnormal fetal middle cerebral
artery Doppler, p<0.05. Abnormal PI of the fetal umbilical and
middle cerebral arteries had a statistically significant association
(p<0.05) with adverse fetal outcomes. Even though more babies with
adverse outcomes had abnormal RI, this was not statistically
significant, p>0.05 (Table 3). Results Eighty participants were enrolled for this study, but four signed
against medical advice and left before they were delivered. The
remaining 76 were analyzed. In this study, the sociodemographic
characteristics showed that the modal age of the participants,
32(42.0%), fell within the age range of 20-29 years. The mean age
of the study population was 29.0±7.0 years. Their mean parity was
2.0, but nulliparous and multiparous women had an equal number of
participants, 32(42.1%). The mean diastolic, systolic, and diastolic
blood pressures were respectively 113.4±13.3 mmHg and
172.3±15.7 mmHg. In Table 4, the umbilical artery Doppler indices showed a
higher sensitivity tool, while the middle cerebral artery indices had
better specificity scores. The fetal umbilical artery, individually, the
RI, PI and SDR had sensitivities of 92.0%, 68.0% and 8.0%,
respectively. The specificities for the PI, and SDR, were,
respectively 84.6%, and 84.6% and negative predictive value (NPV)
was highest for RI 60.0%), while positive predictive value (PPV)
was highest for PI (92.1%) and least for SDR (50.0%). The fetal
middle cerebral artery indices, PI, RI and SDR individually showed
specificities of 98.0%, 92.3% and 92.3%, respectively, for adverse
fetal outcomes. Positive predictive values for PI were 100.0%,
91.7% for SDR and 80.0% for RI. Table 1 shows no statistically significant, p>0.05 association
between Doppler indices and maternal complications. Results While in
Table 2, there was a statistically significant association between Table 1: Doppler results and maternal outcome Table 1: Doppler results and maternal outcome
Maternal complication
Indices (either singly or a combination of pi,ri, SDR, CPR)
Normal Umbilical artery indices (%)
Abnormal (%)
Fisher’s exact test
P-value
Eclampsia
0
4
4.978
0.290
Abruptio placentae
0
2
Pph
2
4
Icu admission
0
6
Maternal death
0
2
Total
2
18
Middle cerebral artery (normal)
Abnormal
8.996
0.109
Eclampsia
0
2
Abruptio placentae
0
2
Pph
2
4
Icu admission
0
6
Maternal death
0
2
Total
2
18
Table 2: Doppler results and neonatal outcome
Indices (pi,ri, SDR, CPR)
Normal doppler n(%)
Abnormal doppler n(%)
Chi-square (x2)
P-value
Neonatal complications
Umbilical artery Doppler
4.978
0.290
Birth asphyxia
6(7.8)
16(21.1)
Low birth weight
4(5.3)
14(18.4)
Nicu admission
10(13.2)
30(39.5)
Perinatal death
5(6.6)
9(11.8)
Middle cerebral artery Doppler
Birth asphyxia
4(5.3)
18(23.7)
30.938
*0.000
Low birth weight
3(3.9)
15(19.7)
Nicu admission
8(10.5)
32(42.1)
Perinatal death
6(7.8)
8(10.5)
*Significant
Table 3: Relationship between abnormal Doppler indices and adverse fetal outcome (birth asphyxia, NICU admission and perinatal death)
Indices
Abnormal indices
Adverse fetal outcome
Good fetal outcome
Chi-square
P-value
Uma
Pi
38
34
4
19.86
0.001*
Ri
66
46
20
4.978
0.290
Sdr
8
4
4
5.556
0.235
Mca
Pi
12
12
0
13.2
0.010*
Ri
10
8
2
2.08
0.720
Sdr
24
22
2
21.64
0.000*
Cpr
40
38
2
35.10
0.000*
Table 4: Accuracy of Doppler indices
Variable
Sensitivity (%)
Specificity (%)
Negative predictive value (%)
Positive predictive value (%)
Umbilical artery
Pi
68.0
84.6
57.9
92.1 Table 1: Doppler results and maternal outcome
Maternal complication
Indices (either singly or a combination of pi,ri, SDR, CPR)
Normal Umbilical artery indices (%)
Abnormal (%)
Fisher’s exact test
P-value
Eclampsia
0
4
4.978
0.290
Abruptio placentae
0
2
Pph
2
4
Icu admission
0
6
Maternal death
0
2
Total
2
18
Middle cerebral artery (normal)
Abnormal
8.996
0.109
Eclampsia
0
2
Abruptio placentae
0
2
Pph
2
4
Icu admission
0
6
Maternal death
0
2
Total
2
18
Table 2: Doppler results and neonatal outcome
Indices (pi,ri, SDR, CPR)
Normal doppler n(%)
Abnormal doppler n(%)
Chi-square (x2)
P-value
Neonatal complications
Umbilical artery Doppler
4.978
0.290
Birth asphyxia
6(7.8)
16(21.1)
Low birth weight
4(5.3)
14(18.4)
Nicu admission
10(13.2)
30(39.5)
Perinatal death
5(6.6)
9(11.8)
Middle cerebral artery Doppler
Birth asphyxia
4(5.3)
18(23.7)
30.938
*0.000
Low birth weight
3(3.9)
15(19.7)
Nicu admission
8(10.5)
32(42.1)
Perinatal death
6(7.8)
8(10.5)
*Significant Total
2
18
Table 2: Doppler results and neonatal outcome
Indices (pi,ri, SDR, CPR)
Normal doppler n(%)
Abnormal doppler n(%)
Chi-square (x2)
P-value
Neonatal complications
Umbilical artery Doppler
4.978
0.290
Birth asphyxia
6(7.8)
16(21.1)
Low birth weight
4(5.3)
14(18.4)
Nicu admission
10(13.2)
30(39.5)
Perinatal death
5(6.6)
9(11.8)
Middle cerebral artery Doppler
Birth asphyxia
4(5.3)
18(23.7)
30.938
*0.000
Low birth weight
3(3.9)
15(19.7)
Nicu admission
8(10.5)
32(42.1)
Perinatal death
6(7.8)
8(10.5)
*Significant Table 2: Doppler results and neonatal outcome Table 2: Doppler results and neonatal outcome www.ijirms.in
377
Table 3: Relationship between abnormal Doppler indices and adverse fetal outcome (birth asphyxia, NICU admission and perinatal death)
Indices
Abnormal indices
Adverse fetal outcome
Good fetal outcome
Chi-square
P-value
Uma
Pi
38
34
4
19.86
0.001*
Ri
66
46
20
4.978
0.290
Sdr
8
4
4
5.556
0.235
Mca
Pi
12
12
0
13.2
0.010*
Ri
10
8
2
2.08
0.720
Sdr
24
22
2
21.64
0.000*
Cpr
40
38
2
35.10
0.000*
Table 4: Accuracy of Doppler indices
Variable
Sensitivity (%)
Specificity (%)
Negative predictive value (%)
Positive predictive value (%)
Umbilical artery
Pi
68.0
84.6
57.9
92.1 Table 3: Relationship between abnormal Doppler indices and adverse fetal outcome (birth asphyxia, NICU admission and perinatal death)
Indices
Abnormal indices
Adverse fetal outcome
Good fetal outcome
Chi-square
P-value
Uma
Pi
38
34
4
19.86
0.001*
Ri
66
46
20
4.978
0.290
Sdr
8
4
4
5.556
0.235
Mca
Pi
12
12
0
13.2
0.010*
Ri
10
8
2
2.08
0.720
Sdr
24
22
2
21.64
0.000*
Cpr
40
38
2
35.10
0.000* 377 International Journal of Innovative Research in Medical Science (IJIRMS) International Journal of Innovative Research in Medical Science (IJIRMS)
Ri
92.0
23.1
60.0
69.7
Sdr
8.0
84.6
32.4
50.0
Middle cerebral artery
Pi
24.0
100.0
40.6
100.0
Ri
16.0
92.3
36.4
80.0
Sdr
44.0
92.3
46.2
91.7
Cpr
78.0
76.0
81.0
58.0 while the RI was most sensitive with 95.2% [18]. Ethical considerations Approval for this study was obtained from the Research and Ethics
Committee of the Federal Teaching Hospital Abakaliki. Patients
signed written and informed consent forms after carefully explaining
the objectives, procedure, and full implications of participation in
the study. This study was conducted in compliance with the ethical
standards of our institution on human subjects and with the Helsinki
Declaration. For the individual indices, we found that abnormal PI of the
fetal umbilical and middle cerebral arteries had a statistically
significant association (p<0.05) with adverse fetal outcomes. Also,
the SDR of the fetal MCA and the CPR showed similar associations. In contrast to our observation, Rani et al., performed a randomized
controlled trial, found a significant association between adverse
pregnancy outcomes and all the indices of Doppler velocimetry [24]. Ours was not a randomized controlled trial. Padmini et al. found a
significant association for only CPR, although more than half of the
patients in his study had mild Preeclampsia [18]. Other studies [25,26]
also found similar associations. Omtzigt did not find any significant
association [27], while Agrawal et al. found that only the SDR of the
umbilical artery had the highest correlation with adverse outcomes
[28] Discussion This is like the findings reported in other studies
[2 12 19 20] I
t
t th
i
ifi
t
i ti
b t We found that the CPR had the best accuracy scores across
the board (76-81%). Just like we reported, a systematic review by
Vollgraff et al. concluded that CPR outperformed other parameters
of fetal Doppler velocimetry to identify at-risk fetuses [29]. This is
more because the CPR represents the events at the umbilical
(downstream) artery and middle cerebral artery (upstream); by the
time the CPR becomes abnormal, centralization would have
occurred, implying that the fetus is already in the compensatory
phase of the pathology [29]. Hence measuring the CPR when
assessing fetal Doppler indices is indispensable for fetal
prognostication [24,28-30]. Conclusion This study found an association between fetal Doppler velocimetry
indices of the umbilical and middle cerebral arteries with adverse
fetal outcomes. Abnormal fetal umbilical artery velocimetry indices
imply fetal jeopardy, which requires further evaluation or
intervention. In contrast, normal middle cerebral artery velocimetry
indices are a reassuring sign which indicates the fetus was coping
fine. An abnormal cerebroplacental ratio signifies a decompensated
fetus; further deterioration will lead to fetal compromise. Results The PPV was
equally high for the PI and RI of the UmA and the PI of MCA. Our
findings differ from Rani et al.’s, where specificity was higher than
Sensitivity for the UmA than MCA indices [24]. The specificities
were>90% for MCA PI, UmA PI and RI, but MCA RI was 37.5%. Lopez-Mendez et al., in their study, found that individually and in
combination, the indices of UmA and MCA had high specificities
and high PPV values (80-90%). However, their sensitivities were at
most 50% [2]. Discussion Doppler interrogation of fetal vessels could be helpful for early pick-
up of signs that could identify fetuses at risk or in jeopardy even
before deleterious harm is done
[12,18]. Although maternal
complications ranging from eclampsia at 5.3%, abruptio placentae
at 2.6%, primary postpartum haemorrgae (PPH) at 7.9%, ICU
admission at 6% to maternal death in 2.6% were recorded in this
study, there was no statistically significant association between
maternal complications and abnormal fetal velocimetry indices. This
was not unexpected because the Doppler indices investigated were
those of the fetus. This is like the findings reported in other studies
[2,12,19,20]. In contrast, there was a significant association between
fetal middle cerebral artery (MCA) velocimetry indices and adverse
fetal outcomes. However, there was no significant association
between abnormal umbilical artery Doppler and adverse fetal
outcomes. Our findings contrast the reports of Veradju et al., wherein
they found a significant association between adverse perinatal
outcome and abnormal fetal umbilical artery (UmA) Doppler indices
[12]. Padmini et al. also studied 80 preeclamptic patients using UmA
and MCA Doppler velocimetry indices. They reported that of the 30
patients with abnormal Doppler indices, 25 had adverse outcomes
ranging from low APGAR scores to perinatal deaths and NICU
admission [16]. Alnakash et al. found a significant association
between abnormal indices of UmA and MCA with the adverse fetal
outcomes of low birth weight, low Apgar score and higher NICU
admission [21]. Udo et al. working at the University of Benin
Teaching Hospital [22], and Ayuba et al. at Aminu Kano Teaching
Hospital [23], found a significant association between abnormal
Doppler indices and adverse pregnancy outcomes. However, their
study population was heterogeneous in population with patients with
hypertensive disorders other than Preeclampsia included. Abnormal
middle cerebral artery doppler is a late event when there is placental
dysfunction due to fetal compensation, so-called centralization. Doppler interrogation of fetal vessels could be helpful for early pick-
up of signs that could identify fetuses at risk or in jeopardy even
before deleterious harm is done
[12,18]. Although maternal
complications ranging from eclampsia at 5.3%, abruptio placentae
at 2.6%, primary postpartum haemorrgae (PPH) at 7.9%, ICU
admission at 6% to maternal death in 2.6% were recorded in this
study, there was no statistically significant association between
maternal complications and abnormal fetal velocimetry indices. This
was not unexpected because the Doppler indices investigated were
those of the fetus. References [1]
Gunasena GG, Jayasundara DM, Salgado SS, Wijesinghe
PS, Biyagama BR, “The placenta in Preeclampsia:
Association of histology with umbilical artery Doppler
Velocimetry”, MOJ Womens Health, 2017, Vol.4, pp.92-
97. [18]
Padmini CP, Priyanka D, Chaitra RM, Adithya S, “Role of
Doppler indices of umbilical and middle cerebral artery in
predicting perinatal outcome in Preeclampsia” Int J Rep
ContracepObstet Gynecol. 2016, Vol.5, pp.845-849. [2]
Lopez-Mendez LA, Martinez-Gaytan V, Cortes-Flores R,
Ramos-Gonzalez, Ochoa-Torres MA, Garza-Veloz I et al.,
“Doppler ultrasound evaluation in Preeclampsia”, BMC
Research Notes, 2013, Vol.6, pp.477-483. [19]
Marshal D, Lindheimer MD Jason G, “Explaining and
Predicting Preeclampsia” N Eng J Med 2006, Vol.355,
pp.1056- 1058. [20]
Khong SL, Kane SC, Brennecke SP, Costa FS, “First-
trimester uterine artery Doppler analysis in the prediction
of later pregnancy complications” Disease Markers. 2015,
Article ID 679730, pp.1-10. [3]
World Health Organization,“Make every mother count”,
The world health report;, 2005 Bulletin, pp.1-125. [4]
Langer AV, Tell K, Villar K et al., “Reducing eclampsia-
related deaths - a call to action” Lancet, 2008,Vol.371,
pp.705 – 706. [21]
Alnakash AH, Mushina ZA, “Relationship between
Preeclampsia umbilical blood flow and perinatal
outcome” Int J Sci Res Pub. 2015, Vol.5, pp.465-470. [5]
World Health Organization, “Neonatal and perinatal
mortality, country, regional and global estimates” Geneva,
2006. [22]
Udo DU, Igbinedion BO, Akhide IA, Enabudosoe E,
“Assessment of uterine and umbilical arteries Doppler
indices in third-trimester pregnancy-induced hypertension
in UBTH, Benin-City.” Niger Med Pract. 2017, pp.71:1-5. [6]
Padmini CP, Priyanka D, Chaitra RM, Adithya S, “Role of
Doppler indices of umbilical and middle cerebral artery in
predicting perinatal outcome in Preeclampsia”, Int J Rep
Contracep Obstet Gynecol, 2016, Vol.5, pp.845–849. [23]
Ayuba R, Abubakar IS, Yakasai IA, “Umbilical artery
Doppler velocimetry study on predicting adverse
pregnancy outcomes among pregnant women with
hypertensive disorders in Kano, Nigeria” Niger J Basic
Clin Sci. 2015, Vol.12, pp.95-104. [7]
Hazra KS, Dash KK, Arunima C, Ghosh MK, Banerjee D,
Guha S, “A prospective study of Doppler velocimetry in
pregnancy-induced hypertension in a rural population of a
developing country”, J Basic Clin Reprod Sci., 2013,
Vol.2, pp.127-31. [24]
Rani S, Anju H, Ravinder K, “Prediction of perinatal
outcome in Preeclampsia using middle cerebral artery and
umbilical artery pulsatility and resistance indices”
Hypertension
in
Pregnancy
2016,
DOI:
10.3109/10641955.2015.1137585. [8]
Henry G. Clinical use of Doppler in obstetrics. In:
Protocols for high-risk pregnancies. JT Queenan, JC
Hobbins, Spong CY (Eds). 5th Edition. Oxford. Funding [13]
Meherishi SS, Mangal S, “Umbilical artery Doppler and
biophysical profile score: A study of their efficacy in
pregnancy-induced hypertension and intrauterine growth
retardation” Int J Res Med Sci., 2017, Vol.5, pp.4047-
4050. The study was funded by the authors. Acknowledgement We are grateful to the hospital staff and patients who participated in
this study. [16]
Tabassum A, Aftab S, Khan T, “Utility of uterine Doppler
ultrasonography
in
predicting
Preeclampsia
in
primigravidae” Ann Pak Inst Med Sci. 2016, Vol.12,
pp.161-165. List of abbreviations mmHg: Milimeter of mercury
CPR: Cerebro-placenatal ration
DBP: Diastolic blood pressure
DIC: Disseminated intravascular coagulation
ICU: Intensive care unit
MCA: Middle cerebral artery
NICU: Newborn intensive care unit
NPV: Negative predictive value
PI: Pulsatility index
PPH: Postpartum haemorrage
PPV: Positive predictive value
RI: Resistive index
SBP: Systolic blood pressure
SDR: Systolic diastolic ration
UmA: Umbilical artery
WHO: World health organization The MCA indices had low sensitivities, 24.0% for PI, while
PI, RI and SDR individually showed specificities of 100.0%, 92.3%
and 92.3%, respectively. Positive predictive values for PI were
100.0%, 91.7% for SDR and 80.0% for RI. The negative predictive
values for all the indices were less than 50%. Our study shows that
the umbilical artery indices are more sensitive; abnormal values
indicate fetuses likely in jeopardy and require further evaluation and
intervention. In contrast, high specificity for the middle cerebral
artery indices implies that typical values are a reassuring sign of fetal
well-being. These findings compare with Padmini et al.’s, where
specificity was higher for UmA PI than Sensitivity (98.3%v75%), 378 www.ijirms.in International Journal of Innovative Research in Medical Science (IJIRMS) International Journal of Innovative Research in Medical Science (IJIRMS) Data availability Data would be available upon reasonable request. [17]
Shahinaj R, Manoku N, Kroi E, Tasha I, “The value of the
middle cerebral to umbilical artery Doppler ratio in
predicting neonatal outcome in patients with Preeclampsia
and gestational hypertension” J Prenatal Med. 2010,
Vol.4,pp.17-21. Authors’ Contributions Johnbosco E. MAMAH conceived the study. Johnbosco E. MAMAH, Darlington-Peter C. UGOJI, and Chichetaram R. OTU
collected the data and performed literature review. Robinson ONOH,
Michael ORJI and Odidika U. J. UMEORA supervised the work. All
the authors wrote the final draft and approved the final manuscript. [14]
Ghosh GS, Gudmundsson S, “Uterine and umbilical artery
Doppler are comparable in predicting the perinatal
outcome of growth-restricted fetuses” BJOG, 2009,
Vol.116, pp.424-430. pp
[15]
Gagnon R, Michiel V., “The use of fetal Doppler in
obstetrics - Society of Obstetrics and Gynecology of
Canada guideline Number 130. 2003” J Obstet Gynecol
Can, 2003, Vol.25, pp.601-7. Conflict of interest [12]
Vedaraju KS, Suresh SK, “USG Doppler study of uterine,
umbilical and fetal middle cerebral arteries among severe
preeclamptic women and their relation to perinatal
outcomes” Int J Anat Radiology Surg, 2016, Vol.5, pp.14-
18. References Wiley-
Blackwell. 2010; Pp.80–91. [25]
Nomura RM, Niigaki JI, Horigome FT, Francisco RP,
Zugaib M, “Doppler velocimetry of the fetal middle
cerebral artery and other parameters of fetal well-being in
neonatal survival during pregnancies with placental
insufficiency” Rev Assoc Med Bra 2013, Vol.59, pp.392-
399. [9]
Williams KP, Galerneau F., “Pathophysiology of
eclampsia. Clinics Mother Child Health” 2015, Vol.12,
pp.197-201. [10]
Jibril UN, Martin SD, Umar A, Aliyu U, Njiida M, Abioye
T, “An assessment of eclampsia management in two health
institutions in Maiduguri, Borno State” Int J Recent Adv
Multidisciplinary Res., 2014, Vol.01, pp.010-013. [26]
Indiramani Y, Ratnakumari V, Jyothirmayi B, “Colour
Doppler study of umbilical artery in antenatal women with
severe Preeclampsia and foetal outcome” Open J Obstet
Gynecol. 2016, Vol.6, pp.129-135. [11]
Ugwu EO, Dim CC, Okonkwo CD, Nwankwo TO,
“Maternal and perinatal outcome of severe Preeclampsia
in Enugu, Nigeria After the introduction of Magnesium
sulphate” Niger J Clin Pract, 2011,Vol.14, pp.418-21. [27]
Omtzigt Wj, Reuwer JH, Bruinse HW, “A randomized
controlled trial on the clinical value of umbilical Doppler 379 www.ijirms.in International Journal of Innovative Research in Medical Science (IJIRMS) velocimetry in antenatal care” Am J Obstet Gynecol. 1994, Vol.170, pp.625-34 Open Access This article is licensed under a
Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and
reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made. The images or other third-party material in this article are
included in the article’s Creative Commons license, unless indicated
otherwise in a credit line to the material. If material is not included
in the article’s Creative Commons license and your intended use is
not permitted by statutory regulation or exceeds the permitted use,
you will need to obtain permission directly from the copyright
holder. To
view
a
copy
of
this
license,
visit
https://creativecommons.org/licenses/by/4.0/. [28]
Agrawal S, Vinita D, Agarwal A, Pandey A, “Fetal
Doppler for prediction of adverse perinatal outcome in
Preeclampsia in a low resource setting” Int J Rep
Contracep Obstet Gynecol. 2016, Vol.5, pp.3439-3443. [29]
Vollgraff HS, De Boer MA, Heymans MW, Schoonmade
LJ, Bossuyt MM, Mol WJ et al., “Prognostic accuracy of
cerebroplacental ratio and middle cerebral artery Doppler
for adverse perinatal outcome: systematic review and
meta-analysis.” Ultrasound Obstet Gynecol. 2018, Vol.51,
pp.313-322. References [30]
Srikumar S, Debnath J, Ravikumar R, Bandhu H, Maurya
V, “Doppler indices of the umbilical and fetal middle
cerebral artery at 18-40weeks of normal gestation: a pilot
study” Med J Armed Forces Ind. 2017, Vol.73, pp.232-
241. © The Author(s) 2023 © The Author(s) 2023 380 www.ijirms.in
|
https://openalex.org/W2998556825
|
https://riubu.ubu.es/bitstream/10259/5216/1/Domi-ijms_2020.pdf
|
English
| null |
Interaction Analysis of Commercial Graphene Oxide Nanoparticles with Unicellular Systems and Biomolecules
|
International journal of molecular sciences
| 2,019
|
cc-by
| 15,820
|
Received: 29 October 2019; Accepted: 24 December 2019; Published: 27 December 2019 Abstract: The ability of commercial monolayer graphene oxide (GO) and graphene oxide nanocolloids
(GOC) to interact with different unicellular systems and biomolecules was studied by analyzing
the response of human alveolar carcinoma epithelial cells, the yeast Saccharomyces cerevisiae and the
bacteria Vibrio fischeri to the presence of different nanoparticle concentrations, and by studying the
binding affinity of different microbial enzymes, like the α-l-rhamnosidase enzyme RhaB1 from the
bacteria Lactobacillus plantarum and the AbG β-d-glucosidase from Agrobacterium sp. (strain ATCC
21400). An analysis of cytotoxicity on human epithelial cell line A549, S. cerevisiae (colony forming
units, ROS induction, genotoxicity) and V. fischeri (luminescence inhibition) cells determined the
potential of both nanoparticle types to damage the selected unicellular systems. Also, the protein
binding affinity of the graphene derivatives at different oxidation levels was analyzed. The reported
results highlight the variability that can exist in terms of toxicological potential and binding affinity
depending on the target organism or protein and the selected nanomaterial. Keywords: graphene; unicellular organisms; toxicity; binding capacity; ATR-FTIR; TEM; ICP-M International Journal of
Molecular Sciences International Journal of
Molecular Sciences Int. J. Mol. Sci. 2020, 21, 205; doi:10.3390/ijms21010205 Interaction Analysis of Commercial Graphene Oxide
Nanoparticles with Unicellular Systems
and Biomolecules Brixhilda Domi 1,†, Carlos Rumbo 1,†, Javier García-Tojal 2, Livia Elena Sima 3,
Gabriela Negroiu 3 and Juan Antonio Tamayo-Ramos 1,* Brixhilda Domi 1,†, Carlos Rumbo 1,†, Javier García-Tojal 2, Livia Elena Sima 3,
Gabriela Negroiu 3 and Juan Antonio Tamayo-Ramos 1,* 1
International Research Centre in Critical Raw Materials-ICCRAM, University of Burgos, Plaza Misael
Bañuelos s/n, 09001 Burgos, Spain; bdomi@ubu.es (B.D.); crumbo@ubu.es (C.R.)
2
Department of Chemistry, University of Burgos, Plaza Misael Bañuelos s/n., 09001 Burgos, Spain;
qipgatoj@ubu.es 1
International Research Centre in Critical Raw Materials-ICCRAM, University of Burgos, Plaza Misael
Bañuelos s/n, 09001 Burgos, Spain; bdomi@ubu.es (B.D.); crumbo@ubu.es (C.R.)
2
Department of Chemistry, University of Burgos, Plaza Misael Bañuelos s/n., 09001 Burgos, Spain;
qipgatoj@ubu.es 3
Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy,
060031 Bucharest, Romania; livia_e_sima@yahoo.com (L.E.S.); gabrielanegroiu@yahoo.com (G.N.) 3
Department of Molecular Cell Biology, Institute of Biochemistry of the Romanian Academy,
060031 Bucharest, Romania; livia_e_sima@yahoo.com (L.E.S.); gabrielanegroiu@yahoo.com (G.N.) *
Correspondence: ja.tamayoramos@gmail.com †
These authors contributed equally to this work. www.mdpi.com/journal/ijms 1. Introduction The interest in the immobilization of microorganisms and microbial enzymes for biotechnological
applications has been continuously rising during the last decades because of several factors, including
the increased availability of microbial strains and biocatalysts tailored to new applications, the
development of new immobilization supports with improved properties, and the need of a shift
toward the use of more sustainable processes in different industrial fields [1–5]. The immobilization of
microorganisms and enzymes on solid carriers leads to a number of benefits. Immobilized biocatalysts
facilitate the efficient recovery and separation of the reaction product, the reutilization of the biocatalyst,
and enhance the safety of the material handling (i.e., preventing the appearance of allergies). The use
of solid supports of microbial cells for the production of high-value compounds (chemicals, enzymes,
etc.) and transformation processes in multiple fields (e.g., agricultural, environmental, food, medical,
etc.) has been explored as well to enhance the microbial biological activity, to facilitate their delivery
and to separate them more easily from the fermentation broth [3,5–8]. Therefore, during the last years
there has been an emerging interest in biocompatibility studies for interfacing biological systems with Int. J. Mol. Sci. 2020, 21, 205; doi:10.3390/ijms21010205 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, 205 Int. J. Mol. Sci. 2020, 21, 205 2 of 19 artificial materials. Unicellular microorganisms, such as bacteria, fungi, and algae, have been utilized
extensively for the encapsulation of whole single cells as well as for the introduction of nanomaterials
onto the living cells. g
During the last 40 years, a range of different materials have been investigated as enzyme and
microbial immobilization matrices: from organic compounds, like natural alginate or carrageenan or
synthetic polymers, to inorganic compounds, such as processed or natural minerals, like silica [3,9]. In the last decade, the focus has been put in the use of nanocomposites as promising immobilization
matrices. This is, in part, due to the enormous functional surface area they provide, which increases
the microbial and enzyme loading. Metal and carbon derived nanomaterials, as well as electrospun
nanofibers have taken the lead in this area [5,8,10,11]. 1. Introduction Regarding the use of nanoparticles, an extensive
number of studies have described the properties of different nanomaterials such as magnetic
nanoparticles, including iron oxide (Fe3O4 and γ-Fe2O3), alloy-based (CoPt3 and FePt), pure metal (Fe
and Co), and spinel-type ferromagnets (MgFe2O4, MnFe2O4, and CoFe2O4) [12], or carbon derived
nanoparticles, namely single and multiwall carbon nanotubes, graphene, graphene oxide, fullerene,
etc. [4,13–15], as suitable carriers for enzymes of industrial interest. Similarly, applications for the use
of these types of nanomaterials for the immobilization of prokaryotic and eukaryotic microorganisms
have been investigated [11,16–18]. Among the different carbon-derived nanomaterials, graphene oxide has received a particular focus
for biological applications because of its vast surface area, electroconductivity, superflexibility, and
thermal stability, which makes this type of nanomaterial a suitable biological carrier [19,20]. Currently,
it is possible to find in the market a portfolio of graphene oxide derivatives, expanding the availability
of possible microbial and biomolecule immobilization materials for different applications. The use of
distinct commercial graphene oxide nanoparticles can influence dramatically the biocatalyst loading,
biochemical properties, and stability. For this reason, the selection of an optimal biocatalyst-carrier
combination makes advisable a thorough screening of the available options [4]. Also, in regard to the
suitability of graphene oxide derivatives as support for microbial immobilization, conflicting results
relating biocompatibility and cytotoxicity induced by these nanomaterials have been reported in the
literature [21], which could be in part due to their heterogeneity in functional groups composition,
the presence of different amounts of trace elements, their size and morphology, etc. The fact that
the materials used in most biocompatibility and toxicology studies are mostly homemade makes it
challenging to achieve highly reproducible results. According to previous reports, graphene oxide
nanoparticles have dose- and size-dependent toxicity toward different cell lines, such as human
fibroblast, human hepatocellular carcinoma, human skin keratinocyte, etc. [22–26]. However, the
amount of literature available focusing on the biocompatibility analysis of graphene with microbial
cells is much scarcer. In this research study we selected two graphene derivatives: monolayer graphene oxide (GO;
supplied by Graphenea) and graphene oxide nanocolloids (GOC; supplied by Sigma-Merk), and both
their toxicological potential against different unicellular organisms and their binding affinity toward
different industrial enzymes was compared. 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on
a mica surface and carbon-coated copper grids respectively. Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on a
mica surface and carbon-coated copper grids respectively. Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC) Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on
a mica surface and carbon-coated copper grids respectively. Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on a
mica surface and carbon-coated copper grids respectively. Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC) AFM topography imaging showed that both nanomaterial types have a wide lateral size
distribution, ranging from the nanometric to the micrometric scale, while the flakes thickness is
around 1–2 nm. Graphene oxide nanomaterials of similar characteristics have been reported to
produce membrane-damaging activity in different unicellular systems [25,27,28]. AFM topography imaging showed that both nanomaterial types have a wide lateral size
distribution, ranging from the nanometric to the micrometric scale, while the flakes thickness is
around 1–2 nm. Graphene oxide nanomaterials of similar characteristics have been reported to produce
membrane-damaging activity in different unicellular systems [25,27,28]. (b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on
a mica surface and carbon-coated copper grids respectively. AFM topography imaging showed that both nanomaterial types have a wide lateral size
distribution, ranging from the nanometric to the micrometric scale, while the flakes thickness is
around 1–2 nm Graphene oxide nanomaterials of similar characteristics have been reported to p
g
g
y
y
[
,
,
]
The FTIR spectra of GO and the new GOC batch was determined as well, and both nanomaterials
showed to be very similar in their oxygen functional groups content (Figure 2). 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives The physical-chemical properties of the graphene oxide derivatives selected for this study were
recently determined [4]. Microscopy analyses using AFM and TEM instruments showed that GO
and GOC flakes were mostly in monolayer state and had a different size, while the analysis of their
composition revealed a high similarity between both nanomaterials. In the present study, the same
commercial nanomaterials’ suspensions were selected, but a new batch of the GOC material was used
(for more details see the Materials and Methods section). Therefore, we decided to perform a new
microscopy and spectroscopy analysis to confirm the physico-chemical properties of the new GOC 3 of 19 Int. J. Mol. Sci. 2020, 21, 205 sample. Surprisingly, new AFM and TEM analyses revealed that the nanoparticles of the new GOC
batch were morphologically very different to the older GOC batch (GOCo) (Supplementary Figure
S1), showing instead a high similarity in morphology and size to that observed on the monolayer GO
particles (Figure 1). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
3 of 20
S1), showing instead a high similarity in morphology and size to that observed on the monolayer GO
particles (Figure 1). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
3 of 20
S1), showing instead a high similarity in morphology and size to that observed on the monolayer GO
particles (Figure 1). Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on
a mica surface and carbon-coated copper grids respectively. Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). Graphene suspensions with a final concentration of 20 mg L−1 were deposited by drop casting on a
mica surface and carbon-coated copper grids respectively. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
3 of 20
S1), showing instead a high similarity in morphology and size to that observed on the monolayer GO
particles (Figure 1). Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC) 3 of 20
the monolayer GO y and size to that observed on t Figure 1. AFM and TEM analysis of graphene oxide (GO) (a) and graphene oxide nanocolloids (GOC)
(b). 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives Following the
tentative assignments given in the figure, the most significant difference found between GO and GOC
was that the former showed a slightly greater content in ether/alcoxy groups than the latter, which
could be related with the increase in the intensity of ν(C–O) stretching modes reported by other
The FTIR spectra of GO and the new GOC batch was determined as well, and both nanomaterials
showed to be very similar in their oxygen functional groups content (Figure 2). Following the tentative
assignments given in the figure, the most significant difference found between GO and GOC was that
the former showed a slightly greater content in ether/alcoxy groups than the latter, which could be
related with the increase in the intensity of ν(C–O) stretching modes reported by other authors [29]. p
p
produce membrane-damaging activity in different unicellular systems [25,27,28]. The FTIR spectra of GO and the new GOC batch was determined as well, and both nanomaterials
showed to be very similar in their oxygen functional groups content (Figure 2). Following the
tentative assignments given in the figure, the most significant difference found between GO and GOC
was that the former showed a slightly greater content in ether/alcoxy groups than the latter, which
could be related with the increase in the intensity of ν(C–O) stretching modes reported by other
th
[29] thors [29]. Figure 2. ATR-IR spectra of different graphene derivatives: GO (red) and GOC (blue), in the 4000‒400
cm‒1 (a) and 2000‒400 cm‒1 regions (b). Figure 2. ATR-IR spectra of different graphene derivatives: GO (red) and GOC (blue), in the
4000–400 cm−1 (a) and 2000–400 cm−1 regions (b). [29]. Figure 2. ATR-IR spectra of different graphene derivatives: GO (red) and GOC (blue), in the 4000‒400
cm‒1 (a) and 2000‒400 cm‒1 regions (b). Figure 2. ATR-IR spectra of different graphene derivatives: GO (red) and GOC (blue), in the
4000–400 cm−1 (a) and 2000–400 cm−1 regions (b). Int. J. Mol. Sci. 2020, 21, 205 4 of 19 The results obtained indicate that the reproducibility in the production of commercial graphene
oxide may still have relevant issues, making essential for the end user to confirm that the purchased
product matches with the expected characteristics. The results obtained indicate that the reproducibility in the production of commercial graphene
oxide may still have relevant issues, making essential for the end user to confirm that the purchased
product matches with the expected characteristics. 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives Additionally,
ICP-MS data suggested the possible presence of S in both nanomaterials, which can be present as well
in graphene oxide prepared through the Hummer´s method, being its content significantly higher
in GO. However, the obtained results in case of GOC were close to the background noise. For this
reason, to get further insight into the possible presence of sulfur species and the differences in their
content between GO and GOC, XPS analysis was performed. Again, the obtained results indicated
that S species were higher in GO (relative atomic percentage: 0.6%) than in GOC, where a reliable
quantitative value could not be determined. The presence of organosulfate groups in graphene oxide
is described, and suggested to be responsible for part of the reactivity of this nanomaterial, such as
in the immobilization of adsorbed species [31]. However, we could not get insights on the type of S
species (e.g., organic or inorganic) present in GO or GOC. e o
e
e ec o
o
e C
S p oce
e
e
so s o
GO (ppm)
GOC (ppm)
Al
0.160 ± 0.113
1.445 ± 0.106
B
<0.004
1.600 ± 0.255
Ba
0.006 ± 0.008
0.214 ± 0.006
Ca
0.063 ± 0.088
0.835 ± 0.035
Cu
0.052 ± 0.039
0.581 ± 0.030
Fe
0.379 ± 0.067
1.899 ± 0.033
Ga
0.004 ± 0.006
0.047 ± 0.000
K
3.770 ± 0.184
2.628 ± 0.252
Mg
0.350 ± 0.028
2.000 ± 0.113
Mn
34.700 ± 0.156
62.405 ± 0.233
Mo
0.029 ± 0.002
0.017 ± 0.001
Na
1.240 ± 0.509
4.810 ± 0.057
Ni
0.027 ± 0.020
0.027 ± 0.007
Pb
0.054 ± 0.023
0.152 ± 0.009
S
43.200 ± 2.786
5.084 ± 2.752
Sn
0.003 ± 0.003
0.034 ± 0.001
Sr
0.008 ± 0.001
0.034 ± 0.001
V
<0.0001
0.006 ± 0.001
W
0.004 ± 0.001
0.006 ± 0.001
Zn
0.068 ± 0.061
1.069 ± 0.740 Overall, the concentration of metallic elements was higher in GOC than in GO. Both nanomaterials
showed to have a high content of Mn (GO: 34.700 ppm; GOC: 62.405 ppm) and K (GO: 3.770;
GOC: 2.628 ppm), which suggests they were obtained through the Hummer’s method, which is the
most common oxidation method currently used for GO production and known to result in residual
manganese accumulation because of the use of permanganate oxidant (KMnO4) [30]. 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives Since the presence of trace metal impurities in graphene derivatives, either contained in the
graphite precursor or transferred by reactants used in the nanomaterial preparation, has been previously
described, a trace element analysis of GO and GOC was done by inductively coupled plasma mass
spectrometry (ICP-MS). As shown in Table 1, the presence of different metallic elements was observed
in GO and GOC, although the concentration of most of them was found to be low. Nevertheless,
significant differences in the concentration of some of the identified metals and metalloids were
observed between both nanomaterials. Table 1. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of GO and GOC. Values
below the detection limit of the ICP-MS procedure are also shown. Table 1. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of GO and GOC. Values
below the detection limit of the ICP-MS procedure are also shown. Table 1. Inductively coupled plasma mass spectrometry (ICP-MS) analysis of GO and GOC. Values
below the detection limit of the ICP-MS procedure are also shown. GO (ppm)
GOC (ppm)
Al
0.160 ± 0.113
1.445 ± 0.106
B
<0.004
1.600 ± 0.255
Ba
0.006 ± 0.008
0.214 ± 0.006
Ca
0.063 ± 0.088
0.835 ± 0.035
Cu
0.052 ± 0.039
0.581 ± 0.030
Fe
0.379 ± 0.067
1.899 ± 0.033
Ga
0.004 ± 0.006
0.047 ± 0.000
K
3.770 ± 0.184
2.628 ± 0.252
Mg
0.350 ± 0.028
2.000 ± 0.113
Mn
34.700 ± 0.156
62.405 ± 0.233
Mo
0.029 ± 0.002
0.017 ± 0.001
Na
1.240 ± 0.509
4.810 ± 0.057
Ni
0.027 ± 0.020
0.027 ± 0.007
Pb
0.054 ± 0.023
0.152 ± 0.009
S
43.200 ± 2.786
5.084 ± 2.752
Sn
0.003 ± 0.003
0.034 ± 0.001
Sr
0.008 ± 0.001
0.034 ± 0.001
V
<0.0001
0.006 ± 0.001
W
0.004 ± 0.001
0.006 ± 0.001
Zn
0.068 ± 0.061
1.069 ± 0.740
Overall, the concentration of metallic elements was higher in GOC than in GO. Both nanomaterials
showed to have a high content of Mn (GO: 34.700 ppm; GOC: 62.405 ppm) and K (GO: 3.770;
GOC: 2.628 ppm), which suggests they were obtained through the Hummer’s method, which is the
most common oxidation method currently used for GO production and known to result in residual
manganese accumulation because of the use of permanganate oxidant (KMnO4) [30]. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC The viability of the human cell line A549 after 24 h of exposure to 40, 80, and 160 mg L−1 of GO
and GOC was analyzed using the neutral red uptake and MTT assays. The neutral red assay is based
on the ability of healthy cells to incorporate and retain the neutral red dye in their lysosomes, which is
an indicator of the cell’s capacity to maintain pH gradients through the production of ATP, and thus a
viability indicator. In Figure 3, the results obtained for neutral red assay are presented. No negative
effects on cell viability was observed in any of the concentrations tested for both nanomaterials, showing
all the studied conditions (negative control and exposed cells) a similar percentage of viable cells. The viability of the human cell line A549 after 24 h of exposure to 40, 80, and 160 mg L−1 of GO
and GOC was analyzed using the neutral red uptake and MTT assays. The neutral red assay is based
on the ability of healthy cells to incorporate and retain the neutral red dye in their lysosomes, which
is an indicator of the cell’s capacity to maintain pH gradients through the production of ATP, and
thus a viability indicator. In Figure 3, the results obtained for neutral red assay are presented. No
negative effects on cell viability was observed in any of the concentrations tested for both
nanomaterials, showing all the studied conditions (negative control and exposed cells) a similar
percentage of viable cells
Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
5 of 20
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
The viability of the human cell line A549 after 24 h of exposure to 40, 80, and 160 mg L−1 of GO
and GOC was analyzed using the neutral red uptake and MTT assays. The neutral red assay is based
on the ability of healthy cells to incorporate and retain the neutral red dye in their lysosomes, which
d
f h
ll
d
h
h h
d
f A
d percentage of viable cells. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
way ANOVA followed by Dunnett post hoc test to compare every mean with the control, and
id
d i
ifi
t t
≤0 05 *
≤0 05
Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-way
ANOVA followed by Dunnett post hoc test to compare every mean with the control, and considered
significant at p ≤0.05. * p ≤0.05. Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one- The MTT assay is based on the ability of viable cells with active metabolism to convert MTT into
a purple colored formazan product that can be measured at OD 590 nm, being this color formation a
useful marker to assess cells viability. The cytotoxicity studies conducted using this assay (Figure 4)
revealed that cells exposed to GOC presented a slight decline in viability at the higher concentrations
tested, being statistically significant in the case of cells exposed to 160 mg L−1, whereas in cells
i
b t d
ith GO
i
ifi
t diff
f
d b t
t
l
d
l
The MTT assay is based on the ability of viable cells with active metabolism to convert MTT into a
purple colored formazan product that can be measured at OD 590 nm, being this color formation a
useful marker to assess cells viability. The cytotoxicity studies conducted using this assay (Figure 4)
revealed that cells exposed to GOC presented a slight decline in viability at the higher concentrations
tested, being statistically significant in the case of cells exposed to 160 mg L−1, whereas in cells incubated
with GO, no significant differences were found between controls and samples. considered significant at p ≤ 0.05. * p ≤ 0.05. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC The MTT assay is based on the ability of viable cells with active metabolism to convert MTT into
a purple colored formazan product that can be measured at OD 590 nm, being this color formation a
useful marker to assess cells viability. The cytotoxicity studies conducted using this assay (Figure 4)
revealed that cells exposed to GOC presented a slight decline in viability at the higher concentrations
tested, being statistically significant in the case of cells exposed to 160 mg L−1, whereas in cells
i
b t d
ith GO
i
ifi
t diff
f
d b t
t
l
d
l ubated with GO, no significant differences were found between controls and samples. Figure 4. Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and
GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
incubated with GO, no significant differences were found between controls and samples. Figure 4. Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and
GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
Figure 4. Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and GOC
(right). Results are expressed as % of control (untreated cells). Data represent the mean (±standard
deviation, SD) of three independent replicates. Differences were established using a one-way ANOVA
followed by Dunnett post hoc test to compare every mean with the control, and considered significant
at p ≤0.05. * p ≤0.05. ubated with GO, no significant differences were found between controls and samples. g
p Figure 4. Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and
GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation SD) of three independent replicates Differences were established using a one
Figure 4. Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and
GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
Figure 4. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
way ANOVA followed by Dunnett post hoc test to compare every mean with the control, and
considered significant at p ≤0 05 * p ≤0 05
Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-way
ANOVA followed by Dunnett post hoc test to compare every mean with the control, and considered
significant at p ≤0.05. * p ≤0.05. is an indicator of the cell’s capacity to maintain pH gradients through the production of ATP, and
thus a viability indicator. In Figure 3, the results obtained for neutral red assay are presented. No
negative effects on cell viability was observed in any of the concentrations tested for both
nanomaterials, showing all the studied conditions (negative control and exposed cells) a similar
percentage of viable cells. Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). Results are expressed as % of control (untreated cells). Data represent the mean
(±standard deviation, SD) of three independent replicates. Differences were established using a one-
ay ANOVA follo
ed by Du
ett
o t ho
te t to
o
a e e e y
ea
ith the
o t ol a d pH gradients through the production of ATP, and
s obtained for neutral red assay are presented. No
d in any of the concentrations tested for both
ons (negative control and exposed cells) a similar is an indicator of the cell s capacity to maintain pH gradients through the production of ATP, and
thus a viability indicator. In Figure 3, the results obtained for neutral red assay are presented. No
negative effects on cell viability was observed in any of the concentrations tested for both
nanomaterials, showing all the studied conditions (negative control and exposed cells) a similar
percentage of viable cells. cator. In Figure 3. Viability of A549 cells (neutral red assay) treated with different concentrations of GO (left)
and GOC (right). 2.1. Characteristics of the Selected Commercial Graphene Oxide Derivatives Additionally,
ICP-MS data suggested the possible presence of S in both nanomaterials, which can be present as well
in graphene oxide prepared through the Hummer´s method, being its content significantly higher
in GO. However, the obtained results in case of GOC were close to the background noise. For this
reason, to get further insight into the possible presence of sulfur species and the differences in their
content between GO and GOC, XPS analysis was performed. Again, the obtained results indicated
that S species were higher in GO (relative atomic percentage: 0.6%) than in GOC, where a reliable
quantitative value could not be determined. The presence of organosulfate groups in graphene oxide
is described, and suggested to be responsible for part of the reactivity of this nanomaterial, such as
in the immobilization of adsorbed species [31]. However, we could not get insights on the type of S
species (e.g., organic or inorganic) present in GO or GOC. Int. J. Mol. Sci. 2020, 21, 205
Int. J. Mol. Sci. 2019, 20, x FOR 5 of 19
5 of 20 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC Viability of A549 cells (MTT assay) treated with different concentrations of GO (left) and GOC
(right). Results are expressed as % of control (untreated cells). Data represent the mean (±standard
deviation, SD) of three independent replicates. Differences were established using a one-way ANOVA
followed by Dunnett post hoc test to compare every mean with the control, and considered significant
at p ≤0.05. * p ≤0.05. Int. J. Mol. Sci. 2020, 21, 205
way ANOVA follow
considered significan 6 of 19
nd The toxicity of graphene oxide in human cell lines has been widely investigated in different studies. However, the results and conclusions reached by them are apparently inconsistent, as evidenced by
some of the recent reviews [21,32]. Several factors, such as the size, the surface chemistry, or the levels
of impurities, critically affect the physico-chemical properties of the nanoparticles and, subsequently,
the interactions with cells, which lead to differences in their inherent cytotoxicity. Moreover, the toxicity
of GO varies greatly depending on the cell line and cell type exposed [33]. In our experiments, only a
slight statistically significant decrease in viability was detected in A549 cells treated with 160 mg L−1 of
GOC (less than 15% of decrease) performing the MTT assay, whereas no negative effect was detected
in the NR assay. It is also important to mention that in both assays a different number of cells per
well were used, being six times lower in the MTT assay. Even in this case, where the nanoparticle/cell
exposure ratio was higher, both GO and GOC demonstrated to be safe in terms of cell viability. These
results are in concordance with the work of Chang et al. [34], which was performed using the same
cell line. These authors described the good biocompatibility of GO, describing only a slight decrease
in the viability after an exposure to high doses. In contrast, other authors observed a negative effect
on the viability caused by these nanoparticles on A549 cells. Gies et al. described a size and dose
dependent effect, showing a high decrease in the percentage of viable cells after 24 h of exposure to high
concentrations of GO (100 and 200 mg L−1) [33]. Likewise, Reshma et al. showed a dose-dependent
decrease in viability of cells treated with reduced GO (rGO) and PEGylated GO [35]. These authors
observed a significant reduction from concentrations of, at least, 25 mg L−1. Mittal et al. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC analyzed
the interaction between three graphene oxide derivatives with A549 cells [36], observing a significant
reduction of viability over 48 h of exposure even at low concentrations, whereas Hu et al. described
only a mild effect in cytotoxicity of A549 cells exposed during 24 h to GO and rGO, being significantly
higher in the case of the latter [37]. This variability between the results obtained using the same cell
line could be attributed to the factors explained above, such as the levels of impurities present in the
nanoparticles, or even the oxidative method through which the nanoparticles were prepared, which
influence their toxicological behavior [38]. The toxicity of graphene oxide in human cell lines has been widely investigated in different
studies. However, the results and conclusions reached by them are apparently inconsistent, as
evidenced by some of the recent reviews [21,32]. Several factors, such as the size, the surface
chemistry, or the levels of impurities, critically affect the physico-chemical properties of the
nanoparticles and, subsequently, the interactions with cells, which lead to differences in their
inherent cytotoxicity. Moreover, the toxicity of GO varies greatly depending on the cell line and cell
type exposed [33]. In our experiments, only a slight statistically significant decrease in viability was
detected in A549 cells treated with 160 mg L−1 of GOC (less than 15% of decrease) performing the
MTT assay, whereas no negative effect was detected in the NR assay. It is also important to mention
that in both assays a different number of cells per well were used, being six times lower in the MTT
assay. Even in this case, where the nanoparticle/cell exposure ratio was higher, both GO and GOC
demonstrated to be safe in terms of cell viability. These results are in concordance with the work of
Chang et al. [34], which was performed using the same cell line. These authors described the good
biocompatibility of GO, describing only a slight decrease in the viability after an exposure to high
doses. In contrast, other authors observed a negative effect on the viability caused by these
nanoparticles on A549 cells. Gies et al. described a size and dose dependent effect, showing a high
decrease in the percentage of viable cells after 24 h of exposure to high concentrations of GO (100 and
200 mg L−1) [33]. Likewise, Reshma et al. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC showed a dose-dependent decrease in viability of cells
treated with reduced GO (rGO) and PEGylated GO [35]. These authors observed a significant
reduction from concentrations of, at least, 25 mg L−1. Mittal et al. analyzed the interaction between
three graphene oxide derivatives with A549 cells [36], observing a significant reduction of viability
over 48 h of exposure even at low concentrations, whereas Hu et al. described only a mild effect in
cytotoxicity of A549 cells exposed during 24 h to GO and rGO, being significantly higher in the case
of the latter [37]. This variability between the results obtained using the same cell line could be
attributed to the factors explained above, such as the levels of impurities present in the nanoparticles,
or even the oxidative method through which the nanoparticles were prepared, which influence their
toxicological behavior [38] In relation to the possible induction of oxidative stress by GO and GOC, the DCFH-DA assay was
used to measure the reactive oxygen species (ROS) levels on the A549 cells after contact with different
concentrations of the nanomaterials. Figure 5 shows that the ROS levels were significantly increased in
A549 cells after 1 h of exposure to both nanoparticles, being this induction much higher in the case of
the cells incubated with GO. toxicological behavior [38]. In relation to the possible induction of oxidative stress by GO and GOC, the DCFH-DA assay
was used to measure the reactive oxygen species (ROS) levels on the A549 cells after contact with
different concentrations of the nanomaterials. Figure 5 shows that the ROS levels were significantly
increased in A549 cells after 1 h of exposure to both nanoparticles, being this induction much higher
in the case of the cells incubated with GO in the case of the cells incubated with GO. Figure 5. Reactive oxygen species (ROS) production of A549 cells treated with different concentrations
of GO (left) and GOC (right). The reported values are expressed in arbitrary units and correspond to
the averages of two biological replicates per culture condition. Data represent the mean of three
replicates (±standard deviation, SD). Differences were established using a one-way ANOVA followed
Figure 5. Reactive oxygen species (ROS) production of A549 cells treated with different concentrations
of GO (left) and GOC (right). The reported values are expressed in arbitrary units and correspond
to the averages of two biological replicates per culture condition. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC Data represent the mean of three
replicates (±standard deviation, SD). Differences were established using a one-way ANOVA followed
by Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤0.05. ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. in the case of the cells incubated with GO. Figure 5. Reactive oxygen species (ROS) production of A549 cells treated with different concentrations
of GO (left) and GOC (right). The reported values are expressed in arbitrary units and correspond to
the averages of two biological replicates per culture condition. Data represent the mean of three
replicates (±standard deviation, SD). Differences were established using a one-way ANOVA followed
Figure 5. Reactive oxygen species (ROS) production of A549 cells treated with different concentrations
of GO (left) and GOC (right). The reported values are expressed in arbitrary units and correspond
to the averages of two biological replicates per culture condition. Data represent the mean of three
replicates (±standard deviation, SD). Differences were established using a one-way ANOVA followed
by Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤0.05. ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. Int. J. Mol. Sci. 2020, 21, 205 7 of 19 Our assays were performed using concentrations of both nanoparticle types up to 40 mg L−1. From that concentration, we have observed that in our experimental procedure the fluorescent
response may be masked by both GO and GOC, leading to an underestimation of the ROS production. Either way, our results demonstrate that the low concentrations tested in our assays are enough to
produce statistically significant levels of oxidative stress after 1 h of incubation, being this much higher
in the case of GO. The induction of oxidative stress after interaction with graphene oxides and their
derivatives have been reported in several works using different cell lines [39–41]. These nanomaterials
can induce cellular damage through the formation of ROS by their interaction with cellular membranes. In the specific case of A549 cell line, several works have demonstrated their ability to induce ROS
release. For example, Chang et al. found that GO exposure can induce oxidative stress at low
concentrations [34]. Mittal et al. observed an overproduction of ROS in A549 cells in contact with GO
and their derivatives, as well as in other human lung cells such as the BEAS-2B cell line [36]. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC In both
studies, the times of exposure tested were longer than the times used in the present work. In any case,
based on our results and in previous reports, it has been evidenced that an acute exposure of human
cells to graphene oxide can induce high oxidative stress levels. High levels of ROS can cause damage to different biomolecules of the cell, such as proteins or
nucleic acids, which can lead to activation of apoptosis. In order to assess whether the levels of ROS
produced by A549 cells after being exposed to GO and GOC can induce an apoptotic response, we
quantified the percentages of apoptotic and necrotic cells using flow cytometry, upon the addition
of different nanoparticles concentrations for 24 h. The obtained results have shown that cells treated
with different GO concentrations (Figure 6b; 40, 80, 160 mg L−1) showed a constant 93–95% of viable
cells, similar to the untreated control sample (Figure 6a). In the case of GOC, we evidenced a stable
6–10% cell death, irrespective of the administered dose (Figure 6b). As a positive control for the assay,
we used cisplatin (a common chemotherapeutic agent) which induced over 40% cell death (Figure 6a). Interestingly, we found that the PI signal was decreasing in a dose-dependent manner in GO-
and GOC-treated cells (Figure 6c). However, despite the signal to noise ratio diminution for the PI
staining, this did not impede the quantification of the PI+ cell subpopulation. The PI signal decrease
is probably caused by the quenching of the dye by the nanoparticles, as previously reported [42,43]. The quenching could be due to the energy transfer from the fluorophore to the metal [42] or in the case
of graphenes, it could be due to the excitation of an exciton too [43]. Wu et al. found that the quenching
efficiency of GO was still around 30% when the distance between dyes and GO was increased to more
than 30 nm [44]. Several studies have described the impact of graphene-based materials on different types of
programmed cell death, including apoptosis [45], in diverse cell lines, through distinct mechanisms such
as caspase activation or DNA fragmentation [46,47]. For example, in the A549 cell line, the implication
of graphene nanopores in the induction of early apoptosis was described and, at concentrations higher
than 250 mg L−1, late apoptosis was observed too [48]. In addition, Adil et al. with increased doses of GO and GOC.
2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC Interestingly, we found that the PI signal was decreasing in a dose-dependent manner in GO-
and GOC-treated cells (Figure 6c). However, despite the signal to noise ratio diminution for the PI
staining, this did not impede the quantification of the PI+ cell subpopulation. The PI signal decrease
is probably caused by the quenching of the dye by the nanoparticles, as previously reported [42,43]
The quenching could be due to the energy transfer from the fluorophore to the metal [42] or in the
case of graphenes, it could be due to the excitation of an exciton too [43]. Wu et al. found that the
quenching efficiency of GO was still around 30% when the distance between dyes and GO was
increased to more than 30 nm [44]. Several studies have described the impact of graphene-based materials on different types of
programmed cell death, including apoptosis [45], in diverse cell lines, through distinct mechanisms
such as caspase activation or DNA fragmentation [46,47]. For example, in the A549 cell line, the
implication of graphene nanopores in the induction of early apoptosis was described and, at
concentrations higher than 250 mg L−1, late apoptosis was observed too [48]. In addition, Adil et al
b
d h
i
b
i
d b
h
i
d
i
f il
d
d
The viability of S. cerevisiae cells exposed to two different GO and GOC concentrations (160
and 800 mg L−1) and exposure times (2 and 24 h) was assessed through colony forming units (CFU)
determination. As displayed in Figure 7, no significant differences in viability were observed in
the selected exposure conditions after 2 h of exposure, except for the condition where a high GOC
concentration was used. However, after 24 h, viability issues could be observed after a longer exposure
time. In case of GO, the nanomaterial reduced S. cerevisiae CFUs after an exposure of 24 h, provoking a
viability loss of 36.5% when the material was present at the lower concentration and 49.7% when the
material was present at the higher concentration. In contrast, GOC showed no significant influence
on the yeast viability at 160 mg L−1, although the viability loss observed at the higher concentration
was very similar for both nanomaterials. The effect on S. 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC observed that apoptosis
can be triggered by green synthesized nanocomposites of silver-decorated highly reduced graphene
oxide [49], while Mbeh et al. described that high concentrations of graphene oxide nanoribbons (100 mg
L−1) can also cause cell apoptosis [50]. However, other authors did not find any evidence of apoptosis
induction in A549 cells after treatment with GO derivatives. For instance, Chang et al. observed
that, independently of dose and size, GO did not induce any apoptosis or necrosis in A549 cells [34]. Moreover, Hu et al. described that apoptosis did not occur in A549 cells treated with GO nanosheets
after a 24-h exposure with 20 and 85 mg L−1 [37]. Finally, Yang et al. found that the exposure to
different graphene quantum dots, even at high concentration (200 mg L−1), did not result in apoptosis
induction [51]. The results described in these latter works are in concordance with our observations,
since, in spite of the fact that both GO and GOC produced oxidative stress in A549 cells, no significant
increase in apoptosis was detected at concentrations up to 160 mg L−1. 8 of 19
8 of 20 Int. J. Mol. Sci. 2020, 21, 205
Int. J. Mol. Sci. 2019, 20, x FO Figure 6. Flow cytometry analysis of apoptosis response of A549 cells treated with different
concentrations of GO (top) and GOC (bottom) upon double staining with Annexin V-FITC and
propidium iodide (PI). Results are displayed as density plots and expressed as percent (%) live (low
left quadrants), apoptotic (low right quadrants), and necrotic (upper right quadrants) cells (a,b) of the
total cell population excluding doublets. Histograms (c) show distribution of PI signal in cells treated
Figure 6. Flow cytometry analysis of apoptosis response of A549 cells treated with different
concentrations of GO (top) and GOC (bottom) upon double staining with Annexin V-FITC and
propidium iodide (PI). Results are displayed as density plots and expressed as percent (%) live (low left
quadrants), apoptotic (low right quadrants), and necrotic (upper right quadrants) cells (a,b) of the total
cell population excluding doublets. Histograms (c) show distribution of PI signal in cells treated with
increased doses of GO and GOC. Figure 6. Flow cytometry analysis of apoptosis response of A549 cells treated with different
concentrations of GO (top) and GOC (bottom) upon double staining with Annexin V-FITC and
propidium iodide (PI). 2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC
2.2. Determination of Human Cancer Cell Line A549 Response to GO and GOC Results are displayed as density plots and expressed as percent (%) live (low
left quadrants), apoptotic (low right quadrants), and necrotic (upper right quadrants) cells (a,b) of the
total cell population excluding doublets. Histograms (c) show distribution of PI signal in cells treated
Figure 6. Flow cytometry analysis of apoptosis response of A549 cells treated with different
concentrations of GO (top) and GOC (bottom) upon double staining with Annexin V-FITC and
propidium iodide (PI). Results are displayed as density plots and expressed as percent (%) live (low left
quadrants), apoptotic (low right quadrants), and necrotic (upper right quadrants) cells (a,b) of the total
cell population excluding doublets. Histograms (c) show distribution of PI signal in cells treated with
increased doses of GO and GOC. with increased doses of GO and GOC. 2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC with increased doses of GO and GOC.
2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤0.05. * p ≤0.05, *** p ≤0.001, **** p ≤0.0001. Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤ 0.05. Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤0.05. * p ≤0.05, *** p ≤0.001, **** p ≤0.0001. p
p
p
To evaluate whether GO and GOC were able to induce oxidative stress in S. cerevisiae, cells
growing at exponential phase were exposed to 160 and 800 mg L−1 of the nanomaterials, for 24 h. As
shown in the Figure 8, the oxidative stress levels were significantly increased in S. cerevisiae in the
To evaluate whether GO and GOC were able to induce oxidative stress in S. cerevisiae, cells growing
at exponential phase were exposed to 160 and 800 mg L−1 of the nanomaterials, for 24 h. As shown in
the Figure 8, the oxidative stress levels were significantly increased in S. cerevisiae in the presence of both
carbon nanoparticles. Carbon derived nanomaterials have shown previously to induce oxidative stress
in yeast. Non-commercial grade GO and O-SWCNT, also induced ROS with a similar concentration to
the one tested here, although the exposure time tested in both cases was 24 h instead of 2 h [52,54]. However, the oxidative stress provoked by MWCNT in yeast seem to be lower than that observed in the
present study for GO and GOC or that previously observed for other carbon derived nanoparticles [53]. Int. J. Mol. Sci. with increased doses of GO and GOC.
2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC 2019, 20, x FOR PEER REVIEW
10 of 20
presence of both carbon nanoparticles. Carbon derived nanomaterials have shown previously to
induce oxidative stress in yeast. Non-commercial grade GO and O-SWCNT, also induced ROS with
a similar concentration to the one tested here, although the exposure time tested in both cases was 24
h instead of 2 h [52,54]. However, the oxidative stress provoked by MWCNT in yeast seem to be lower
than that observed in the present study for GO and GOC or that previously observed for other carbon
derived nanoparticles [53] p
[
]
Figure 8. Oxidative stress (ROS) determination of S. cerevisiae cells exposed to 160 mg L−1 of GO and
GOC during 2 h. The reported values are expressed in arbitrary units and correspond to the averages
of two biological replicates per culture condition. Differences were established using a one-way
ANOVA followed by Dunnett post hoc test to compare every mean with the control, and considered
significant at p ≤ 0.05. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figure 8. Oxidative stress (ROS) determination of S. cerevisiae cells exposed to 160 mg L−1 of GO and
GOC during 2 h. The reported values are expressed in arbitrary units and correspond to the averages of
two biological replicates per culture condition. Differences were established using a one-way ANOVA
followed by Dunnett post hoc test to compare every mean with the control, and considered significant
at p ≤0.05. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. p
[
] Figure 8. Oxidative stress (ROS) determination of S. cerevisiae cells exposed to 160 mg L−1 of GO and
GOC during 2 h. The reported values are expressed in arbitrary units and correspond to the averages
of two biological replicates per culture condition. Differences were established using a one-way
ANOVA followed by Dunnett post hoc test to compare every mean with the control, and considered
significant at p ≤ 0.05. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Figure 8. Oxidative stress (ROS) determination of S. cerevisiae cells exposed to 160 mg L−1 of GO and
GOC during 2 h. The reported values are expressed in arbitrary units and correspond to the averages of
two biological replicates per culture condition. with increased doses of GO and GOC.
2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC cerevisiae viability of non-commercial grade
graphene oxide nanoparticles was also tested in a recent study, and the fungus mortality was found to
be close to 20% in the presence of 600 mg L−1 [52]. Also, the toxicological potential of other carbon
nanomaterials toward S. cerevisiae was reported, such as multi-walled carbon nanotubes (MWCNTs) or 9 of 19
g
logical
ll d Int. J. Mol. Sci. 2020, 21, 205
g
mortality was found to oxidized single-walled carbon nanotubes (O-SWCNTs), which induced significant yeast mortality at
400 mg L−1 (6.1%) and 188.2 mg L−1 (approximately 11%) respectively [53,54]. carbon nanotubes (MWCNTs) or oxidized single-walled carbon nanotubes (O-SWCNTs), which
induced significant yeast mortality at 400 mg L−1 (6.1%) and 188.2 mg L−1 (approximately 11%)
respectively [53,54]. p
y [
,
] p
y [
,
]
Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤ 0.05. *
≤0 05 ***
≤0 001 ****
≤0 0001
Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤0.05. * p ≤0.05, *** p ≤0.001, **** p ≤0.0001. Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). The reported values are the averages of three biological
replicates per culture condition. Differences were established using a one-way ANOVA followed by
Dunnett post hoc test to compare every mean with the control, and considered significant at p ≤ 0.05. Figure 7. Colony forming units (CFUs) determination of S. cerevisiae cells exposed to 160 and 800 mg
L−1 of GO and GOC during 2 h (a) and 24 h (b). 2.4. Determination of Vibrio Fischeri Bioluminescence Inhibition to GO and GOC .4. Determination of Vibrio Fischeri Bioluminescence Inhibition to GO and GOC The marine bacteria Vibrio fischeri was also used to compare the toxicological potential of both
graphene oxide suspensions. The V. fischeri luminescence assay is an environmental monitoring tool to
determine the toxicity in sediments and leachates that may be a source of contamination in aquatic
ecosystems. The ability of the nanomaterials to inhibit the microorganism luminescence was measured
at two concentrations (160 and 800 mg L−1) and exposure times (10 and 30 min). When the lower
concentration of GO and GOC was present in the media, we did not observe a V. fischeri significant
luminescence inhibition. The bacteria luminescence decreased in the presence of a higher concentration
of the nanomaterials, with significant difference between both nanomaterial types (Figure 9). In case
of GO, the presence of 800 mg L−1 induced a 100% of luminescence inhibition, already after 10 min
of exposure. In contrast, the same concentration of GOC showed a significantly lower luminescence
inhibition capacity at both exposure times (p < 0.001 and p < 0.01 respectively). Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
11 of 20 Figure 9. Luminescence inhibition assay of V. fischeri cells exposed to 800 mg L−1 of GO and GOC
during 30 min. The reported values are the averages of four biological replicates per culture condition. Figure 9. Luminescence inhibition assay of V. fischeri cells exposed to 800 mg L−1 of GO and GOC
during 30 min. The reported values are the averages of four biological replicates per culture condition. Figure 9. Luminescence inhibition assay of V. fischeri cells exposed to 800 mg L−1 of GO and GOC
during 30 min. The reported values are the averages of four biological replicates per culture condition. Figure 9. Luminescence inhibition assay of V. fischeri cells exposed to 800 mg L−1 of GO and GOC
during 30 min. The reported values are the averages of four biological replicates per culture condition. Previous studies have evaluated the luminescence inhibition of V. fischeri promoted by
nanomaterials, such as nano-metal oxides, nanoscale cationic polymers, silica nanoparticles, catechol-
based submicron particles or functionalized reduced graphene oxide nanoparticles [56–59]. Interestingly, the toxicity of reduced graphene oxide functionalized with Fe3O4 [57], was similar to
that observed for GOC in the present study
Previous studies have evaluated the luminescence inhibition of V. with increased doses of GO and GOC.
2.3. Determination of Saccharomyces Cerevisiae Cells Response to GO and GOC Differences were established using a one-way ANOVA
followed by Dunnett post hoc test to compare every mean with the control, and considered significant
at p ≤0.05. * p ≤0.05, ** p ≤0.01, *** p ≤0.001, **** p ≤0.0001. Int. J. Mol. Sci. 2020, 21, 205 10 of 19 We also aimed to determine the possible genotoxic effect of the selected graphene oxide
nanomaterials on S. cerevisiae using the comet assay protocol previously described [55]. However,
because of the nanomaterials’ morphology, graphene oxide concentrations higher than 20 mg L−1
prevented the proper visualization and analysis of the cell nuclei under the fluorescence microscope,
making the comet assay an unsuitable method for the determination of genotoxiciy in yeast with two
dimensional nanoparticles of a big lateral size. 2.4. Determination of Vibrio Fischeri Bioluminescence Inhibition to GO and GOC fischeri promoted by
nanomaterials, such as nano-metal oxides, nanoscale cationic polymers, silica nanoparticles,
catechol-based submicron particles or functionalized reduced graphene oxide nanoparticles [56–59]. Interestingly, the toxicity of reduced graphene oxide functionalized with Fe3O4 [57], was similar to
that observed for GOC in the present study. 2 5 Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes
2.5. Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes 2.5. Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes
Biotechnological and biomedical applications of graphene oxide rely on nanomaterial-
biomolecule interactions. The protein binding capacity of nanomaterials determines possible
biological applications and their toxicological potential too [60,61]. In case of commercial GO and
GOC, both nanomaterial suspensions showed a high protein loading capacity and a good potential
as enzyme immobilization supports [4] However their maximum protein binding capacity was not
Biotechnological and biomedical applications of graphene oxide rely on nanomaterial-biomolecule
interactions. The protein binding capacity of nanomaterials determines possible biological applications
and their toxicological potential too [60,61]. In case of commercial GO and GOC, both nanomaterial
suspensions showed a high protein loading capacity and a good potential as enzyme immobilization
supports [4]. However, their maximum protein binding capacity was not determined, and their 11 of 19 Int. J. Mol. Sci. 2020, 21, 205 polypeptide binding properties were determined using a single enzyme. Also, having into account
that the protein binding efficiency of the new GOC lot (MKCD9594) was unknown, we decided
to characterize the nanomaterial-enzyme binding efficiency of GO and GOC. In addition, to assess
whether a variation on the GO and GOC oxidation state could further increase their enzyme loading
capacity, the nanomaterials were partially reduced and their protein binding capacity was compared
with that of the untreated nanomaterials. The partial reduction of GO and GOC was performed using
a concentrated solution (50 mM) of the mild reductant mercaptoethylamine-HCl (further details are
described in the Materials and Methods section). The reduction of the nanocarbon derivatives was
confirmed by ATR-FTIR analysis (Figure 10). The spectrum of GOC exhibited drastic changes after the
nanomaterials’ treatment with the mercaptoethylamine-HCl. Basically, the intensity of the absorptions
sharply decreased, in good agreement with the reduction of the described functional groups. In the
case of rGO, an analogous trend to that shown by the rGOC spectrum was observed. Int. J. Mol. Sci. 2019, 20, x FOR PEER REVIEW
12 of 20 Figure 10. IR spectra of GOC and rGOC (a) and GO and rGO (b) in the 4000‒400 cm‒1 region. Figure 10. IR spectra of GOC and rGOC (a) and GO and rGO (b) in the 4000–400 cm−1 region. Figure 10. IR spectra of GOC and rGOC (a) and GO and rGO (b) in the 4000‒400 cm‒1 region. Figure 10. 2 5 Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes
2.5. Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes IR spectra of GOC and rGOC (a) and GO and rGO (b) in the 4000–400 cm−1 region. The maximum enzyme loading capacity of chemically reduced GO (rGO) and GOC (rGOC) was
analyzed and compared with that of the non-modified nanoparticles, using the bacterial enzymes α-
L-rhamnosidase enzyme RhaB1, from Lactobacillus plantarum, and the β-D-glucosidase AbG, from
Agrobacterium sp. (strain ATCC 21400), following the immobilization protocol described previously
[4]. As displayed in Table 2, the binding capacity of GO and GOC was different for both enzymes
d i
ifi
tl
hi h
th
th t b
d i
th
d
d
i
f th
ti l
The maximum enzyme loading capacity of chemically reduced GO (rGO) and GOC (rGOC) was
analyzed and compared with that of the non-modified nanoparticles, using the bacterial enzymes
α-l-rhamnosidase enzyme RhaB1, from Lactobacillus plantarum, and the β-d-glucosidase AbG, from
Agrobacterium sp. (strain ATCC 21400), following the immobilization protocol described previously [4]. As displayed in Table 2, the binding capacity of GO and GOC was different for both enzymes and
significantly higher than that observed in the reduced versions of the nanoparticles. sig i ica
y
ig e
a
a obse
e
i
e e uce
e sio s o
e
a opa ic es
Table 2. Maximum binding capacity (%) of GO, GOC, rGO, and rGOC using different carbohydrate
Table 2. Maximum binding capacity (%) of GO, GOC, rGO, and rGOC using different carbohydrate
active enzymes. g
y
g
p
Table 2. Maximum binding capacity (%) of GO, GOC, rGO, and rGOC using different carbohydrate
active enzymes. Carbon Nanomaterial
RhaB1 Binding (mg mg−1)
AgB Binding (mg mg−1)
GO
4.88 ± 0.17
1.65 ± 0.04
GOC
5.90 ± 0.11
1.22 ± 0.14
rGO
1.98 ± 0.11
1.00 ± 0.03
rGOC
1 99 ± 0 23
0 70 ± 0 08
Table 2. Maximum binding capacity (%) of GO, GOC, rGO, and rGOC using different carbohydrate
active enzymes. Carbon Nanomaterial
RhaB1 Binding (mg mg−1)
AgB Binding (mg mg−1)
GO
4.88 ± 0.17
1.65 ± 0.04
GOC
5.90 ± 0.11
1.22 ± 0.14
rGO
1.98 ± 0.11
1.00 ± 0.03
rGOC
1.99 ± 0.23
0.70 ± 0.08 active enzymes. 2 5 Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes
2.5. Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes Carbon Nanomaterial
RhaB1 Binding (mg mg−1)
AgB Binding (mg mg−1)
GO
4.88 ± 0.17
1.65 ± 0.04
GOC
5.90 ± 0.11
1.22 ± 0.14
rGO
1.98 ± 0.11
1.00 ± 0.03
rGOC
1 99 ± 0 23
0 70 ± 0 08
Carbon Nanomaterial
RhaB1 Binding (mg mg−1)
AgB Binding (mg mg−1)
GO
4.88 ± 0.17
1.65 ± 0.04
GOC
5.90 ± 0.11
1.22 ± 0.14
rGO
1.98 ± 0.11
1.00 ± 0.03
rGOC
1.99 ± 0.23
0.70 ± 0.08 Although π–π stacking and hydrophobic effects are considered the predominant mechanisms
of protein binding with graphene-based materials, and both phenomena should be more dominant
after the reduction of graphene oxide, the reduced versions of GO and GOC did not improve the
enzyme binding capacity of the untreated nanomaterials. Previous studies reporting the influence of
graphene oxide reduction on protein binding capacity show controversial results [60,62–64]. As
recently described by Qi and collaborators [64], changes on graphene-based nanomaterials’ surface
properties affect as well their aggregation properties, which may become a crucial factor influencing
Although π–π stacking and hydrophobic effects are considered the predominant mechanisms
of protein binding with graphene-based materials, and both phenomena should be more dominant
after the reduction of graphene oxide, the reduced versions of GO and GOC did not improve the
enzyme binding capacity of the untreated nanomaterials. Previous studies reporting the influence of
graphene oxide reduction on protein binding capacity show controversial results [60,62–64]. As recently
described by Qi and collaborators [64], changes on graphene-based nanomaterials’ surface properties
affect as well their aggregation properties, which may become a crucial factor influencing their protein Int. J. Mol. Sci. 2020, 21, 205 12 of 19 adsorption capacity. The obtained result also showed that the maximum loading capacity of GO and
GOC was significantly higher for the α-rhamnosidase RhaB1. A similar result was observed when
using the reduced versions. Different enzymes could exhibit different enzyme loadings and stabilities
when bound to graphene oxide because of the differences in the charge status of their surface functional
groups [65]. The obtained results using distinct unicellular models and biomolecules display significant
changes in the toxicological potential of GO and GOC: the former had a higher ability to induce
oxidative stress in human alveolar carcinoma epithelial cells A549, and the yeast Saccharomyces cerevisiae,
while provoking a higher luminescence inhibition capacity on the bacteria Vibrio fischeri too. 3.3. ICP-MS Samples (0.1 g) were subjected to a digestion process with 7 mL of HNO3 Suprapur (Merck KGaA,
Darmstadt, Germany) (65% v/v) and 1 mL of H2O2 (30% v/v), while being subjected to the following
thermal treatment: a temperature gradient from room temperature up to 80 ◦C in 4 min, followed
by a second temperature gradient, from 80 to 120 ◦C in 4 min, and by a third temperature gradient,
from 120 to 190 ◦C in 5 min. Then, temperature was kept constant at 190 ◦C for 30 min, and finally
samples were cooled down for 1 h. The analysis of the digested samples was done with an Agilent
8900 ICP-QQQ instrument. 3.4. XPS Analysis X-ray photoelectron spectroscopy (XPS) was done by the SGIker unit at the University of the
Basque Country (UPV/EHU) using a SPECS system equipped with a Phoibos 150 on powders deposited
into glass slides. 3.1. Materials and Reagents Most of the chemicals and reagents were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt,
Germany) and Acros Organics (Thermo Fisher Scientific Inc., Madrid, Spain). The graphene derivatives
were obtained from different suppliers as well; graphene oxide nanocolloids (GOC; ref: 795534; old lot:
MKBT5205V; new lot: MKCD9594) were purchased from Sigma-Aldrich, and monolayer graphene oxide
(GO; C309/GORB014/D1) was purchased from Graphenea (San Sebastian, Spain). The α-l-rhamnosidase
RhaB1 from Lactobacillus plantarum and the AbG β-d-glucosidase from Agrobacterium sp. (strain ATCC
21400) were obtained from Megazyme Ltd. (Biocon S.L., Barcelona, Spain). 2 5 Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes
2.5. Determination of GO and GOC Binding Efficiency on Different Microbial Enzymes Also,
both products behaved differently in their enzyme binding capacity. The lateral dimension, surface
structure, functional groups, purity and protein corona, strongly influence the toxicity of graphene
oxide in biological systems [66]. Since GO and GOC are distinct in terms of their apparent particle size
distribution, elemental composition and in the presence of oxygen functional groups, identifying the
most relevant factors determining the differences observed regarding their toxicological potential is
difficult. Nevertheless, the present work contributes to have a better understanding on the biological
impact and biotechnological potential of commercial grade graphene oxide. 3.2. ATR-FTIR Analysis IR spectra were recorded on dry solid samples in the 4000–400 cm−1 region by a JASCO FT-IR
4200 spectrophotometer equipped with a Single Reflection ATR PRO ONE device. Each of the graphics
is the result of overlapping 128 scans with a 4 cm−1 resolution. 3.6.2. MTT Assay A549 cells were seeded in 96 well plates at 5 × 103 cells per well and treated with 40, 80,
and 160 mg L−1 of the materials diluted in DMEM 1% FCS. Cells incubated with medium alone
were used as controls. Plates were then incubated for 24 h and, after exposure, cell culture
medium with materials was discarded, wells were washed with DPBS, and a solution of MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (0.5 mg L−1) was added to each well
and incubated for 3 h, followed by adding 100 µL DMSO to dissolve the MTT crystals. After 15 min
of gentle shaking, the absorbance was measured with a microplate reader (BioTek Synergy HT, OD
590 nm). Results were expressed as percentage of control (absorbance of cells in absence of materials). Each assay included three independent replicates. 3.6.1. Neutral Red Assay A549 cells were seeded in 96 well plates at 3 × 104 cells per well and treated with 40, 80, and
160 mg L−1 of the materials diluted in DMEM 1% FCS. After 24 h of exposure, cells were washed
and incubated with 100 µL of the neutral red solution which was prepared as follows: neutral red
stock (4 mg L−1) was diluted 1/100 in treatment media, and incubated in the dark for 24 h at 37 ◦C
before use. At that time, the solution was centrifuged to remove debris from neutral red powder. After
2.5 h incubation, neutral red solution was discarded, cells were washed once with DPBS (Dulbecco’s
phosphate-buffered saline), and subsequently fixed with formaldehyde 4%. Cells were washed again
with DPBS and a dye release solution (50% ethanol 96◦, 49% distilled H2O, and 1% acetic acid) was
added to each well. After 10 min of gentle shaking, this solution was transferred to a new opaque
96-well plate, and fluorescence was measured with a microplate reader (BioTek Synergy HT, excitation
wavelength, 530/25; emission wavelength 645/40). Results were expressed as percentage of control
(absorbance of cells in absence of materials). Each assay included three independent replicates. 3.6. Assays in A549 Cells The human alveolar carcinoma epithelial cell line A549 (ATCC, CCL-185) was utilized for toxicity
evaluation. Cells were grown in DMEM medium (Dulbecco’s Modified Eagle Medium) supplemented
with 10% fetal calf serum (FCS), 1% penicillin/streptomycin and grown in a humidified incubator at 37
◦C in the presence of 5% CO2. 3.5. AFM and TEM Analysis AFM and TEM analyses were performed at the Microscopy Unit from the University of Valladolid. Samples were deposited on Lacey Carbon Type-A, 300 mesh, copper grids, and visualized and 13 of 19 Int. J. Mol. Sci. 2020, 21, 205 photographed using a JEOL JEM-1011 HR TEM coupled with a Gatan Erlangshen ES1000W camera. For AMF analysis, samples were deposited on a mica surface from aqueous solutions by drop casting. Images were recorded in AC mode (tapping mode) with a CYPHER ES instrument from Asylum
Research (Oxford Instruments, Abingdon, UK), using silicon cantilevers AC160TS-R3 with aluminum
reflex coating (Olympus) and tip radius <10 nm. The analysis was done using a set point of 500, 72 mV,
a drive amplitude of 791.16, a drive frequency of 268.639, and integral gain of 268.639. Data acquisition
and control was done with IGOR Pro 6.2 (Asylum Research, Oxford Instruments, Abingdon, UK). Images analysis was done with ARgyle (Argyle Software Ltd., Bath, UK). 3.6.3. ROS Determination in Human Cells The quantitative measurement of intracellular reactive oxygen species (ROS) was investigated
using 2,7-dichlorofluorescin diacetate (DCFH-DA). A549 cells were seeded in a 96 micro-well plate at 3
× 104 cells per well and labelled with 50 µM DCFH-DA in Hanks’ Balanced Salt Solution (HBSS) for
30 min. After the incubation, cells were washed once with HBSS, and different concentrations of the
materials diluted in HBSS were added to each well. Fluorescence was measured with a microplate
reader (BioTek Synergy HT, excitation wavelength, 530/25; emission wavelength 645/40) after 1 h
of incubation. Int. J. Mol. Sci. 2020, 21, 205 14 of 19 14 of 19 3.6.4. Apoptosis Assay Flow cytometry was used for the quantitative assessment of apoptosis. A549 cells were seeded
in 24 well plates at 10 × 104 cells per well and treated with 40, 80, and 160 mg L−1 of the materials
diluted in DMEM 1%FCS. Cells incubated with medium alone were used as negative controls while
cells treated with 50 µM cisplatin served as positive control for the staining. After 24 h of incubation,
cells in suspension were harvested and collected together with the monolayers detached using
trypsin-EDTA solution (Invitrogen), for each sample. After centrifugation, cells were resuspended
in buffer and stained using a dead cell apoptosis kit with Annexin V-FITC and propidium iodide
(Molecular Probes) according with manufacturer’s protocol. Samples were filtered through 70-µm
nylon meshes (Miltenyi Biotec) and acquired on a BD FACSVerse analyzer controlled by FACSuite
software (BD Biosciences, Franklin Lakes, United States). Analysis was performed on the Cytobank
platform (https:$\delimiter"026E30F$$\delimiter"026E30F$community.cytobank.org). Single stained
controls, using Triton-X-100 permeabilized (0.2% in PBS, 10 min) and untreated cells, respectively were
generated for compensation purposes and gating thresholding. Results are depicted as color density
plots and histograms. 3.7. Assays in Saccharomyces Cerevisiae The S. cerevisiae BY4741 strain was grown and maintained in standard liquid YPD medium (1%
yeast extract, 1% yeast bacto-peptone, 2% glucose). Cell cultures in liquid media were done on a rotary
shaker at 185 rpm at 30 ◦C. 3.7.2. ROS Determination in S. cerevisiae Intracellular levels of reactive oxygen species were determined using the reagent CM-H2DCFDA
following a protocol similar to that reported by James et al. (2015) [67]. S. cerevisiae cells growing in
exponential phase were pelleted, washed, and incubated with CM-H2DCFDA (7 µM) in DPBS for
60 min at 30 ◦C and 185 rpm. Afterwards, yeast cells were washed again, resuspended in YPD and
subsequently exposed to the graphene oxide nanomaterials (160 mg L−1) for 2 h. Then, yeast cells were
washed two times with DPBS, incubated 2 min in a solution containing AcLi 2M, and subsequently
washed and incubated again for 2 min in a solution containing SDS (0.01%) and chloroform (0.4%). Finally, cells were pelleted and the supernatant was transferred to a black opaque 96-micro-well plate,
where the fluorescence was measured (excitation = 485; emission = 528) using a microplate reader
(Synergy-HT, BioTek). 3.7.1. Colony Forming Units Determination Yeast cells in exponential growth phase (OD600 = 1) were exposed to GO and GOC at 160 and
800 mg L−1 in 1 mL cultures performed in 24-well plates. Samples were obtained after 2 and 24 h
of cells exposure. To determine yeast colony forming units after the two exposure times, cells were
inoculated on solid YPD medium (6% agar) and incubated at 30 ◦C. 3.8. Vibrio Fischeri Luminescence Inhibition Assay V. fischeri NRRL B-11177 cells were inoculated in 5 mL of Marine Broth 2216 and grown at 15 ◦C
for 48 h. The bacterial suspension was pelleted, resuspended in 5 mL of NaCl 2% (w/v) at 15 ◦C
and maintained at 10 ◦C for 30 min. The exposure experiment was started by pipetting 10 µL of the
bacterial suspension in black opaque microplate wells containing 90 µL of GO and GOC (160 and
800 mg L−1) in a water suspension containing NaCl 2% (w/v). The 96-well plate was incubated in a
Thermomixer at 800 rpm and 15 ◦C, and V. fischeri luminescence was measured for 30 min using a
microplate reader (Synergy-HT, BioTek). The luminescence inhibition (using as reference the negative
control condition) was calculated using the values obtained at 10 (M10) and 30 (M30) min using the
following formula, adapted from Jarque et al. (2016) [68], where CF is a correction factor (the Mt/peak 15 of 19 Int. J. Mol. Sci. 2020, 21, 205 ratio in negative controls) reflecting natural attenuation of bacterial luminescence after 30 min of
incubation in non-exposed conditions: ratio in negative controls) reflecting natural attenuation of bacterial luminescence after 30 min of
incubation in non-exposed conditions: INH% = 100 −
Mt
CF × peak × 100 3.9. Preparation of rGO and rGOC The mild reductant mercaptoethylamine-HCl was used to reduce commercial GO and GOC
nanoparticles. Water suspensions of GO and GOC (1000 mg L−1) containing 50 mM of the reducing
agent concentrated were incubated overnight at 4 ◦C. Afterwards, rGO and rGOC were pelleted, using
a Thermo ST 16R Sorvall centrifuge (5000 rpm; acceleration: 9, deceleration: 9), and subsequently
washed with a sodium phosphate buffer (12.5 mM; pH 6.5) solution, three times. Finally, the reduced
nanomaterials water suspensions were kept at a final concentration of 1000 mg L−1 in sodium phosphate
buffer (12.5 mM; pH 6.5), and stored at 4 ◦C. References 1. Bayat, Z.; Hassanshahian, M.; Cappello, S. Immobilization of microbes for bioremediation of crude oil
polluted environments: A mini review. Open Microbiol. J. 2015, 9, 48–54. [PubMed] 1. Bayat, Z.; Hassanshahian, M.; Cappello, S. Immobilization of microbes for bioremediation of crude oil
polluted environments: A mini review. Open Microbiol. J. 2015, 9, 48–54. [PubMed] 2. Gurung, N.; Ray, S.; Bose, S.; Rai, V. A broader view: Microbial enzymes and their relevance in industries,
medicine, and beyond. Biomed. Res. Int. 2013, 2013, 329121. [CrossRef] [PubMed] 2. Gurung, N.; Ray, S.; Bose, S.; Rai, V. A broader view: Microbial enzymes and their relevance in industries,
medicine, and beyond. Biomed. Res. Int. 2013, 2013, 329121. [CrossRef] [PubMed] 3. Robatjazi, S.M.; Shojaosadati, S.A.; Khalilzadeh, R.; Farahani, E.V.; Balochi, N. Immobilization of magnetic
modified Flavobacterium ATCC 27551 using magnetic field and evaluation of the enzyme stability of
immobilized bacteria. Bioresour. Technol. 2012, 104, 6–11. [CrossRef] [PubMed] 3. Robatjazi, S.M.; Shojaosadati, S.A.; Khalilzadeh, R.; Farahani, E.V.; Balochi, N. Immobilization of magnetic
modified Flavobacterium ATCC 27551 using magnetic field and evaluation of the enzyme stability of
immobilized bacteria. Bioresour. Technol. 2012, 104, 6–11. [CrossRef] [PubMed] 4. Antón-Millán, N.; García-Tojal, J.; Marty-Roda, M.; Garroni, S.; Cuesta-López, S.; Tamayo-Ramos, J.A. Influence of three commercial graphene derivatives on the catalytic properties of a Lactobacillus plantarum
α-l-rhamnosidase when used as immobilization matrices. ACS Appl. Mater. Interfaces 2018, 10, 18170–18182. [CrossRef] 4. Antón-Millán, N.; García-Tojal, J.; Marty-Roda, M.; Garroni, S.; Cuesta-López, S.; Tamayo-Ramos, J.A. Influence of three commercial graphene derivatives on the catalytic properties of a Lactobacillus plantarum
α-l-rhamnosidase when used as immobilization matrices. ACS Appl. Mater. Interfaces 2018, 10, 18170–18182. [CrossRef] 5. Tamayo-Ramos, J.A.; Rumbo, C.; Caso, F.; Rinaldi, A.; Garroni, S.; Notargiacomo, A.; Romero-Santacreu, L.;
Cuesta-López, S. Analysis of polycaprolactone microfibers as biofilm carriers for biotechnologically relevant
bacteria. ACS Appl. Mater. Interfaces 2018, 10, 32773–32781. [CrossRef] 6. Li, Y.-G.; Gao, H.-S.; Li, W.-L.; Xing, J.-M.; Liu, H.-Z. In situ magnetic separation and immobilization of
dibenzothiophene-desulfurizing bacteria. Bioresour. Technol. 2009, 100, 5092–5096. [CrossRef] 7. Kureˇciˇc, M.; Rijavec, T.; Hribernik, S.; Lapanje, A.; Kleinschek, K.S.; Maver, U. Novel electrospun fibers with
incorporated commensal bacteria for potential preventive treatment of the diabetic foot. Nanomedicine 2018,
13, 1583–1594. [CrossRef] 8. Zhang, J.; Zhao, W.; Zhang, H.; Wang, Z.; Fan, C.; Zang, L. Recent achievements in enhancing anaerobic
digestion with carbon-based functional materials. Bioresour. Technol. 2018, 266, 555–567. [CrossRef] 9. 4. Conclusions 2020, 21, 205 SD
Standard deviation
PI
Propidium iodide
MWCNs
Multiwalled carbon nanotubes
O-SWCNTs
Oxidized single-walled carbon nanotubes
rGO
Reduced monolayer graphene oxide
rGOC
Reduced graphene oxide nanocolloids
DMSO
Dimethyl sulfoxide SD
Standard deviation
PI
Propidium iodide
MWCNs
Multiwalled carbon nanotubes
O-SWCNTs
Oxidized single-walled carbon nanotubes
rGO
Reduced monolayer graphene oxide
rGOC
Reduced graphene oxide nanocolloids
DMSO
Dimethyl sulfoxide 4. Conclusions The results obtained in the present study show the potential of different commercial graphene
oxide nanomaterials to interact with distinct unicellular systems and biomolecules, pointing out the
variability that can be found in terms of toxicological potential and binding affinity depending on
the target organism or protein, and the selected nanomaterial. GO showed a higher capacity than
GOC to induce oxidative stress in both S. cerevisiae and human cells. In the same line, GO showed a
significantly higher V. fischeri luminescence inhibition too. Also, differences in the binding capacity of
both nanomaterials were observed, being their maximum loading capacity different as well, in function
of the enzyme tested. Therefore, the presented results clearly indicate the usefulness of this type of
studies in order to determine the actual toxicological and biochemical potential for specific commercial
graphene oxide products. plementary Materials: Supplementary Materials can be found at http://www.mdpi.com/1422-0067/21/1/205/s1 Author Contributions: J.A.T.-R. conceived and designed the work. J.A.T.-R., B.D., C.R., J.G.-T., and L.E.S. performed the experiments. J.A.T.-R., B.D., C.R., J.G.-T., L.E.S., and G.N. analyzed and interpreted the data. J.A.T.-R. and C.R. drafted the manuscript. J.A.T.-R., C.R., and G.N. critically revised the manuscript for intellectual
content. All authors have read and agreed to the submission of the manuscript. All authors have read and agreed
to the published version of the manuscript. Funding: This work was supported by the European Union’s H2020 research and innovation programme
under the Marie Skłodowska-Curie grant agreements Nº 691095, Nº 721642 and Nº 734873; Junta de Castilla y
Leon-FEDER under grants Nº BU079U16, BU291P18 and BU022G18, and Ministerio de Economía y Competitividad
CTQ2016-75023-C2-1-P and CTQ2015-70371-REDT MetDrugs Network (Spain). We thank Anca Filimon for her
invaluable assistance and SITEX 45, particularly to Dumitru Ulieru, for the support. Conflicts of Interest: The authors declare that they have no conflict of interests. Abbreviations
GO
Monolayer graphene oxide
GOC
Graphene oxide nanocolloids
ROS
Reactive oxygen species
AFM
Atomic force microscopy
TEM
Transmission electron microscopy
FTIR
Fourier-transform infrared spectroscopy
ICP-MS
Inductively coupled plasma mass spectrometry
ppm
parts-per-million
ATP
Adenosine triphosphate Abbreviations
GO
Monolayer graphene oxide
GOC
Graphene oxide nanocolloids
ROS
Reactive oxygen species
AFM
Atomic force microscopy
TEM
Transmission electron microscopy
FTIR
Fourier-transform infrared spectroscopy
ICP-MS
Inductively coupled plasma mass spectrometry
ppm
parts-per-million
ATP
Adenosine triphosphate 16 of 19 Int. J. Mol. Sci. References Jesionowski, T.; Zdarta, J.; Krajewska, B. Enzyme immobilization by adsorption: A review. Adsorption 2014,
20, 801–821. [CrossRef] 10. Putzbach, W.; Ronkainen, N.J. Immobilization techniques in the fabrication of nanomaterial-based
electrochemical biosensors: A review. Sensors 2013, 13, 4811–4840. [CrossRef] 11. Yang, S.H.; Lee, T.; Seo, E.; Ko, E.H.; Choi, I.S.; Kim, B.-S. Interfacing living yeast cells with graphene oxide
nanosheaths. Macromol. Biosci. 2012, 12, 61–66. [CrossRef] [PubMed] 12. Bilal, M.; Zhao, Y.; Rasheed, T.; Iqbal, H.M.N. Magnetic nanoparticles as versatile carriers for enzymes
immobilization: A review. Int. J. Biol. Macromol. 2018, 120, 2530–2544. [CrossRef] [PubMed] 13. Lin, L.-H.; Shih, J.-S. Immobilized fullerene C60-enzyme-based electrochemical glucose sensor. J. Chin. Chem. Soc. 2011, 58, 228–235. [CrossRef] 14. Pagán, M.; Suazo, D.; Del Toro, N.; Griebenow, K. A comparative study of different protein immobilization
methods for the construction of an efficient nano-structured lactate oxidase-SWCNT-biosensor. Biosens. Bioelectron. 2015, 64, 138–146. [CrossRef] 15. Mubarak, N.M.; Wong, J.R.; Tan, K.W.; Sahu, J.N.; Abdullah, E.C.; Jayakumar, N.S.; Ganesan, P. Immobilization
of cellulase enzyme on functionalized multiwall carbon nanotubes. J. Mol. Catal. B Enzym. 2014, 107, 124–131. [CrossRef] 16. Fraga-García, P.; Kubbutat, P.; Brammen, M.; Schwaminger, S.; Berensmeier, S. Bare iron oxide nanoparticles
for magnetic harvesting of microalgae: From interaction behavior to process realization. Nanomaterials 2018,
8, 292. [CrossRef] 17. Ranmadugala, D.; Ebrahiminezhad, A.; Manley-Harris, M.; Ghasemi, Y.; Berenjian, A. Magnetic
immobilization of bacteria using iron oxide nanoparticles. Biotechnol. Lett. 2018, 40, 237–248. [CrossRef] Int. J. Mol. Sci. 2020, 21, 205 17 of 19 17 of 19 18. Banerjee, P.; Barman, S.R.; Swarnakar, S.; Mukhopadhyay, A.; Das, P. Treatment of textile effluent using
bacteria-immobilized graphene oxide nanocomposites: Evaluation of effluent detoxification using Bellamya
bengalensis. Clean Technol. Environ. Policy 2018, 20, 2287–2298. [CrossRef] 19. Durán, N.; Martinez, D.S.T.; Silveira, C.P.; Durán, M.; De Moraes, A.C.M.; Simões, M.B.; Alves, O.L.;
Fávaro, W.J. Graphene oxide: A carrier for pharmaceuticals and a scaffold for cell interactions. Curr. Top. Med. Chem. 2015, 15, 309–327. [CrossRef] 20. Poulin, P.; Jalili, R.; Neri, W.; Nallet, F.; Divoux, T.; Colin, A.; Aboutalebi, S.H.; Wallace, G.; Zakri, C. Superflexibility of graphene oxide. Proc. Natl. Acad. Sci. USA 2016, 113, 11088–11093. [CrossRef] 21. Liao, C.; Li, Y.; Tjong, S. Graphene nanomaterials: Synthesis, biocompatibility, and cytotoxicity. Int. J. Mol. Sci. 2018, 19, 3564. [CrossRef] [PubMed] 22. Lammel, T.; Boisseaux, P.; Fernández-Cruz, M.-L.; Navas, J.M. References Internalization and cytotoxicity of graphene
oxide and carboxyl graphene nanoplatelets in the human hepatocellular carcinoma cell line Hep G2. Part. Fibre Toxicol. 2013, 10, 27. [CrossRef] [PubMed] 23. Wang, K.; Ruan, J.; Song, H.; Zhang, J.; Wo, Y.; Guo, S.; Cui, D. Biocompatibility of graphene oxide. Nanoscale Res. Lett. 2011, 6, 8. [CrossRef] [PubMed] 24. Ge, L.; Chen, A.; Pei, J.; Zhao, L.; Fang, X.; Ding, G.; Wang, Z.; Xiao, W.; Tang, F. Enhancing the thermostability
of α-l-rhamnosidase from Aspergillus terreus and the enzymatic conversion of rutin to isoquercitrin by adding
sorbitol. BMC Biotechnol. 2017, 17, 21. [CrossRef] 25. Li, R.; Guiney, L.M.; Chang, C.H.; Mansukhani, N.D.; Ji, Z.; Wang, X.; Liao, Y.-P.; Jiang, W.; Sun, B.;
Hersam, M.C.; et al. Surface oxidation of graphene oxide determines membrane damage, lipid peroxidation,
and cytotoxicity in macrophages in a pulmonary toxicity model. ACS Nano 2018, 12, 1390–1402. [CrossRef] 26. Pelin, M.; Fusco, L.; Martín, C.; Sosa, S.; Frontiñán-Rubio, J.; González-Domínguez, J.M.; Durán-Prado, M.;
Vázquez, E.; Prato, M.; Tubaro, A. Graphene and graphene oxide induce ROS production in human HaCaT
skin keratinocytes: The role of xanthine oxidase and NADH dehydrogenase. Nanoscale 2018, 10, 11820–11830. [CrossRef] 27. Malina, T.; Maršálková, E.; Holá, K.; Tuˇcek, J.; Scheibe, M.; Zboˇril, R.; Maršálek, B. Toxicity of graphene oxide
against algae and cyanobacteria: Nanoblade-morphology-induced mechanical injury and self-protection
mechanism. Carbon N. Y. 2019, 155, 386–396. [CrossRef] 28. Zhang, M.; Yu, Q.; Liang, C.; Liu, Z.; Zhang, B.; Li, M. Graphene oxide induces plasma membrane damage,
reactive oxygen species accumulation and fatty acid profiles change in Pichia pastoris. Ecotoxicol. Environ. Saf. 2016, 132, 372–378. [CrossRef] 29. Arrais, A.; Diana, E.; Boccaleri, E. A study on the carbon soot derived from the wood combustion and on the
relative alkali-extractable fraction. J. Mater. Sci. 2006, 41, 6035–6045. [CrossRef] 30. Wong, C.H.A.; Sofer, Z.; Kubešová, M.; Kuˇcera, J.; Matˇejková, S.; Pumera, M. Synthetic routes contaminate
graphene materials with a whole spectrum of unanticipated metallic elements. Proc. Natl. Acad. Sci. USA
2014, 111, 13774–13779. [CrossRef] 31. Eigler, S.; Dotzer, C.; Hof, F.; Bauer, W.; Hirsch, A. Sulfur species in graphene oxide. Chem. A Eur. J. 2013, 19,
9490–9496. [CrossRef] [PubMed] 32. Seabra, A.B.; Paula, A.J.; De Lima, R.; Alves, O.L.; Durán, N. Nanotoxicity of graphene and graphene oxide. Chem. Res. Toxicol. 2014, 27, 159–168. [CrossRef] [PubMed] 3. Gies, V.; Zou, S. References Langmuir 2018, 34, 603–611. [CrossRef] 45. Ou, L.; Lin, S.; Song, B.; Liu, J.; Lai, R.; Shao, L. The mechanisms of graphene-based materials-induced
programmed cell death: A review of apoptosis, autophagy, and programmed necrosis. Int. J. Nanomed. 2017,
12, 6633–6646. [CrossRef] 46. Zhang, Y.; Ali, S.F.; Dervishi, E.; Xu, Y.; Li, Z.; Casciano, D.; Biris, A.S. Cytotoxicity effects of graphene
and single-wall carbon nanotubes in neural phaeochromocytoma-derived PC12 cells. ACS Nano 2010, 4,
3181–3186. [CrossRef] 47. Markovic, Z.M.; Ristic, B.Z.; Arsikin, K.M.; Klisic, D.G.; Harhaji-Trajkovic, L.M.; Todorovic-Markovic, B.M.;
Kepic, D.P.; Kravic-Stevovic, T.K.; Jovanovic, S.P.; Milenkovic, M.M.; et al. Graphene quantum dots as
autophagy-inducing photodynamic agents. Biomaterials 2012, 33, 7084–7092. [CrossRef] 48. Tabish, T.A.; Pranjol, M.Z.I.; Jabeen, F.; Abdullah, T.; Latif, A.; Khalid, A.; Ali, M.; Hayat, H.; Winyard, P.G.;
Whatmore, J.L.; et al. Investigation into the toxic effects of graphene nanopores on lung cancer cells and
biological tissues. Appl. Mater. Today 2018, 12, 389–401. [CrossRef] 49. Adil, S.; Khan, M.; Khan, M.; Al-Marri, A.; Al-Warthan, A.; Alkhathlan, H.; Siddiqui, M.; Nayak, V.; Kamal, A. Apoptosis inducing ability of silver decorated highly reduced graphene oxide nanocomposites in A549 lung
cancer. Int. J. Nanomed. 2016, 11, 873. [CrossRef] 50. Mbeh, D.A.; Akhavan, O.; Javanbakht, T.; Mahmoudi, M.; Yahia, L. Cytotoxicity of protein corona-graphene
oxide nanoribbons on human epithelial cells. Appl. Surf. Sci. 2014, 320, 596–601. [CrossRef] 51. Yuan, X.; Liu, Z.; Guo, Z.; Ji, Y.; Jin, M.; Wang, X. Cellular distribution and cytotoxicity of graphene quantum
dots with different functional groups. Nanoscale Res. Lett. 2014, 9, 108. [CrossRef] [PubMed] 52. Zhu, S.; Luo, F.; Zhu, B.; Wang, G.-X. Toxicological effects of graphene oxide on Saccharomyces cerevisiae. Toxicol. Res. (Camb.) 2017, 6, 535–543. [CrossRef] 53. Zhu, S.; Zhu, B.; Huang, A.; Hu, Y.; Wang, G.; Ling, F. Toxicological effects of multi-walled carbon nanotubes
on Saccharomyces cerevisiae: The uptake kinetics and mechanisms and the toxic responses. J. Hazard. Mater. 2016, 318, 650–662. [CrossRef] 54. Zhu, S.; Luo, F.; Li, J.; Zhu, B.; Wang, G.-X. Biocompatibility assessment of single-walled carbon nanotubes
using Saccharomyces cerevisiae as a model organism. J. Nanobiotechnol. 2018, 16, 44. [CrossRef] [PubMed] 55. Oliveira, R.; Johansson, B. Quantitative DNA damage and repair measurement with the yeast comet assay. Methods Mol. Biol. 2012, 920, 101–109. [PubMed] 56. Mortimer, M.; Kasemets, K.; Heinlaan, M.; Kurvet, I.; Kahru, A. High throughput kinetic Vibrio fischeri
bioluminescence inhibition assay for study of toxic effects of nanoparticles. Toxicol. References Systematic toxicity investigation of graphene oxide: Evaluation of assay selection, cell t
exposure period and flake size. Toxicol. Res. (Camb.) 2018, 7, 93–101. [CrossRef] [PubMed] 34. Chang, Y.; Yang, S.-T.; Liu, J.-H.; Dong, E.; Wang, Y.; Cao, A.; Liu, Y.; Wang, H. In vitro toxicity evaluation of
graphene oxide on A549 cells. Toxicol. Lett. 2011, 200, 201–210. [CrossRef] 35. Reshma, S.C.; Syama, S.; Mohanan, P.V. Nano-biointeractions of PEGylated and bare reduced graphene
oxide on lung alveolar epithelial cells: A comparative in vitro study. Colloids Surf. B Biointerfaces 2016, 140,
104–116. [CrossRef] 36. Mittal, S.; Kumar, V.; Dhiman, N.; Chauhan, L.K.S.; Pasricha, R.; Pandey, A.K. Physico-chemical properties
based differential toxicity of graphene oxide/reduced graphene oxide in human lung cells mediated through
oxidative stress. Sci. Rep. 2016, 6, 39548. [CrossRef] 37. Hu, W.; Peng, C.; Luo, W.; Lv, M.; Li, X.; Li, D.; Huang, Q.; Fan, C. Graphene-based antibacterial paper. ACS Nano 2010, 4, 4317–4323. [CrossRef] Int. J. Mol. Sci. 2020, 21, 205 18 of 19 18 of 19 38. Chng, E.L.K.; Pumera, M. The toxicity of graphene oxides: Dependence on the oxidative methods used. Chem. A Eur. J. 2013, 19, 8227–8235. [CrossRef] 39. Das, S.; Singh, S.; Singh, V.; Joung, D.; Dowding, J.M.; Reid, D.; Anderson, J.; Zhai, L.; Khondaker, S.I.;
Self, W.T.; et al. Oxygenated functional group density on graphene oxide: Its effect on cell toxicity. Part. Part. Syst. Charact. 2013, 30, 148–157. [CrossRef] y
40. Wu, W.; Yan, L.; Wu, Q.; Li, Y.; Li, Q.; Chen, S.; Yang, Y.; Gu, Z.; Xu, H.; Yin, Z.Q. Evaluation of the toxicity of
graphene oxide exposure to the eye. Nanotoxicology 2016, 10, 1329–1340. [CrossRef] 41. Mendonça, M.C.P.; Soares, E.S.; De Jesus, M.B.; Ceragioli, H.J.; Batista, Â.G.; Nyúl-Tóth, Á.; Molnár, J.;
Wilhelm, I.; Maróstica, M.R.; Krizbai, I.; et al. PEGylation of reduced graphene oxide induces toxicity in
cells of the blood–brain barrier: An in vitro and in vivo study. Mol. Pharm. 2016, 13, 3913–3924. [CrossRef]
[PubMed] 42. Pustovit, V.N.; Shahbazyan, T.V. Fluorescence quenching near small metal nanoparticles. J. Chem. Phys. 2012,
136, 204701. [CrossRef] [PubMed] 43. Kasry, A.; Ardakani, A.A.; Tulevski, G.S.; Menges, B.; Copel, M.; Vyklicky, L. Highly efficient fluorescence
quenching with graphene. J. Phys. Chem. C 2012, 116, 2858–2862. [CrossRef] 44. Wu, X.; Xing, Y.; Zeng, K.; Huber, K.; Zhao, J.X. Study of fluorescence quenching ability of graphene oxide
with a layer of rigid and tunable silica spacer. References Vitr. 2008, 22, 1412–1417. [CrossRef] [PubMed] 57. Patel, S.K.S.; Choi, S.H.; Kang, Y.C.; Lee, J.-K. Eco-friendly composite of Fe3O4-reduced graphene oxide
particles for efficient enzyme immobilization. ACS Appl. Mater. Interfaces 2017, 9, 2213–2222. [CrossRef]
[PubMed] Int. J. Mol. Sci. 2020, 21, 205 19 of 19 19 of 19 58. Contreras Rodríguez, A.R.; Saiz-Poseu, J.; García-Pardo, J.; García, B.; Lorenzo, J.; Ojea-Jiménez, I.; Komilis, D.;
Sedó, J.; Busqué, F.; Sánchez, A.; et al. Biocompatible polydopamine-like particles for the removal of heavy
metals at extremely low concentrations. RSC Adv. 2016, 6, 40058–40066. [CrossRef] 9. Ríos, F.; Fernández-Arteaga, A.; Fernández-Serrano, M.; Jurado, E.; Lechuga, M. Silica micro-and nanopart
reduce the toxicity of surfactant solutions. J. Hazard. Mater. 2018, 353, 436–443. [CrossRef] [PubMed] 60. Zhang, Y.; Zhang, J.; Huang, X.; Zhou, X.; Wu, H.; Guo, S. Assembly of graphene oxide-enzyme conjugates
through hydrophobic interaction. Small 2012, 8, 154–159. [CrossRef] [PubMed] 61. Barbero, F.; Russo, L.; Vitali, M.; Piella, J.; Salvo, I.; Borrajo, M.L.; Busquets-Fité, M.; Grandori, R.; Bastús, N.G.;
Casals, E.; et al. Formation of the protein corona: The interface between nanoparticles and the immune
system. Semin. Immunol. 2017, 34, 52–60. [CrossRef] [PubMed] 62. Chong, Y.; Ge, C.; Yang, Z.; Garate, J.A.; Gu, Z.; Weber, J.K.; Liu, J.; Zhou, R. Reduced cytotoxicity of graphene
nanosheets mediated by blood-protein coating. ACS Nano 2015, 9, 5713–5724. [CrossRef] [PubMed] 63. Wei, X.-Q.; Hao, L.-Y.; Shao, X.-R.; Zhang, Q.; Jia, X.-Q.; Zhang, Z.-R.; Lin, Y.-F.; Peng, Q. Insight into
the interaction of graphene oxide with serum proteins and the impact of the degree of reduction and
concentration. ACS Appl. Mater. Interfaces 2015, 7, 13367–13374. [CrossRef] [PubMed] 64. Qi, Y.; Chen, W.; Liu, F.; Liu, J.; Zhang, T.; Chen, W. Aggregation morphology is a key factor determining
protein adsorption on graphene oxide and reduced graphene oxide nanomaterials. Environ. Sci. Nano 2019,
6, 1303–1309. [CrossRef] 65. Zhang, Y.; Wu, C.; Guo, S.; Zhang, J. Interactions of graphene and graphene oxide with proteins and peptides. Nanotechnol. Rev. 2013, 2, 27–45. [CrossRef] 66. Ou, L.; Song, B.; Liang, H.; Liu, J.; Feng, X.; Deng, B.; Sun, T.; Shao, L. Toxicity of graphene-family
nanoparticles: A general review of the origins and mechanisms. Part. Fibre Toxicol. 2016, 13, 57. [CrossRef] 67. James, J.; Fiji, N.; Roy, D.; Andrew MG, D.; Shihabudeen, M.S.; Chattopadhyay, D.; Thirumurugan, K. A rapid
method to assess reactive oxygen species in yeast using H2DCF-DA. Anal. Methods 2015, 7, 8572–8575. © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/). References [CrossRef] 68. Jarque, S.; Masner, P.; Klánová, J.; Prokeš, R.; Bláha, L. Bioluminescent Vibrio fischeri assays in the assessment
of seasonal and spatial patterns in toxicity of contaminated river sediments. Front. Microbiol. 2016, 7, 1738. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
|
https://openalex.org/W3192873433
|
https://link.springer.com/content/pdf/10.1007/s40037-012-0005-4.pdf
|
English
| null |
Workplace learning
|
Perspectives on medical education
| 2,012
|
cc-by
| 4,790
|
T. Dornan (&)
Department of Educational Development and Research, Maastricht University,
PO Box 616, 6200 MD Maastricht, The Netherlands
e-mail: t.dornan@maastrichtuniversity.nl Abstract This critical review found Dutch research to be strong at the undergraduate and
residency levels and more or less absent in continuing medical education. It confirms
the importance of coaching medical students, giving constructive feedback, and
ensuring practice environments are conducive to learning though it has proved hard
to improve them. Residents learn primarily from experiences encountered in the
course of clinical work but the fine balance between delivering clinical services and
learning can easily be upset by work pressure. More intervention studies are needed. Qualitative research designs need to be more methodologically sophisticated and use
a wider range of data sources including direct observation, audio-diaries, and text
analysis. Areas for improvement are clear but achieving results will require
persistence and patience. Keywords
Workplace learning Undergraduate medical education Residency
Continuing medical education Qualitative research Published online: 7 February 2012
The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 7 February 2012
The Author(s) 2012. This article is published with open access at Springerlink.com Published online: 7 February 2012
The Author(s) 2012. This article is published with open access at Springerlink.com Perspect Med Educ (2012) 1:15–23
DOI 10.1007/s40037-012-0005-4 Perspect Med Educ (2012) 1:15–23
DOI 10.1007/s40037-012-0005-4 REVIEW ARTICLE Workplace learning Tim Dornan The practice of workplace learning I am struck by differences between my Dutch experiences and what I have seen in
Britain and some other parts of the world. Dutch medical students—like ones in
North America—have rich workplace learning opportunities. Particularly in the
senior clerkship years, they participate in practice in a way that is rarely seen in
contemporary Britain. Work I did with Maastricht University and the Open
University of the Netherlands showed that participation is central to students’
identity development, and identity development is at the centre of their learning [2]. So there is much to commend contemporary Dutch undergraduate workplace
learning. But there is a potential cost to patients. de Feijter et al. [3] showed how
learning from participation can confront medical students with difficult choices. A
nurse might, for example, ask a student to do something they are not fully trained or
authorised to do. On the one hand, they should refuse for the sake of patient safety. But on the other, their identity formation is strongly linked to performing tasks, so
there is an incentive to perform the unsafe act. That tension could be a usefully
formative one in a well-supported learning environment but there is a fine balance
between providing too much and too little support. The fact that such a tension
emerged in recent Dutch research suggests the balance may not always be achieved. Much UK health care is delivered by the National Health Service and patient safety is
such a politically sensitive issue that the tension described by de Feijter et al. [3] is
much less apparent in contemporary Britain (though I was wholly familiar with it
30 years ago) because students now have such limited opportunities to participate in
practice. A very positive feature of Dutch medical education is how the transition [4]
between senior medical student and qualified doctor has been deliberately blurred. Dutch medical students are progressively exposed to the tension of practice, which
may tip the balance towards a more favourable transition [4]. Contemporary
Dutch undergraduate curriculum design is a nice example of what Kennedy and
colleagues termed ‘progressive independence’ [5]. For the reasons given above, the
trend in the UK has been in just the opposite direction, though efforts are now being
made to reverse it. Research in Manchester showed how the abruptness of newly
qualified UK doctors’ entry to practice can compromise patient safety by
contributing to prescribing errors [6]. Introduction Workplace learning is as old as medicine itself. Before the Flexner report of 1910 [1],
the term tended to mean working for more senior doctors who were accountable to
nobody for the quality of their work, which could be very poor. Flexner was
commissioned to write his report to improve that state of affairs. One result of the
report was that medical education came to be delivered (or at least supervised) by
universities. Another was that educational standards rose. Teachers were now 12 123 16 T. Dornan academic practitioners but the tradition of learning through service persisted. Other
articles in this issue describe newer developments in medical education—in student
selection,
faculty
development,
assessment,
simulation,
and
competency
development. Old-fashioned workplace learning, however, remains important
because practice has to be learned by practising. Workplace learning exists in
medical curricula in many different guises: Early clinical experience, clerkships,
residency, and continuing medical education. I start with some personal observations
on how workplace medical education is practised in Dutch-speaking Europe and then
review Dutch education research in the field. Throughout the article, I use the
adjective ‘Dutch’ to refer to medical education in Flanders as well as the Netherlands. 123 The practice of workplace learning That is a counterpart to the situations 123 17 Workplace learning analyzed by de Feijter et al. [3]. It shows just how important it is to achieve safe,
legal, progressive immersion in practice. Despite the concerns I expressed in the
previous paragraph, I think Dutch progressive immersion is a better approach to
workplace learning than the UK’s relative exclusion of medical students from
practice until the moment of qualification, followed by abrupt immersion. My international comparison is less favourable towards the Netherlands and
Flanders when I view the period after qualification. Newly qualified UK doctors
complete a 2-year rotation through different specialities, enter a common core
rotation (e.g. surgery or internal medicine), and then specialize (e.g. neurology,
rheumatology). Medical graduates in the Dutch-speaking world do not have a period
of general professional education so they develop their CanMEDS competencies [7]
within a speciality-specific milieu. Even if there is more participation in practice in
Dutch than UK undergraduate medical education, I doubt it can fully provide the
insights into other fields of practice that are so essential to interdisciplinary
collaboration. I do, however, have a positive observation about Dutch postgraduate
education to offset that negative one. Boor et al. [8–10] demonstrated that a widely
used measure of postgraduate education environments lacked validity evidence,
teased out the dimensions of the learning environment construct, then developed and
validated the D-RECT instrument, whose 11-subscale structure makes it a powerful
tool for formative and summative evaluation and quality development. They have
made a valuable contribution to international scholarship by developing a valid
means of measuring the quality of postgraduate learning environments. Finally, a reflection on continuing professional development (CPD; or continuing
medical education, CME). It is a very prominent part of the medical education
continuum in Britain, the USA, Canada, and Australia, which does not seem so much
the case in the Netherlands and Flanders. But is that all bad? I have argued elsewhere
that the UK discourse of CPD is a disempowering, regulatory discourse rather than a
discourse that empowers lifelong workplace learning [11]. CPD is an important
topic, because it concerns maintaining the quality of expert professional practice. It is
under-researched compared with other aspects of medical education and presents
good opportunities for education research that can impact on the quality of health
care. Medical student education Medical student education I use a piece of my own research, a realist synthesis [12] of how medical students
learn in workplaces [13], to show how Dutch publications have contributed to the
scholarship of workplace learning. Our team, which includes two Dutch researchers,
six others, and myself identified papers published between 2000 and 2006 that form
an evidence-base of how medical students learn in workplaces. Forty-seven percent
of the 168 papers originated from the USA, 19% from Britain, 11% from mainland
Europe and the Nordic countries, 7% from Canada, 9% from Australia or
New Zealand, and 7% from other parts of the world. Looking more closely at the 23 18 T. Dornan Dutch contribution, 14 papers (8%) were conducted solely in the Netherlands or
Belgium, or had Dutch institutions as collaborators. After excluding the four papers
of my own which were co-published with Maastricht University but conducted on
British soil, 10 Dutch contributions remained [14–23]. Fieldwork was done at the VU
University, Amsterdam in four studies [15, 17, 18], at Maastricht University in three
studies [16, 19, 20], and at the Erasmus University, Rotterdam [21], Catholic
University of Leuven [22], and the University of Antwerp [14] (one study each). Eight studies concerned clerkship learning [14–18, 21–23], one concerned the
transition to clerkship [20], and one reported a longitudinal experience in primary
care during the early curriculum years [19]. Five of the clerkship studies were purely
observational and three had an element of intervention [18, 19, 23]. Nine were purely
qualitative or used mixed methods whilst one used structural equations modelling to
analyze numerical data [21]. The findings of the Dutch studies were quite consistent with one another and
similar to findings in other countries. The two main determinants of learning during
clerkships were the quality of supervision and casemix [16, 21]. Better supervision
could influence and compensate for limited casemix [21]. Supervision directly
enhanced academic performance [21]. Feedback was most effective when given by
someone who knew the student and whom the student knew [17]. Sympathetic and
warm feedback had important positive effects on students’ emotions and harsh or
absent feedback had negative effects [17, 20]. Learning environments that were more
orientated towards education (rather than pure service provision) were motivating,
whereas learning environments in which education was not a priority left students
feeling abandoned [22]. Students did not always receive high-quality supervision and
feedback [15, 17, 18, 20]. Medical student education When given, feedback was not always based on
observation of their performance [17]. The ‘learning by trial and error’ [15] that
resulted left students in doubt about their proficiency and whether they were attaining
curriculum objectives. Being given clear learning outcomes [19] and being coached
in clinical skills [14] helped students learn. The three studies that had an
interventional component are very informative in that the interventions made little
difference. The introduction of an in-training assessment scheme had little, if any,
effect on supervision and feedback because residents were unclear about their roles
and students were reluctant to reveal their weaknesses to their assessors [23]. Attempts to improve the quality of supervision and feedback in a surgical clerkship
had a limited impact on students’ hit and miss exposure to relevant casemix and the
supervisory support to their learning [17, 18]. The fieldwork on which the findings in the previous paragraph are based is now
somewhat out of date so they may not reflect what is happening on the ground today. I suspect, however, they do. Changing the ‘tea-steeping’ model (blocks of
experiential learning by immersion within functioning clinical units) to a more
outcome-focused, structured, instructed, and supervised model means overcoming a
lot of inertia, as discussed in Cooke and colleagues’ Flexner centenary monograph
[24]. Likewise, a recent review concluded that constructive feedback based on
personal knowledge of students is generally absent in workplaces [25]. So, the
findings of our review ring true despite their age. 123 123 19 Workplace learning I have looked for Dutch lines of inquiry into undergraduate medical education since
2006. My (doubtless incomplete) scan identified several. One, conducted in general
practice, explored the consequences of placing learners in supportive environments
[26, 27]. It is well established that two important dimensions of workplace
instructional quality are high-quality supervision and access to appropriate patients. Promotion of independence, this research showed, is an important third dimension. High-quality supervision helps students learn independently from the casemix they
have access to [27]. The same authors explored medical students’ learning in primary
care from a sociocultural perspective and found that students form their professional
identities within a private ‘developmental space’ under the combined influence of their
workplace context, personal interactions, and professional ones [26]. p
p
p
Contemporary research into communication education again shows inertia. Communication skills training—mostly provided in the pre-clerkship years—aims to
equip students with tools for patient-centred practice. Medical student education Bombeke et al. [28] found
exposure to hospital environments in the clerkship years counteracted patient-
centred orientations developed in the earlier years. Lack of student self-efficacy,
pressures of working environments and negative role models contributed to this
decline of patient-centredness. A lack of patient-centred, self-caring, and self-aware
role models in clerkship learning environments, their research suggests, may be
responsible. The findings of a second study by the same researchers, which compared
students who had received communication skills training with students who had not,
were really rather alarming [29]. Students trained in communication skills showed a
greater decline in patient-centredness during clerkships than students who had not
been trained in communication skills. Communication skills training, the study
suggested, may accentuate the clash between student idealism and workplace reality,
which led to a decline in patient-centredness. Contemporary medical practice, it
seems, is not patient-centred enough to serve as an educational model. One wonders,
then, how it will ever be possible to make doctors more patient-centred. The study
certainly suggests that communication skills education confined to the early
curriculum years will not do the trick. A third cluster of recent studies, from Groningen, concerned transition from pre-
clerkship to clerkship education [30], the influence of learning environments, [31,
32] and how students learned within them [33, 34]. van Hell et al. [31, 32] found that
feedback was most valued by students when it came from a doctor rather than an
allied professional, was based on direct observation of their behaviour, and/or was
initiated by themselves. Students’ ratings of the value of learning environments were
higher when they spent more time in them and were more active participants [32]. Clerkship students used diverse learning strategies [33] and were motivated by
comparing themselves with higher performing members of their peer group [34]. Residency I recently searched the international literature for empirical research into how
residents learn. Remarkably little has been published. I judged two lines of enquiry to
be particularly informative. Both were qualitative and both were Dutch [35, 36]. Residents’ learning, according to those papers, always starts from experiences 12 123 3 20 T. Dornan encountered in the course of clinical work [35, 37], although the sheer pressure of
clinical workload can easily reduce the value of workplace learning [36]. So,
residents’ most important learning is ‘informal’ [36], as has been shown in other
professions [38]. One of those two studies [36] was into how residents learn from
deliberate practice while the other [35] explored how residents gave personal
meaning to their workplace experiences, supported by their supervisors [37]. Teunissen et al. continued their research into personal meaning with two further
studies. One was an experiment, which showed how ‘priming’ junior residents with
an extraneous line of thought influenced their germane thinking about clinical
problems [39]. This experiment supported their theory that residents’ interpretations
of workplace experiences are influenced by personal knowledge and showed that
extraneous factors have a stronger influence in junior than senior residents [39]. A
second study by the same group evaluated two ‘dispositions’ of trainees and how
they related to one another: One was being disposed to learn versus being disposed to
make a good impression on others. The other disposition was towards seeking or not
seeking feedback, given its perceived benefits and costs to the resident. The paper
makes two important points: One is that residents are not passive recipients of
feedback; feedback is an active discourse between supervisor and supervisee. The
second point is that specialists’ style of giving feedback influences residents’
learning. Supportive specialists give feedback in a way that helps residents perceive
more benefits and fewer costs [40]. Returning to my international review of research into how residents learn, one of
the four remaining papers—which contributed consensus data about important
factors in workplace learning environments—was Dutch [41]. The remaining three
non-Dutch papers examined factors that influence residents’ participatory learning
[42], the exchange of tacit knowledge between anaesthesiologists [43], and tensions
between patient care and learning [44]. Conclusion According to this survey, the Dutch contribution to international scholarship in
workplace learning is strong at the undergraduate and residency levels and absent at
the CME level. A positive feature of the Dutch effort is the amount of high-quality
research into residency education. A methodological weakness of the workplace
learning research I have reviewed—in common research from other countries—is an
excess of observational over interventional/experimental research. Qualitative
workplace research tends towards focus groups and interviews in which researchers
take respondents’ words as truth, rather than being critical about why respondents say
the things they do in the research context. There are qualitative methodologies that
address those concerns. The analytical heuristics of phenomenology and discourse
analysis, for example, address that epistemological problem. Workplace learning
research could benefit from alternative methods of data collection: Direct (participant)
observation and audio-diary techniques, for example, give near contemporaneous
accounts of learning, which reduce the problem of respondents’ experiences being
reconstructed in retrospect to fit the research. Even without using phenomenology or 123 21 Workplace learning discourse analysis, analytical approaches could be more sophisticated. Grounded
theory had a strong influence on the early years of qualitative research, leaving a
legacy of work that starts from no identified theoretical position and never reaches one. Grounded theory has a clear place, particularly in ‘scoping’ a field of research, when it
generates new theory. ‘Thematic analysis’, in my view, has a more limited place,
because it too rarely states its epistemological position and too often assumes that
some
objective
truth
resides
within
research
respondents’
spoken
words. Constructivist grounded theory is showing promise as a methodology that addresses
some of the concerns expressed above. By using prior theory to provide ‘sensitizing
insights’ that can be applied to data interpretation, it allows new theory to be built on
pre-existing theory. There are examples of this in the Dutch work I have reviewed. Teunissen et al. [35], for example, allowed one grounded theory study to inform a
second one [37], and then elaborated their theory programmatically by means of
well-theorised experimental [39] and quantitative survey research [40]. Bombeke
et al. derived sensitizing concepts from an ‘Attitude-Social influence-Self efficacy
model’ and used them to analyze their patient-centredness data. de Feijter et al. [3]
used Activity Theory in an informative way to reveal tensions in patient safety
education while van der Zwet et al. Conclusion concept of ‘developmental space’ was informed by
sociocultural learning theory [26]. So what, finally, can we conclude about the state of the art in workplace learning? Workplaces afford rich learning opportunities, which are integral to their primary
role—getting jobs done—but in constant tension with it. That tension is responsible for
both the greatest successes and the greatest failings of workplace learning. Learning is
mediated by the relationships that exist between learners, peers, more experienced
practitioners, other health professionals, and patients. Participation in the activities of
workplaces is a discourse, in which all participants play active parts. Supervision,
feedback, and other teaching and learning activities are, likewise, discourses in which
learners play important parts. Each workplace has its own rich cultural history, which
means they respond slowly to efforts to change them. Humanistic qualities of
practitioners, which have not traditionally been given the importance they have now
assumed, are the essential ingredient of effective workplace learning environments. Education research has given clear direction about how those environments can be
improved, but improving them will require persistence and patience. References 1. Flexner A. Medical education in the United States and Canada. A report to the Carnegie foundation f
the advancement of teaching. Boston: Updyke; 1910. 2. Dornan T, Boshuizen H, King N, Scherpbier A. Experience-based learning: a model linking th
processes and outcomes of medical students’ workplace learning. Med Educ. 2007;41:84–91. 3. de Feijter JM, de Grave WS, Dornan T, Koopmans RP, Scherpbier AJJA. Students’ perceptions of
patient safety during the transition from undergraduate to postgraduate training: activity theory
analysis. Adv Health Sci Educ Theory Pract. 2010. doi:10.1007/s10459-010-9266-z. 4. Teunissen P, Westerman M. Opportunity or threat; ambiguity in the consequences of transitions
medical education. Med Educ. 2011;45:51–9. 5. Kennedy TJT, Regehr G, Baker GR, Lingard LA. Progressive independence in clinical training:
tradition worth defending? Acad Med. 2005;80(Suppl):S106–11. 6. Dornan T, Ashcroft D, Heathfield H, et al. An in depth investigation into causes of prescribing errors
by foundation trainees in relation to their medical education. EQUIP study. London: General Medical
Council; 2009. 7. Frank JR, editor. The CanMEDS 2005 physician competency framework. Ottawa: Office
Education, The Royal College of Physicians and Surgeons of Canada; 2005. 8. Boor K, Scheele F, van der Vleuten CP, Scherpbier AJ, Teunissen PW, Sijtsma K. Psychometr
properties of an instrument to measure the clinical learning environment. Med Educ. 2007;41:92– 9. Boor K, Scheele F, van der Vleuten CP, Teunissen PW, den Breejen EM, Scherpbier AJ. How
undergraduate clinical learning climates differ: a multi method case study. Med Educ. 2008;42:1029–36. 10. Boor K, van der Vleuten C, Teunissen P, Scherpbier A, Scheele F. Development and analysis of
D-RECT, an instrument measuring residents’ learning climate. Med Teach. 2011;33:820–7. 11. Dornan T. Self-assessment in CPD: lessons from the UK undergraduate and postgraduate education
domains. J Cont Educ Health Prof. 2008;28:32–7. 12. Wong G, Greenhalgh T, Westhorp G, Pawson R. Realist methods in medical education research: what
are they and what can they contribute? Med Educ. 2012;46:89–96. 13. Dornan T, Tan N, Boshuizen H et al. Experience based learning (eXBL). Realist synthesis of the
conditions, processes, and outcomes of medical students’ workplace learning. BEME. 2012; (In peer
review). 14. Remmen R, Denekens J, Scherpbier A, et al. An evaluation study of the didactic quality of clerkship
Med Educ. 2000;34:460–4. 15. van der Hem-Stokroos HH, Scherpbier AJJA, van der Vleuten CPPM, de Vries H, Haarman HJTHM. How effective is a clerkship as a learning environment. Med Teach. 2001;23:599–604. 16. Essentials •
Conscious effort is needed to make working environments conducive to learning
as well as ‘getting the job done’. •
Conscious effort is needed to make working environments conducive to learning
as well as ‘getting the job done’. •
Constructive feedback from a supportive practitioner who is known to a learne
aids learning. •
Excessive workload makes it hard for residents to learn from practice. •
Continuing education is a phase of the lifelong learning continuum that tends t
be neglected. •
There is more to qualitative research than transcribing what people say in
interviews or group discussions and analysing it thematically. 12 123 22 T. Dornan 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. the source are credited. References Dolmans DHJM, Wolfhagen IHAP, Essed GGM, Scherpbier AJJA, van der Vleuten CPM. The
impacts of supervision, patient mix, and numbers of students on the effectiveness of clinical rotations. Acad Med. 2002;77:332–5. 17. van der Hem-Stokroos HH, Daelmans HEM, van der Vleuten CPM, Haarman HJThM, Scherpbier
AJJA. A qualitative study of constructive clinical learning experiences. Med Teach. 2003;25:120–6. 18. van der Hem-Stokroos H, Daelmans H, van der Vleuten C, Haarman H, Scherpbier A. The impact of
multifaceted educational structuring on learning effectiveness in a surgical clerkship. Med Educ. 2004;38:879–86. 19. Mainhard MT, van den Hurk MM, van de Wiel MWJ, Crebolder HFJM, Scherpbier AJJA. Learning in
a clinical education programme in primary care: the Maastricht Adoption Programme. Med Educ. 2004;38:1236–43. 20. Prince KJAH, Boshuizen HPA, van der Vleuten CPM, Scherpbier AJJA. Students’ opinions about
their preparation for clinical practice. Med Educ. 2005;39:704–12. 21. Wimmers PF, Schmidt HG, Splinter TAW. Influence of clerkship experiences on clinical competence. Med Educ. 2006;40:450–8. 22. Deketelaere A, Kelchtermans G, Struyf E, De Leyn P. Disentangling clinical learning experiences: an
exploratory study on the dynamic tensions in internship. Med Educ. 2006;40:908–15. 123 123 Workplace learning 23 23. Daelmans HEM, Overmeer RM, van der Hem-Stokroos HH, Scherpbier AJJA, Stehouwer CDA, van
der Vleuten CPM. In-training assessment: qualitative study of effects on supervision and feedback in
an undergraduate clinical rotation. Med Educ. 2006;40:51–8. g
24. Cooke M, Irby DM, O’Brien BC. Educating physicians: a call for reform of medical school and
residency. Carnegie foundation for the advancement of teaching. San Francisco: Jossey-Bass; 2010. 25. Teunissen P, Wilkinson T. Learning and teaching in workplaces. In: Mann K, Scherpbier A, Spencer J,
Dornan T, editors. Medical education. Theory and practice. Edinburgh: Churchill Livingstone; 2011. p. 193–209. 26. van der Zwet J, Zwietering PJ, Teunissen PW, van der Vleuten CPM, Scherpbier AJJA. Workplace
learning from a socio-cultural perspective: creating developmental space during the general practice
clerkship. Adv Health Sci Educ Theory Pract. 2010. doi:10.1007/s10459-010-9268-x. 27. van der Zwet J, Hanssen VGA, Zwietering PJ, et al. Workplace learning in general practice:
supervision, patient mix and independence emerge from the black box once again. Med Teach. 2010;32:e294–9. 28. Bombeke K, Symons L, Debaene L, de Winter B, Schol S, van Royen P. Help, I’m losing patien
centredness! Experiences of medical students and their teachers. Med Educ. 2010;44:662–73. 29. Bombeke K, et al. References 2004;26:247–73. 39. Teunissen PW, Stapel DA, Scheele F, et al. The influence of context on residents’ evaluations: effects
of priming on clinical judgment and affect. Adv Health Sci Educ Theory Pract. 2009;14:23–41. 40. Teunissen PW, Stapel DA, van der Vleuten C, Scherpbier A, Boor K, Scheele F. Who wants feedback? An investigation of the variables influencing residents’ feedback-seeking behavior in relation to night
shifts. Acad Med. 2009;84:910–7. 41. Stok-Koch L, Bolhuis S, Koopmans R. Identifying factors that influence workplace learning in
postgraduate medical education. Educ Health. 2007;20:8. 42. Sheehan D, Wilkinson T, Billett S. Interns’ participation and learning in clinical environments in a
New Zealand hospital. Acad Med. 2005;80:302–8. 43. Pope C, Smith A, Goodwin D, Mort M. Passing on tacit knowledge in anaesthesia: a qualitative study. Med Educ. 2003;37:650–5. 44. Hoffman KG, Donaldson JF. Contextual tensions of the clinical environment and their influence o
teaching and learning. Med Educ. 2004;38:448–54. References Medical students trained in communication skills show a decline in patient-centred
attitudes: an observational study comparing two cohorts during clinical clerkships. Patient Educ
Couns. 2011;84:310–8. 30. van Hell EA, Kuks JBM, Scho¨nrock-Adema J, van Lohuizen MT, Cohen-Schotanus J. Transition to
clinical training: influence of pre-clinical knowledge and skills, and consequences for clinical
performance. Med Educ. 2008;42:830–7. 31. van Hell EA, Kuks JBM, Raat J, van Lohuizen MT, Cohen-Schotanus J. Instructiveness of feedback
during clerkships: influence of supervisor, observation and student initiative. Med Teach. 2008;31:45–50. 31. van Hell EA, Kuks JBM, Raat J, van Lohuizen MT, Cohen-Schotanus J. Instructiveness of feedback
during clerkships: influence of supervisor, observation and student initiative. Med Teach. 2008;31:45–50. 32. van Hell EA, Kuks JBM, Cohen-Schotanus J. Time spent on clerkship activities by students in relation
h i
i
f l
i
i
li
M d Ed
2009 43 674 9 g
p
p
,
;
32. van Hell EA, Kuks JBM, Cohen-Schotanus J. Time spent on clerkship activities by students in relation
to their perceptions of learning environment quality. Med Educ. 2009;43:674–9. 33. van Lohuizen MT, Kuks JBM, van Hell EA, Raat AN, Cohen-Schotanus J. Learning strategies durin
clerkships and their effects on clinical performance. Med Teach. 2009;31:e494–9. 34. Raat J, Kuks J, Cohen-Schotanus J. Learning in clinical practice: stimulating and discouragin
response to social comparison. Med Teach. 2010;32:899–904. 35. Teunissen PW, Scheele F, Scherpbier AJ, et al. How residents learn: qualitative evidence for the
pivotal role of clinical activities. Med Educ. 2007;41:763–70. 36. van de Wiel MWJ, Van den Bossche P, Janssen S, Jossberger H. Exploring deliberate practice in
medicine: howdo physicians learnin theworkplace?Adv Health Sci Educ Theory Pract.2011;16:81–95. 36. van de Wiel MWJ, Van den Bossche P, Janssen S, Jossberger H. Exploring deliberate practice in
medicine: howdo physicians learnin theworkplace?Adv Health Sci Educ Theory Pract.2011;16:81–95. 37. Teunissen PW, Boor K, Scherpbier AJ, et al. Attending doctors’ perspectives on how residents learn. M d Ed
2007 41 1050 8 36. van de Wiel MWJ, Van den Bossche P, Janssen S, Jossberger H. Exploring deliberate practice in
medicine: howdo physicians learnin theworkplace?Adv Health Sci Educ Theory Pract.2011;16:81–95. 37. Teunissen PW, Boor K, Scherpbier AJ, et al. Attending doctors’ perspectives on how residents learn. Med Educ. 2007;41:1050–8. 37. Teunissen PW, Boor K, Scherpbier AJ, et al. Attending doctors’ perspectives on how residents learn. Med Educ. 2007;41:1050–8. 38. Eraut M. Informal learning in the workplace. Stud Contin Educ. Author Biography Tim Dornan now works as an education researcher at Maastricht University having trained as an internist
and endocrinologist and worked in the UK national health service for over 30 years. His interests include
clinical workplace learning, sociocultural theory, qualitative research, and bibliographic methodology. 12 3
|
https://openalex.org/W2949435526
|
https://bmcwomenshealth.biomedcentral.com/track/pdf/10.1186/s12905-019-0775-5
|
English
| null |
Estimating the proportion of Medicaid-eligible pregnant women in Louisiana who do not get abortions when Medicaid does not cover abortion
|
BMC women's health
| 2,019
|
cc-by
| 6,515
|
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Open Access Estimating the proportion of Medicaid-
eligible pregnant women in Louisiana who
do not get abortions when Medicaid does
not cover abortion Estimating the proportion of Medicaid-
eligible pregnant women in Louisiana who
do not get abortions when Medicaid does
not cover abortion Sarah C. M. Roberts1*
, Nicole E. Johns1,3, Valerie Williams2, Erin Wingo1 and Ushma D. Upadh Sarah C. M. Roberts1*
, Nicole E. Johns1,3, Valerie Williams2, Erin Wingo1 and Ushma D. Upadhyay1 Abstract Background: To estimate the proportion of pregnant women in Louisiana who do not obtain abortions because
Medicaid does not cover abortion. Methods: Two hundred sixty nine women presenting at first prenatal visits in Southern Louisiana, 2015–2017,
completed self-administered iPad surveys and structured interviews. Women reporting having considered abortion
were asked whether Medicaid not paying for abortion was a reason they had not had an abortion. Using study
data and published estimates of births, abortions, and Medicaid-covered births in Louisiana, we projected the
proportion of Medicaid births that would instead be abortions if Medicaid covered abortion in Louisiana. Results: 28% considered abortion. Among women with Medicaid, 7.2% [95% CI 4.1–12.3] reported Medicaid not
paying as a reason they did not have an abortion. Existing estimates suggest 10% of Louisiana pregnancies end in
abortion. If Medicaid covered abortion, this would increase to 14% [95% CI 12, 16]. 29% [95% CI 19, 41] of Medicaid
eligible pregnant women who would have an abortion with Medicaid coverage, instead give birth. Conclusions: For a substantial proportion of pregnant women in Louisiana, the lack of Medicaid funding remains
an insurmountable barrier to obtaining an abortion. Forty years after the Hyde Amendment was passed, lack of
Medicaid funding for abortion continues to have substantial impacts on women’s ability to obtain abortions. Keywords: Abortion, Medicaid, Policy, Pregnancy, women’s health, Barriers to care Lack of Medicaid funding impacts the three-fourths of
women obtaining abortions in the U.S. who are of low-
income [6]. Out-of-pocket costs for abortion are over
one-third of monthly personal income for about half of
abortion patients [7]. Having to pay out of pocket has fi-
nancial implications for women, including lost wages
and delay in paying bills [8]. Background The Hyde Amendment, which restricts use of federal
Medicaid dollars to pay for abortion, is one of the lon-
gest running abortion restrictions [1]. Seventeen states
use state funding to pay for abortion for Medicaid eli-
gible women [2], meaning that in most U.S. states, there
is no public funding to pay for abortion for low-income
women. Even in the midst of hundreds of new restrictive
abortion policies enacted between 2011 and 2017 [3],
policy discussions continue to focus on Medicaid cover-
age for abortion [1, 2, 4, 5]. Research about impacts of the Hyde Amendment has
been conducted for almost as long as the policy has been
in effect. Prior to 2009, most research focused on the ex-
tent to which restricting Medicaid funding for abortion
affected women’s ability to obtain abortions [9]. A sys-
tematic review of that body of literature noted methodo-
logical flaws, but concluded that about one-fourth (18–
37%) of women who would have had Medicaid-covered
abortions
instead
gave
birth
when
funding
was * Correspondence: sarah.roberts@ucsf.edu
1Advancing New Standards in Reproductive Health (ANSIRH), Department of
Obstetrics, Gynecology, and Reproductive Sciences, University of California,
San Francisco, 1330 Broadway, Suite 1100, Oakland, CA 94612, USA
Full list of author information is available at the end of the article * Correspondence: sarah.roberts@ucsf.edu
1Advancing New Standards in Reproductive Health (ANSIRH), Department of
Obstetrics, Gynecology, and Reproductive Sciences, University of California,
San Francisco, 1330 Broadway, Suite 1100, Oakland, CA 94612, USA
Full list of author information is available at the end of the article Roberts et al. BMC Women's Health (2019) 19:78
https://doi.org/10.1186/s12905-019-0775-5 Roberts et al. BMC Women's Health (2019) 19:78
https://doi.org/10.1186/s12905-019-0775-5 Study design The Louisiana Abortion Prenatal Study was designed to
study impacts of Louisiana’s abortion restrictions [16]. We recruited participants at three university-affiliated
prenatal care facilities in Southern Louisiana that serve
pregnant women who have or are eligible for Medicaid. We describe the study methods in detail elsewhere [17]. Briefly, between June 2015 and May 2017, we recruited
women at their first prenatal care visit. Participants first
completed self-administered iPad surveys; they then
completed in-clinic structured interviews with a research
coordinator. The Institutional Review Boards at the Uni-
versity of California, San Francisco and The Louisiana
State University Health Sciences Campus granted ethical
approval for this study. Research coordinators first obtained informed consent. They then instructed participants on how to complete
self-administered iPad surveys and left them to complete
surveys independently. After participants completed iPad
surveys, the research coordinator conducted brief in-
clinic structured interviews with participants. Roberts et al. BMC Women's Health (2019) 19:78 Page 2 of 8 Page 2 of 8 unavailable [9]. A small number of studies examined
whether restricting Medicaid funding for abortion led to
delays in obtaining abortions and impacted other out-
comes – such as complications from illegal abortions
and birth outcomes [9]. of abortions per year) and others are small (covering
only a few abortions per year) [19]. The local abortion
fund in Southern Louisiana only covers a small portion
of costs at the abortion clinics in Southern Louisiana. At
the national level, the price for an abortion is more than
$500 and the adjusted prices are higher in states that
have more restrictive abortion policies, such as Louisiana
[20]. Average out-of-pocket costs for abortion (including
abortion funds or clinic discounts) is more than $300 for
first trimester and close to $600 across all gestations [7]. Women in states where abortion for low-income women
is covered by state funds pay, on average, $0 out of
pocket [7]. Since 2009, researchers have continued to study im-
pacts of restricted Medicaid funding for abortion. Meth-
odologically
sophisticated
studies
have
documented
Medicaid funding restrictions’ impact on maternal mor-
bidity and infant mortality [10, 11]. Other research ex-
amined
women’s
and
provider’s
experiences
with
Medicaid coverage and found that, even when Medicaid
can pay for abortion, it sometimes does not, leading to
delays and financial and emotional impacts on women
obtaining abortions [8, 12–15]. At the time we began the study in 2015, Louisiana had
five abortion clinics [21], three in the southern part of
the state. By the time we finished recruitment in 2017,
Louisiana had three abortion clinics, with two in the
southern part of the state. Neither the prenatal care
clinics where we recruited nor the local Planned Parent-
hood facilities provide abortions. Recent literature has not estimated the impact of lack
of Medicaid coverage for abortion. While the systematic
review that produced the one-fourth estimate of those
who would have had a Medicaid covered abortion if
coverage was available was published in 2009, much of
the research behind that estimate was published in the
1980s and 1990s [9]. A key question is whether this esti-
mate is still relevant. Another key unanswered question
is what are characteristics of women who do not obtain
abortions when Medicaid restrictions are in effect? Study procedures
I
h
i In each recruitment facility, a research coordinator
approached all women over 18 who presented for their
first prenatal care appointment during the study time
period and who spoke English. During the first year of
recruitment,
we
began
recruiting
Spanish-speaking
women. Women who were ineligible included those who
were under 18, not pregnant, receiving a noninitial pre-
natal visit, not English or Spanish speaking, or incarcer-
ated. As reported previously, of eligible individuals, 86%
consented to participate [17]. Measures The primary outcome was whether Medicaid not paying
for abortion was a reason a pregnant woman had not
had an abortion. To assess this outcome, we asked mul-
tiple questions. As a first step towards assessing whether
Medicaid was a reason for not having an abortion, the
iPad survey asked, “Have you considered abortion for
this pregnancy even for just one second?” In the in-
clinic interview, the research coordinator repeated this
question verbatim. As described previously, reporting
having considered abortion for this pregnancy was con-
sistent across modes; 94% of participants reported con-
sistently
across
modes
[17]. To
assess
the
main
outcome, in in-clinic interviews, participants who re-
ported considering abortion in the in-clinic interviews In this manuscript, we aim to estimate the proportion
of women who give birth instead of have an abortion be-
cause neither federal Medicaid nor state funds covers
abortion for low-income women in Louisiana. We chose
Louisiana because Louisiana state Medicaid does not
cover abortion [2]. Abortion funds are a set of private
organizations that seek to address limitations in insur-
ance coverage and geographic access to abortion [18]. To help pay for the costs of a low-income woman’s
abortion, these abortion funds provide subsidies to
health care facilities to cover some or all of the costs of
the abortion. Some funds are large (covering thousands Page 3 of 8 Page 3 of 8 Roberts et al. BMC Women's Health (2019) 19:78 Page 3 of 8 were asked: “Medicaid in Louisiana does not pay for
abortion. Was Medicaid not paying for abortion part of
why you have not had an abortion?” Those who
responded yes were considered to have not had an abor-
tion because Medicaid did not cover it. we asked “Are you still considering having an abortion?”,
after the open-ended questions about reasons for not
having an abortion. As people sometimes report more than one reason for
not having an abortion after considering one [17], we
used responses to the open-ended questions about rea-
sons for not having an abortion that we previously coded
into personal reason, interpersonal reason, healthcare/
other organization interaction, and policy-related reason. Specifically, responses coded as policy-related reasons
were used as a check on Medicaid-related reason. Re-
sponses could fall into more than one category. Measures As a secondary measure of the outcome, we used data
from open-ended responses to questions about reason(s)
for not having an abortion and the main reason for not
having an abortion. In the in-clinic interview, the re-
search coordinator asked participants who reported they
had considered abortion “even for just one second” a
series of questions on concrete actions they may have
taken to seek an abortion. Specifically, the research co-
ordinator asked about the following concrete actions,
whether they had: called an abortion clinic, made an ap-
pointment for an abortion, and went to the state-
mandated abortion counseling visit and the abortion ap-
pointment. Once a participant responded that she had
not taken the next concrete action in the series of pos-
sible actions, the research coordinator asked an open-
ended question about her reason(s) for not having taken
that step and then asked her to specify her main reason
for not having an abortion. We trained research coordi-
nators to: document responses verbatim, use neutral
probes for clarity, and obtain more detail from partici-
pants. We classified responses that included “fund”,
“money”, “price”, “insurance”, “dollars”, “$”, “cost” as fi-
nancial reasons for not having an abortion. We did face
validity checks to ensure responses were related to fi-
nancial reasons. We assessed characteristics, including age (continu-
ous), race/ethnicity (categorical), parity (categorical),
education
(categorical),
employment
(dichotomous),
public assistance receipt (dichotomous), food insecurity
(dichotomous), housing insecurity (dichotomous), insur-
ance status (categorical), relationship with man involved
in the pregnancy (categorical), past-year alcohol use dis-
order risk (dichotomous from AUDIT-C scale, number
of drinks modified from 6 to 4 [24]), past-year drug use
(dichotomous), and past-year tobacco use (dichotomous)
. Births and abortions We used published estimates of the number of births
and abortions in Louisiana in 2015 as well as guidance
on estimating the number of miscarriages based on birth
and abortion data [25–27] to estimate the number of
abortions, births, and miscarriages in Louisiana in
2015.We obtained published estimates of the proportion
of Louisiana births paid for by Medicaid in 2015 [28]. We used additional variables as validity checks for
reporting Medicaid as a reason for not having an abor-
tion. We asked which pregnancy outcome women pre-
ferred upon pregnancy discovery and which pregnancy
outcome they preferred now (upon prenatal care entry). In the iPad survey, we asked: “Please think back to the
week right after you found out you were pregnant. Please tell me which option you preferred the week
right after you found out you were pregnant. Having
the baby; Adoption or having someone else raise it; Hav-
ing an abortion.” Then, with the same answer options,
we asked, “Next, please tell us which option you prefer
now.”
We
assessed
pregnancy
planning
using
the
London Measure of Unplanned Pregnancy; for ease of
interpretation, we categorized the scale as unplanned,
ambivalent, or planned [22]. We measured decisional
certainty using the Decisional Conflict Scale, a 16-item
scale used in multiple areas of health care to measure
people’s certainty around different health care decisions;
for ease of interpretation, we categorized the scale as
high certainty, medium certainty, and low certainty [23]. To assess whether participants who reported Medicaid
not paying as a reason for not having an abortion may
have proceeded to have an abortion after the interview, Analysis We estimated the proportion of participants who re-
ported that they did not have an abortion because Me-
dicaid did not pay, including 95% Confidence Intervals
(CIs). We assessed whether this estimate varied if we in-
stead used coded responses from open-ended questions. We then estimated this proportion among women with
Medicaid insurance, including 95% CIs. For validity checks, we examined associations between
Medicaid not paying as a reason and pregnancy outcome
preference at pregnancy discovery, pregnancy outcome
preference at prenatal care entry, pregnancy planning,
and decisional certainty using chi-square tests and Fish-
er’s exact tests. We then estimated the proportion of women who gave
birth instead of having an abortion due to Medicaid not
covering abortion. We used data on the number of abor-
tions and births to Louisiana residents in 2015 as well as
guidance on estimating the number of miscarriages
based on abortions and births to estimate total number
of Louisiana births, miscarriages, and abortions in 2015. Page 4 of 8 Page 4 of 8 Roberts et al. BMC Women's Health (2019) 19:78 Roberts et al. BMC Women's Health (2019) 19:78 Roberts et al. BMC Women's Health (2019) 19:78 women with Medicaid insurance (n = 167), 7.2%, [95%
CI 4.1, 12.3] reported Medicaid not paying as a reason. We used published estimates of births paid for by Lou-
isiana Medicaid and study estimates of the proportion
with Medicaid insurance who reported not having an
abortion because Medicaid would not pay to estimate
the number of births paid for by Medicaid that would
instead be abortions if Medicaid covered abortion. We
added this number to published estimates of abortions
to estimate projected number of abortions in Louisiana
if Medicaid covered abortion. We then calculated pro-
portion of women who give birth instead of having an
abortion because Medicaid does not cover it through the
equation (Projected abortions – Actual abortions)/Pro-
jected abortions. We repeated these steps, replacing esti-
mates of proportions of women who reported that they
did not have an abortion because Medicaid did not pay
with lower and upper bounds of our estimate of the pro-
portion who reported not having an abortion due to Me-
dicaid not paying, to get a 95% CI. Sample Two hundred eighty five participants consented to par-
ticipate. 269 completed structured interviews and 265
responded to the question about whether lack of Medic-
aid coverage for abortion was a reason for not having an
abortion. Having considered abortion was not associated
with interview completion [17]. Study population char-
acteristics are in Table 1. Most participants were Black
(72%), low socio-economic status (65%), received public
assistance in the past year; 50% were food insecure, 33%
housing insecure, and 65% had given birth previously. About one-fourth reported past-year alcohol use dis-
order risk, 16% past-year drug use, and 28% past-year to-
bacco use. Few pregnancies were planned (25%), more
than ten percent preferred abortion upon pregnancy dis-
covery (14%) and most were certain of their decision to
continue pregnancy by the time they entered prenatal
care (98%). [See Table 1]. Proportion who do not obtain abortions due to Medicaid
not paying 5.3% of participants [95% CI 2.9, 8.7] reported Medicaid
not paying for abortion as a reason for not having an
abortion. As a validity check, using the secondary indica-
tor of women whose open-ended responses mentioned
funding, this would be 4.1% [95% CI 2.1, 7.2]. Among Characteristics of those who do not obtain abortions due to
Medicaid not paying Among women with Medicaid insurance, age, race/eth-
nicity, parity, and most measures of socioeconomic sta-
tus were not associated with reporting Medicaid as a
reason for not having an abortion. More women who re-
ported Medicaid as a reason were not in a romantic rela-
tionship with the man involved in the pregnancy (58% v. 18%), had less than high school education (42% v. 22%),
had alcohol use disorder risk (75% v. 21%), used drugs
(42% v. 16%), and used tobacco (67% v. 28%). [See Table
1]. Analysis As validity checks, among women with Medicaid in-
surance, 92% reporting Medicaid not paying as a reason
for not having an abortion preferred abortion at preg-
nancy discovery, compared to 10% who did not report
this reason. 17% of those reporting Medicaid not paying
as a reason preferred abortion at prenatal care entry,
compared to 1% who did not report this reason. 58% of
those reporting Medicaid as a reason for not having an
abortion had unplanned pregnancies, compared to 11%
unplanned pregnancies among those who did not report
this as a reason. 17% of those reporting Medicaid not
paying as a reason reported low certainty about their
pregnancy outcome decision, compared to 5% not
reporting this reason. [See Table 2]. Three participants who reported Medicaid as a reason
reported that they were still considering abortion for this
pregnancy. All three of these participants were in the
first trimester. In addition, most participants who re-
ported Medicaid as a reason also reported a policy-
related reason in response to the open-ended questions. Among those with Medicaid insurance, 4.2% reported
both Medicaid as a reason in response to the direct
question and a policy-related reason in the open-ended
questions. We then described characteristics of women who re-
port not having an abortion because Medicaid did not
pay. We conducted bivariate analyses using t-tests for
continuous and chi-square tests or Fisher’s exact tests
for dichotomous and categorical variables to identify
characteristics associated with not having an abortion
because Medicaid did not pay among those who had
Medicaid insurance. Based on published numbers, approximately 10% of
pregnancies in Louisiana end in abortion. If Medicaid
paid for abortion, this would increase to 14% [95% CI
12, 16]. [See Fig. 1] This means about 29% [95% CI 19,
41] of Medicaid-eligible pregnant women who would
have an abortion if Medicaid covered abortion instead
give birth. Applying 7.2% to the number of Medicaid
births in Louisiana in 2015 (41,931), approximately 3000
[95% CI 1700, 5200] Louisiana women with Medicaid
give birth per year instead of having an abortion because
Medicaid does not cover abortion. Discussion A previous systematic review estimated that about one-
fourth of Medicaid-eligible pregnant women give birth Roberts et al. Discussion BMC Women's Health (2019) 19:78 Page 6 of 8 Table 2 Validity checks
Total
Medicaid population
Medicaid as a reason (Medicaid population denominator)
P-value
among
Medicaid
population
N
265
167
12
155
Yes
No
Pregnancy intentions (lmup)
< 0.001
Unplanned
12% (31)
15% (24)
58% (7)
11% (17)
Ambivalent
64% (168)
63% (105)
33% (4)
66% (101)
Planned
25% (65)
22% (37)
8% (1)
23% (36)
Decisional certainty
0.001
High certainty
77% (196)
78% (129)
33% (4)
82% (125)
Medium certainty
19% (48)
16% (26)
50% (6)
13% (20)
Low certainty
5% (12)
6% (10)
17% (2)
5% (8)
Preferred abortion at pregnancy discovery
< 0.001
No
86% (191)
84% (141)
8% (1)
90% (140)
Yes
14% (74)
16% (26)
92% (11)
10% (15)
Prefer abortion now
0.03
No
98% (228)
98% (162)
83% (10)
99% (152)
Yes
2% (37)
2% (4)
17% (2)
1% (2) instead of having an abortion due to Medicaid not cov-
ering abortion [9]. Using a different methodological ap-
proach,
we
arrived
at
an
estimate
for
Louisiana
substantially similar to the overall estimate from a
decade-old systematic literature review [9]. The earlier
estimate was based on literature published primarily the
1980s through early 2000s and which used primarily
econometric methods and included multiple studies
across multiple geographies [9]. This suggests pregnant
women may accurately report both about considering
abortion and Medicaid as a barrier to abortion care; this is consistent with research findings that women’s im-
pressions of abortion costs and Medicaid coverage for
abortion are generally accurate [15]. instead of having an abortion due to Medicaid not cov-
ering abortion [9]. Using a different methodological ap-
proach,
we
arrived
at
an
estimate
for
Louisiana
substantially similar to the overall estimate from a
decade-old systematic literature review [9]. The earlier
estimate was based on literature published primarily the
1980s through early 2000s and which used primarily
econometric methods and included multiple studies
across multiple geographies [9]. This suggests pregnant
women may accurately report both about considering
abortion and Medicaid as a barrier to abortion care; this Recent research has paid considerable attention to
how laws that seek to dissuade women from having
abortions (such as waiting period and ultrasound laws)
affect women’s ability to obtain and experiences obtain-
ing abortions [29, 30]. Discussion BMC Women's Health (2019) 19:78 Page 5 of 8 Table 1 Sample characteristics and characteristics associated with reporting Medicaid as a
Total
% (N)
Medicaid population
% (N)
Medicaid as a reason (Med
% (N)
Yes
N
265
167
12
Age
Age (mean)
27
27
26
Race/ethnicity
White
8% (22)
11% (19)
8% (1)
Black
72% (190)
74% (124)
75% (9)
Hispanic/Latina
15% (39)
10% (16)
8% (1)
Other/Multi
5% (14)
5% (8)
8% (1)
Parity
0
35% (94)
33% (55)
17% (2)
1
26% (68)
25% (41)
33% (4)
2 or more
39% (103)
42% (71)
50% (6)
Education
Less than HS
25% (65)
23% (39)
42% (5)
HS or GED
48% (127)
48% (80)
58% (7)
Some or completed College
28% (73)
29% (48)
0% (0)
Currently employed
No
52% (137)
55% (91)
75% (9)
Yes
48% (126)
45% (75)
25% (3)
Public assistance
No
35% (92)
26% (44)
33% (4)
Yes
65% (169)
74% (123)
67% (8)
Food insecure
No
50% (133)
52% (87)
25% (3)
Yes
50% (131)
48% (80)
75% (9)
Housing insecure
No
67% (176)
68% (114)
50% (6)
Yes
33% (88)
32% (53)
50% (6)
Relationship
Husband/fiancé
29% (76)
26% (44)
0% (0)
Boyfriend/partner
51% (135)
53% (88)
42% (5)
Ex/friend/none/don’t know
20% (53)
21% (35)
58% (7)
Alcohol use disorder risk
No
76% (200)
75% (125)
25% (3)
Yes
24% (64)
25% (42)
75% (9)
Drug use
No
84% (219)
82% (137)
58% (7)
Yes
16% (43)
18% (30)
42% (5)
Tobacco use
No
72% (188)
69% (115)
33% (4)
Yes
28% (74)
31% (51)
67% (8) Table 1 Sample characteristics and characteristics associated with reporting Medicaid as a reason for not having an abortion
Total
% (N)
Medicaid population
% (N)
Medicaid as a reason (Medicaid population denominator)
% (N)
P-value
among
Medicaid
population
Yes
No
N
265
167
12
155
Age
0.50
Age (mean)
27
27
26
27
Race/ethnicity
0.87
White
8% (22)
11% (19)
8% (1)
12% (18)
Black
72% (190)
74% (124)
75% (9)
74% (115)
Hispanic/Latina
15% (39)
10% (16)
8% (1)
10% (15)
Other/Multi
5% (14)
5% (8)
8% (1)
4% (7)
Parity
0.51
0
35% (94)
33% (55)
17% (2)
34% (53)
1
26% (68)
25% (41)
33% (4)
24% (37)
2 or more
39% (103)
42% (71)
50% (6)
42% (65)
Education
0.03
Less than HS
25% (65)
23% (39)
42% (5)
22% (34)
HS or GED
48% (127)
48% (80)
58% (7)
47% (73)
Some or completed College
28% (73)
29% (48)
0% (0)
31% (48)
Currently employed
0.23
No
52% (137)
55% (91)
75% (9)
53% (82)
Yes
48% (126)
45% (75)
25% (3)
47% (72)
Public assistance
0.52
No
35% (92)
26% (44)
33% (4)
26% (40)
Yes
65% (169)
74% (123)
67% (8)
74% (115)
Food insecure
0.07
No
50% (133)
52% (87)
25% (3)
54% (84)
Yes
50% (131)
48% (80)
75% (9)
46% (71)
Housing insecure
0.20
No
67% (176)
68% (114)
50% (6)
70% (108)
Yes
33% (88)
32% (53)
50% (6)
30% (47)
Relationship
0.003
Husband/fiancé
29% (76)
26% (44)
0% (0)
28% (44)
Boyfriend/partner
51% (135)
53% (88)
42% (5)
54% (83)
Ex/friend/none/don’t know
20% (53)
21% (35)
58% (7)
18% (28)
Alcohol use disorder risk
< 0.001
No
76% (200)
75% (125)
25% (3)
79% (122)
Yes
24% (64)
25% (42)
75% (9)
21% (33)
Drug use
0.03
No
84% (219)
82% (137)
58% (7)
84% (130)
Yes
16% (43)
18% (30)
42% (5)
16% (25)
Tobacco use
0.01
No
72% (188)
69% (115)
33% (4)
71% (111)
Yes
28% (74)
31% (51)
67% (8)
28% (43) acteristics associated with reporting Medicaid as a reason for not having an abortion Roberts et al. Discussion This research has found that these
laws do little to change women’s minds, but do increase
financial costs, have emotional and social costs, and lead
to care delays [29]. While recent Medicaid funding Fig. 1 Estimated Pregnancy Outcomes in Louisiana Roberts et al. BMC Women's Health (2019) 19:78 Page 7 of 8 Page 7 of 8 an abortion. To check for possible underreporting from
self-report data, we performed validity checks using data
from open-ended responses and checking whether our
outcome was associated with expected predictor vari-
ables, pregnancy intentions, and decisional conflict. Third, the association between substance use and report-
ing Medicaid as a reason could be due to self-report bias
[33], with women more willing to report one also more
willing to report the other. However, pregnant women
who use alcohol and drugs face considerable barriers to
prenatal care [34]. They may face similar barriers to
abortion. Fourth, our estimates are likely imprecise. We
have a somewhat wide confidence interval for reporting
Medicaid as a reason for not having an abortion. How-
ever, our confidence interval overlaps with confidence
intervals from the decade old systematic review [9] esti-
mate, suggesting plausible accuracy. restrictions research has documented financial and emo-
tional hardships associated with having to raise money
to pay for abortion [7, 12, 14, 15], it has not focused on
Medicaid restrictions as a barrier to obtaining an abor-
tion. This study confirms that Medicaid funding restric-
tions
for
abortion
continue
to
function
as
an
insurmountable barrier to obtaining an abortion, specif-
ically for women in Louisiana. We also note that women with Medicaid insurance
with alcohol use disorder risk, who used drugs, and who
used tobacco were more likely to report lack of Medicaid
coverage as a reason for not having an abortion. It is un-
clear whether this is due to being more likely to consider
abortion or more likely to have difficulty overcoming
funding barriers to obtaining abortions. Other research
indicates that being unable to obtain an abortion is un-
likely to contribute to sustained reduction in problem-
atic alcohol use or in drug or tobacco use during
pregnancy or the postpartum period [31]. This study also has strengths. First, we had high par-
ticipation (86%). Second, we used an innovative ap-
proach to derive an up-to-date estimate of the impact of
lack of Medicaid funding for abortion. Funding This study has limitations. First, this study was con-
ducted at three prenatal clinics in one region of one
state. Findings may not be generalizable to other states
with different demographics, different numbers of abor-
tion providers, different local abortion fund practices,
and different overall policy climate. Second, estimates
are based, in part, on self-report data about considering
abortion during pregnancy and reasons for not having This study was funded by the David and Lucile Packard Foundation (grant:
2016–64232) and an anonymous foundation. The sponsors had no
involvement in study design; in the collection, analysis and interpretation of
data; in the writing of the report; or in the decision to submit the article for
publication. Abbreviations
GED G
l Ed GED: General Education Diploma; HS: High School; LMUP: London Measure
of Unplanned Pregnancy Discussion This approach
yields a finding consistent with previous estimates, sug-
gesting the previous estimate is still valid. There are several assumptions in this analysis that
could affect the accuracy of these findings. However,
examining these assumptions does not indicate that the
estimate of the proportion of pregnancies among low-
income women that end in birth rather than abortion
when Medicaid does not pay is likely to be outside of
our 95% CI. Specifically, the main question asked
whether lack of Medicaid funding was a reason for hav-
ing an abortion, not the only reason. Some participants
who reported Medicaid as a reason also reported per-
sonal or interpersonal reasons for not having an abor-
tion. However, even if we restrict the Medicaid as a
reason proportion to those who reported a policy-
related reason in response to open-ended questions, the
estimate is 4.2%, within the 95% CI of our estimate. Similarly, three participants who reported Medicaid as a
reason were still considering abortion upon prenatal care
entry. Even if all three proceeded to have an abortion,
this would still be within the 95% CI of our estimate. The sample excludes women who did not receive pre-
natal care. Nationally, about 1.4% of women do not re-
ceive any prenatal care [32]. Even if all the women who
did not receive prenatal care would have had an abortion
had Medicaid paid, this would increase our estimate of
those who reported Medicaid as a reason to 8.6%, which
is still within the upper limit of our 95% CI for this
estimate. Conclusions Forty years after the Hyde Amendment was passed, lack
of Medicaid funding for abortion continues to have sub-
stantial impacts on women’s ability to obtain abortions
in Louisiana. Authors’ contributions
l
d h SCMR conceptualized the study design. VW provided clinical research
expertise and oversight of study implementation at local recruitment sites. SCMR led the data analysis and interpretation of data. NJ and EW assisted
with analysis and participated interpretation of the data. UDU participated in
interpretation of the data. SCMR drafted the manuscript. All authors read the
manuscript, provided critical feedback on the intellectual content of the
manuscript, and approved the final manuscript. Acknowledgements
h
h
h
k
l The authors thank Finley Baba, Elise Belusa, Anna Bernstein, Mattie Boehler-
Tatman, Ivette Gomez, Heather Gould, Jenny Holl, Rebecca Kriz, Heather Lip-
kovich, Nicole Nguyen, Brenly Rowland, Alison Swiatlo for research and pro-
ject assistance and the facilities in Louisiana their collaboration. References Jones RK, Upadhyay UD, Weitz TA. At what cost? Payment for abortion care
by U.S. women. Womens Health Issues. 2013;23(3):e173–8. 32. Osterman MJK, Martin JA: Timing and Adequacy of Prenatal Care in the
United States, 2016. National Vital Statistics Reports. vol. 67. Hyattsville, MD:
National Center for Health Statistics; 2018. 32. Osterman MJK, Martin JA: Timing and Adequacy of Prenatal Care in the
United States, 2016. National Vital Statistics Reports. vol. 67. Hyattsville, MD:
National Center for Health Statistics; 2018. 9. Henshaw SK, Joyce TJ, Dennis A, Finer LB, Blanchard K. Restrictions on
Medicaid funding for abortions: a literature review. New York: Guttmacher
Institute; 2009. 33. Macleod J, Hickman M, Smith GD. Reporting bias and self-reported drug
use. Addiction. 2005;100(4):562–3. 33. Macleod J, Hickman M, Smith GD. Reporting bias and self-reported drug
use. Addiction. 2005;100(4):562–3. 10. Jarlenski M, Hutcheon JA, Bodnar LM, Simhan HN. State Medicaid coverage
of medically necessary abortions and severe maternal morbidity and
maternal mortality. Obstet Gynecol. 2017;129(5):786–94. 34. Roberts SC, Pies C. Complex calculations: how drug use during pregnancy
becomes a barrier to prenatal care. Mat Child Health J. 2011;15(3):333–41. 11. Krieger N, Gruskin S, Singh N, Kiang MV, Chen JT, Waterman PD, Beckfield J,
Coull BA. Reproductive justice & preventable deaths: state funding, family
planning, abortion, and infant mortality, US 1980-2010. SSM Popul Health. 2016;2:277–93. Availability of data and materials
h d
d
d/
l The datasets generated and/or analyzed during the current study are not
publicly available due to the terms of participant consent but are available
from the corresponding author on reasonable request. Page 8 of 8 Page 8 of 8 Roberts et al. BMC Women's Health (2019) 19:78 16. Guttmacher Institute. State policies in brief: targeted regulation of abortion
providers. 2019. https://www.guttmacher.org/statecenter/spibs/spib_TRAP. pdf . Accessed 6 June 2019. Author details
1 1Advancing New Standards in Reproductive Health (ANSIRH), Department of
Obstetrics, Gynecology, and Reproductive Sciences, University of California,
San Francisco, 1330 Broadway, Suite 1100, Oakland, CA 94612, USA. 2Department of Obstetrics and Gynecology, Louisiana State University School
of Medicine, 3700 St. Charles Avenue, 5th floor, New Orleans, LA 70115, USA. 3Present address: Center on Gender Equity and Health, University of
California, San Diego, 9500 Gilman Dr, La Jolla, CA 92093, USA. 21. Guttmacher institute: state facts about abortion: Louisiana. 2018. https://
www.guttmacher.org/fact-sheet/state-facts-about-abortion-louisiana . Accessed 6 June 2019. 21. Guttmacher institute: state facts about abortion: Louisiana. 2018. https://
www.guttmacher.org/fact-sheet/state-facts-about-abortion-louisiana . Accessed 6 June 2019. 22. Barrett G, Smith SC, Wellings K. Conceptualisation, development, and
evaluation of a measure of unplanned pregnancy. J Epidemiol Community
Health. 2004;58(5):426–33. 22. Barrett G, Smith SC, Wellings K. Conceptualisation, development, and
evaluation of a measure of unplanned pregnancy. J Epidemiol Community
Health. 2004;58(5):426–33. 23. O'Connor A. User manual -- decisional conflict scale (16 item statement
format). Ottawa: hospital research institute; 1993. 23. O'Connor A. User manual -- decisional conflict scale (16 item statem
format). Ottawa: hospital research institute; 1993. Received: 2 May 2019 Accepted: 31 May 2019 Received: 2 May 2019 Accepted: 31 May 2019 24. Bush K, Kivlahan DR, McDonell MB, Fihn SD, Bradley KA. The AUDIT alcohol
consumption questions (AUDIT-C): an effective brief screening test for
problem drinking. Ambulatory care quality improvement project (ACQUIP). Alcohol Use Disorders Identification Test. Arch Intern Med. 1998;158(16):
1789–95. Received: 2 May 2019 Accepted: 31 May Consent for publication
Not applicable. Consent for publication
Not applicable. 18. Jones RK, Kooistra K. Abortion incidence and access to services in the
United States, 2008. Perspect Sex Reprod Health. 2011;43(1):41–50. 18. Jones RK, Kooistra K. Abortion incidence and access to services in the
United States, 2008. Perspect Sex Reprod Health. 2011;43(1):41–50. 19. Towey S, Poggi S, Roth R. Abortion funding: a matter of justice. Report. Boston, MA: The National Network of Abortion Funds; 2005. Competing interests The authors declare that they have no competing interests. 20. Jones RK, Ingerick M, Jerman J. Differences in abortion service delivery in
hostile, middle-ground, and supportive states in 2014. Womens Health
Issues. 2018;28(3):212–8. References 1. Adashi EY, Occhiogrosso RH. The Hyde amendment at 40 years and
reproductive rights in the United States: perennial and panoptic. JAMA. 2017;317(15):1523–4. 1. Adashi EY, Occhiogrosso RH. The Hyde amendment at 40 years and
reproductive rights in the United States: perennial and panoptic. JAMA. 2017;317(15):1523–4. 25. Finer LB, Kost K. Unintended pregnancy rates at the state level. Perspect Sex
Reprod Health. 2011;43(2):78–87. 26. Correction and Republication. Abortion surveillance - United States, 2014. MMWR Morb Mortal Wkly Rep. 2018;67(46):1302. 26. Correction and Republication. Abortion surveillance
MMWR Morb Mortal Wkly Rep. 2018;67(46):1302. 2. Guttmacher Institute: State funding of abortion under Medicaid. 2018. https://www.guttmacher.org/state-policy/explore/state-funding-abortion-
under-medicaid. Accessed 6 June 2019. 2. Guttmacher Institute: State funding of abortion under Medicaid. 2018. https://www.guttmacher.org/state-policy/explore/state-funding-abortion-
under-medicaid. Accessed 6 June 2019. 27. Louisiana State Registrar and Vital Records. Birth Data. http://ldh.la.gov/
index.cfm/page/704 . Accessed 17 Apr 2019. index.cfm/page/704 . Accessed 17 Apr 2019. 3. Nash E, Gold RB, Mohammed L, Ansari-Thomas Z, Cappello O. Policy trends
in the states, vol. 2017. New York: Guttmacher Institute; 2018. 3. Nash E, Gold RB, Mohammed L, Ansari-Thomas Z, Cappello O. Policy trends
in the states, vol. 2017. New York: Guttmacher Institute; 2018. 28. Births Financed by Medicaid. https://www.kff.org/medicaid/state-indicator/
births-financed-by-medicaid/?currentTimeframe=0&selectedRows=
%7B%22states%22:%7B%22louisiana%22:%7B%7D%7D%7D&sortModel=
%7B%22colId%22:%22Location%22,%22sort%22:%22asc%22%7D. Accessed
17 Apr 2019. 4. Illinois governor agrees to allow Medicaid for abortions. https://www. apnews.com/e502aa90c6d24403bd5eafb7c467061a/Illinois-governor-agrees-
to-allow-Medicaid-for-abortions. Accessed 6 June 2019. 4. Illinois governor agrees to allow Medicaid for abortions. https://www. apnews.com/e502aa90c6d24403bd5eafb7c467061a/Illinois-governor-agrees-
to-allow-Medicaid-for-abortions. Accessed 6 June 2019. 5. Medicaid will now cover abortion for low-income women in Illinois. Take
That, Hyde! https://www.aclu.org/blog/reproductive-freedom/abortion/
medicaid-will-now-cover-abortion-low-income-women-illinois-take. Accessed 6 June 2019. 29. Roberts SCM, Turok DK, Belusa E, Combellick S, Upadhyay UD. Utah’s 72-
hour waiting period for abortion: experiences among a clinic-based sample
of women. Perspect Sex Reprod Health. 2016;48(4):179–87. 30. Upadhyay UD, Kimport K, Belusa EKO, Johns NE, Laube DW, Roberts SCM. Evaluating the impact of a mandatory pre-abortion ultrasound viewing law:
a mixed methods study. PLoS One. 2017;12(7):e0178871. 6. Jerman J, Jones RK, Onda T. Characteristics of U.S. abortion patients in 2014
and changes since 2008. New York: Guttmacher Institute; 2016. 7. Roberts SC, Gould H, Kimport K, Weitz TA, Foster DG. Out-of-pocket costs
and insurance coverage for abortion in the United States. Womens Health
Issues. 2014;24(2):e211–8. 31. Roberts SCM, Foster DG, Gould H, Biggs MA. Changes in alcohol, tobacco,
and other drug use over five years after receiving versus being denied a
pregnancy termination. J Stud Alcohol Drugs. 2018;79(2):293–301. 8. Ethics approval and consent to participate The study protocol was approved by the institutional review boards of the
University of California, San Francisco and Louisiana State University. Written
consent was obtained from all individual participants included in the study. 17. Roberts SCM, Kimport K, Kriz R, Holl J, Mark K, Williams V. Consideration of
and reasons for not obtaining abortion among women entering prenatal
care in southern Louisiana and Baltimore, Maryland. Sex Res Social Policy
2018. Epub ahead of print. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 12. Dennis A, Manski R, Blanchard K. A qualitative exploration of low-income
Women's experiences accessing abortion in Massachusetts. Womens Health
Issues. 2015;25(5):463–9. 13. Dennis A, Blanchard K. Abortion providers' experiences with Medicaid
abortion coverage policies: a qualitative multistate study. Health Serv Res. 2013;48(1):236–52. 14. Dennis A, Blanchard K, Cordova D. Strategies for securing funding for
abortion under the Hyde amendment: a multistate study of abortion
providers' experiences managing Medicaid. Am J Public Health. 2011;
101(11):2124–9. 15. Dennis A, Manski R, Blanchard K. Does medicaid coverage matter?: a
qualitative multi-state study of abortion affordability for low-income
women. J Health Care Poor Underserved. 2014;25(4):1571–85.
|
https://openalex.org/W4390545486
|
https://link.springer.com/content/pdf/10.1007/978-981-99-8405-3_23.pdf
|
English
| null |
Modeling on Outdoor Thermal Comfort in Traditional Residential Neighborhoods in Beijing Based on GAN
| null | 2,024
|
cc-by
| 4,023
|
© The Author(s) 2024
C. Yan et al. (Eds.): CDRF 2023, Phygital Intelligence, pp. 273–283, 2024.
https://doi.org/10.1007/978-981-99-8405-3_23 Modeling on Outdoor Thermal Comfort
in Traditional Residential Neighborhoods
in Beijing Based on GAN Pixin Gong, Xiaoran Huang(B), Chenyu Huang, and Shiliang Wang School of Architecture and Art, North China University of Technology, Beijing 100144, China
xiaoran.huang@ncut.edu.cn School of Architecture and Art, North China University of Technology, Beijing 100144, China
xiaoran.huang@ncut.edu.cn Abstract. With the support of new urban science and technology, the bottom-up
and human-centered space quality research has become the key to delicacy urban
governance, of which the Universal Thermal Climate Index (UTCI) have a severe
influence. However, in the studies of actual UTCI, datasets are mostly obtained
from on-site measurement data or simulation data, which is costly and ineffec-
tive. So, how to efficiently and rapidly conduct a large-scale and fine-grained
outdoor environmental comfort evaluation based on the outdoor environment is
the problem to be solved in this study. Compared to the conventional qualitative
analysis methods, the rapidly developing algorithm-supported data acquisition and
machine learning modelling are more efficient and accurate. Goodfellow proposed
Generative Adversarial Nets (GANs) in 2014, which can successfully be applied
to image generation with insufficient training data. In this paper, we propose an
approach based on a generative adversarial network (GAN) to predict UTCI in
traditional blocks. 36000 data samples were obtained from the simulations, to
train a pix2pix model based on the TensorFlow framework. After more than 300
thousand iterations, the model gradually converges, where the loss of the function
gradually decreases with the increase of the number of iterations. Overall, the
model has been able to understand the overall semantic information behind the
UTCI graphs to a high degree. Study in this paper deeply integrates the method of
data augmentation based on GAN and machine learning modeling, which can be
integrated into the workflow of detailed urban design and sustainable construction
in the future. Keywords: UTCI · Machine learning · GAN · Data augmentation Keywords: UTCI · Machine learning · GAN · Data augmentation 1.1
Background Currently, most urban dwellers suffered from the urban heatwave events, including both
systematic changes in climate such as warmer summers, and severity of extreme events
such as heat waves [1]. A considerable number of studies have shown that the number of
summer heat stress days suffered by Chinese urban residents has increased year by year P. Gong et al. 274 in the past half century, which has caused significant urban health problems, especially
for the life and health of the elderly. At the same time, research shows that the high
temperature in the city even has a significant negative impact on the baby birth rate and
the pregnancy safety of women [2]. It is estimated that there are about 24966 deaths
related to heatwave in 2021, according to report of the Lancet [3]. Besides, heat-related
labor loss, indirectly resulted in a loss of 1.68% of gross domestic product (GDP) in
2021 [3]. Inthefaceofthegrowingurbanheatproblem,thenumberofgovernmentpublications
has risen each year in recent years. In 2021 alone, the number of papers related to climate
health has increased by 3.7 times compared to the average annual number of papers
issued in the past decades [3]. Academia has also paid close attention to the issue of
urban climate comfort, especially the study of outdoor thermal comfort for hot summer
climates. Over the past century, various models and metrics for thermal environment
evaluation have been proposed for the study of urban environment as well as thermal
comfort. Among them, the UTCI model, based on the human heat exchange mechanism
and combined with the dressing model, integrates a variety of climate elements such as
temperature, humidity and wind speed, and has the characteristics of multi-scale, multi-
area and multi-climate generalization, thus becoming the mainstream evaluation index
of outdoor thermal comfort today. However, outdoor thermal comfort at the human scale has long been neglected in
urban construction [1], especially in traditional neighborhood spaces, which is difficult to
consider at the beginning of design. However, Urban neighborhood spaces are essential
for residents by providing spaces for daily activities, of which the Universal Thermal
Climate Index (UTCI) has an influence on space quality, where positive physical thermal
comfort leads to more lingering and interactive activities, promoting healthy travel and
improving quality of life [6]. 1.1
Background Therefore, in this context, how to efficiently and rapidly conduct a large-scale and
fine-grained outdoor environmental comfort evaluation based on the outdoor environ-
ment of urban traditional neighborhood spaces is the problem to be solved in this
study. 1.3
Research Gaps Although the current researches on outdoor thermal comfort are extensive, there are
corresponding knowledge gaps, mainly as follows: 1. Data sparsity problems are common: data sparsity problems caused by the limitation
of the number of weather stations, cannot highlight spatial climate characteristics
at the meso-micro scale; most studies use mid-term reanalysis data from climate
websites, with data accuracy and confidence are difficult to ensure; 2. The calculation of outdoor thermal comfort relying on numerical simulation is ineffi-
cient, for it takes a long time to run the model to calculate the equivalent temperature
of UTCI, which makes it difficult to carry out the evaluation of UTCI at both urban
scale and human scale. In the study of actual UTCI, data are mostly obtained from
on-site measurement data or simulation data, which is costly and ineffective; 3. The relationship between outdoor thermal comfort and built environment elements
in microclimate environments is relatively underexplored, and some of the relevant
findings are valid only for the sample areas; 4. The small size sample-based measurement modeling lacks diversity, and the study
findings are difficult to be applied on a larger scale. Because location-specific predic-
tions, rather than probabilistic predictions of entire urban fields, limits its operational
utility and usefulness [7]. 1.2
Research Overview on UTCI in Outdoor Environment In general, research on urban-level outdoor UTCI is still in its initial stage in China,
focusing mostly on macroscopic urban space, with relatively little research on micro-
scopic human scale. The current research on thermal environmental comfort in urban
space can be summarized from three aspects: research themes, research methods, and
experimental means. 1. In terms of research topics, the main areas are as follows: researches on the spatial
distribution and temporal trends of UTCI; evaluation of the applicability of UTCI and
try to make corrections on this indicator; researches on performance-driven design
with UTCI as the goal; researches on the influencing factors related to outdoor thermal
climate comfort; 2. In terms of research methods, there are: descriptive statistics of computational results
based on statistics; spatial distribution patterns and temporal trends based on GIS;
modeling studies based on machine learning [6]. 2. In terms of research methods, there are: descriptive statistics of computational results
based on statistics; spatial distribution patterns and temporal trends based on GIS;
modeling studies based on machine learning [6]. Modeling on Outdoor Thermal Comfort 275 3. In terms of experimental methods, there are three main types of research: building
a laboratory for human perception research [4], which allows for small samples of
experimental research, more accurate and easier to control variables, but limited by the
data samples and data sources; on-site measurement to collect climate environmental
data, including temperature, humidity, wind speed, etc., and then calculate the UTCI
values with the help of software, where this model usually limited to expensive cost
such as time and money; the use of simulation software to simulate the virtual model
of the site numerically, and then validated in the field, which is limited by the length
of time consuming on software simulation. 1.4
Research Framework Compared to the conventional qualitative urban morphology analysis methods, the
rapidly developing algorithm-supported data acquisition and machine learning mod-
elling are more efficient and accurate, easing the problems of under-representation and
interference by episodic factors in traditional research methods, and better model tra-
ditionally difficult non-linear phenomena [7]. However, machine learning models with
superior generalization performance need sufficient data samples for training, in order
to get more accurate prediction results. For dealing with the above problems, we try to train a GAN model to replace
numerical simulations, and related studies show that using GAN instead of numeri-
cal simulations for UTCI can improve the speedup by 120–240 times [8]. We propose
a Grasshopper-based workflow (Fig. 1), combined with data simulation, augmentation
and estimation. 276 P. Gong et al. Fig. 1. Workflow of this study Fig. 1. Workflow of this study Specifically, we build a classical city model based on authoritative mapping data
in Rhino/Grasshopper platform, and use Ladybug and Eddy 3D plug-ins to perform
human-scale Micro-environment climate simulation; then Ladybug tools was used to
calculate and generate UTCI images. Finally, based on the deep learning framework, we
train a GAN model for future overall UTCI mapping of the city. The remainder of this paper is organized as follows: Sect. 2 is a literature review of
related researches, including the definition of UTCI and its application and researches
about GAN. Section 3 illustrates the methods in this project about how to prepare the
datasetrequiredandhowtotraintheGANmodel.Section4describestheGANprediction
results and make discussions. Section 5 summarizes the main aspects of this article and
proposes the possible application of the proposed model as well as the limitations and
expectations. 2.1
The Definition of UTCI and Its Application The Universal Thermal Climate Index (UTCI), based on the multi-node dynamic thermo-
physiological UTCI-Fialamodel [9], is usedtopredict humantemperatureandregulatory
responses for combinations of the prevailing outdoor climate conditions. The UTCI
is defined as the air temperature of the reference condition causing the same model
response as actual conditions [10], which provides a human-based representation of the
environment temperature, covering the whole climate range from heat to cold [11]. Compared with the physical temperature information, UTCI can more accurately
distinguish the degree of human body’s perception of cold and hot discomfort, which
was widely used to be applied in tourism, urban planning, construction, etc., in different
scales and climate zones [12, 13]. With the deepening of researches, some studies have
been carried out in recent years on the regional applicability of UTCI [14, 15]. At the 277 Modeling on Outdoor Thermal Comfort same time, researches on UTCI-related impact factors are also emerging. In general,
these impact factors include climate factors, urban traffic factors, urban development
intensity factors, micro-environmental landscape factors [18], etc. 2.2
Researches About GAN Generative Adversarial Network (GAN), proposed by Goodfellow in 2014 [18], has
rapidly created a research boom in the field of deep learning and image generation, and
has been applied in various research areas. Based on this, various variants have been
developed since then, such as DCGAN, WGAN, StyleGAN, etc. The GAN trains a
generator network and a discriminator, where goal of the generator is to map a random
vector to a realistic image, whereas the goal of the discriminator is to distinguish the
generated and the real images [19]. Duetotheadvantagesofallowingfastnumericalgenerationbyimagetransformation,
GAN is applied in more studies, such as residential floor plan generation, building
layout generation, garden layout generation, NDVI/NDRE prediction [20], precipitation
nowcasting [7] and so on. Among them, the Digital Futures Workshop led by YAO et al. has explored GAN with generative urban design in numerous ways, and found that GAN
has good applications in alternative environmental performance models [8]. 3.3
GAN-Based Image Generation GANs were used to predict the outdoor environment comfort with full information, with
learning global features instead of the detailed features of each object [8]. Based on the
Tensorflow framework, we train a pix2pix adversarial network model for fast prediction
of UTCI values, which can effectively reduce the time of environmental performance
simulation. Pix2pix, one of the GAN models, conduct image-to-image translation with
paired training data. Finally, we perform data enhancement on the dataset, and images are panned and
cut in four directions to achieve an 8-fold data enhancement, resulting in 36,000 data
samples. The pre-trained model is then invoked to train the pix2pix generative adver-
sarial network model, based on the TensorFlow framework. We divided the data set into
training, test and validation set, in the ratio of 7:1.5:1.5, where the model was trained
on the training set, and the robustness of the validation set and the model performance
of the test set were evaluated. 3.1
Model Generation Based on Rhino/Grasshopper Platform Rhino/Grasshopper, a parametric modeling platform, is the main modeling tool used
by architects nowadays, which can effectively perform rapid model generation. GAN
training requires a large amount of data, but the building of refined urban models is
usually a complex process. At the same time, there was a problem of different scales
in the collection of previous datasets, as the actual scales reflected by the input two-
dimensional images were uneven, resulting in inaccurate model predictions. In Huang’s
study [8], they proposed a fine method of “Prototype summary-Type derivation”, to
obtain a large number of city models analogous to the study area in a short period of
time. However, the traditional numerical simulation of datasets involves simulating the
environment of independent plots in a wind box, neglecting the correlation between the
selected area and the surrounding environment. Therefore, unlike Huang’s study [8], we take into account the realistic characteristics
and associative features of urban scenes. So, we choose 35 typical tracts (250 m * 250 m)
for modeling based on authoritative mapping data, and each tract satisfies the diverse
characteristics of building layout forms. Our research area is the traditional historic
district within the second ring road of Beijing, where we focus on this area for two
reasons: on one hand, the study of the historic district, with its complex morphology, is
relatively less studied on UTCI; and the outdoor thermal comfort of the historic district
can influence the pedestrian spatial experience and promote the vitality of the historic
district. P. Gong et al. 278 3.2
Simulation and Calculation of UTCI Based on Ladybug Tools Ladybug software package, a collection of tools for environmental performance simula-
tion on the Rhino/grasshopper platform, allows for the simulation of wind, light, heat and
other climate parameters, in which outdoor comfort was evaluated using microclimatic
and energy modelling with OpenFOAM and EnergyPlus, respectively. In this project,
based on each simulation parameter, the final UTCI values were calculated using the
Ladybug software to generate 35 slices of overall UTCI images. Referring to the prin-
ciple of convolutional neural network, the UTCI images of the 35 whole city slices are
then segmented using different sizes of convolutional kernels with different step sizes to
ensure that the image dataset can satisfy the characteristics of multi-scale and front-back
connectivity. Finally, we obtain 4500 paired picture datasets. 4
Results and Discussion In this study, the information in the Fig. 2 shows that the training process of the pix2pix
model gradually converges with the increase of the number of training iterations, and the
mutual game process between the discriminator and the generator in the model training
process can be seen from it. The generator loss increases slightly in the initial stage,
and between 280 and 600 K iterations, the generator loss fluctuates up and down around
0.308, but increases after thousand iterations. The loss of the discriminator function
gradually decreases with the increase of the number of iterations, and the model gradually
converges after about 280 thousand iterations, with loss of the discriminator as around
0.3, but decreases after 400 thousand iterations. From the whole process, the model
began to converge when the model iterated to 280 K, and after 600 K generation, the
model appeared overfitting. From this, it can be seen that setting the iteration number
to 300 thousand generations is more appropriate, so resetting the total number to 400 K
generations for model training. The entire training process uses RTX3090 GPU, and the
training process takes about 12.5 h. Modeling on Outdoor Thermal Comfort 279 Fig. 2. Training loss curves of generator and discriminator Fig. 2. Training loss curves of generator and discriminator Figure3showstheoutputresultsofthemodelonthetestset,fromwhichitcanbeseen
that the predicted images can almost meet the performance and fineness requirements
of the project, and the GAN model has good results in grasping the relationship and
structural pulse of the building layout and UTCI as a whole. The model has excellent
prediction performance for the layout of the enclosed building compound in the selected
area, especially for the UTCI prediction of the north side of the building and the larger
building courtyard. However, the prediction ability needs to be improved for the highly
dense and overly complex building layout scenarios. In order to further compare the effectiveness of the pix2pix model, we trained the
cycleGAN model using the same dataset. The deployment method of the model dataset
was the same as above, and a total of 20.4 h was spent with the using of RTX3090 GPU. The model eventually converged after 100 epochs, and model prediction results on the
test set are shown in Fig. 4. Overall, compared to pix2pix model, there is a certain gap in
details, which also proves that strict image-to-image transformation method of pix2pix
has better performance. 5
Conclusions With the support of new urban science and technology, the bottom-up and human-
centered street quality research has become the key to delicacy urban governance. In
this paper, we propose an approach based on a generative adversarial network (GAN)
to predict UTCI in traditional blocks. 36000 data samples were obtained from the sim-
ulations, to train a pix2pix based on the TensorFlow framework. After more than 300
thousand iterations, the model gradually converges, where the loss of the function grad-
ually decreases with the increase of the number of iterations. We can clearly see that
the pix2pix model has a high grasp of the relationship between the architectural form
of historical ancient city blocks and UTCI. With the help of this model, we can quickly
predict the fine-scale UTCI at the urban block scale in Beijing. Based on this, on the
one hand, we can identify the overheated and uncomfortable areas in the ancient city
in the historical ancient city and formulate more accurate policies. On the other hand,
data mining can be used to explore the relationship with other urban factors. Overall, the
model, learning from data to depict non-linear relationships between input parameters
and output metrics, has been able to understand the overall semantic information behind
the UTCI graphs to a high. Compared to other studies on the use of GAN in built environments, the contribution
of this article lies in: firstly, the processing of the dataset used for training first involves
modeling historical ancient cities based on official data; secondly, each sample data is
captured from fragments on a large-scale simulation result image, so each sample takes
into account the influence of the surrounding environment; thirdly, the data interception
method uses simple segmentation and sliding window interception to ensure the conti-
nuity of the dataset; to ensure the consistency of input data, each image proxy a 50 m
* 50 m block. Therefore, the model built in this study is limited to predicting at a 50 m
scale to ensure persuasiveness. Of course, the drawback of this study is also very obvious: the model training process
lacks the necessary correction mechanism, and there is still a risk of overfitting the model
without actually collecting data for testing. This is also the focus of our next research,
including the control process of model training, the improvement of data types, and
model correction based on actual data. 4
Results and Discussion Overall, the pix2pix model has been able to understand the overall semantic infor-
mation behind the UTCI graphs to a high degree. Although this study is limited by time
and computing power, and no more iterations are set, the model converges well so far,
while reducing the over-convergence of the model caused by over-training. 280 P. Gong et al. Fig. 3. Examples of model performance on test set Fig. 3. Examples of model performance on test set Fig. 4. Examples of cycleGAN model performance on test set Fig. 4. Examples of cycleGAN model performance on test set 281 Modeling on Outdoor Thermal Comfort 5
Conclusions Moreover, if the computing power allows, we
can check whether there are fluctuations in the convergence of the model under more
training iterations. The key to future research lies in model evaluation. In addition to the Ineption
score/FID (Fréchet Inception Distance), the next step is to construct a scale that can be
easily understood by subjective experience. 1. Naboni, E., Natanian, J., Brizzi, G.: A digital workflow to quantify regenerative urban design
in the context of a changing climate. Renew. Sustain. Energy Rev. 113, 109255 (2019). https://
doi.org/10.1016/j.rser.2019.109255 2. Khodadadi, N., Dastoorpoor, M., Khanjani, N.: Universal thermal climate index (UTCI) and
adverse pregnancy outcomes in Ahvaz, Iran. Reprod. Health 19(1), 33 (2022). https://doi.org/
10.1186/s12978-022-01344-7 References P. Gong et al. 282 3. Cai, W., Zhang, C., Zhang, S.: The 2022 China report of the Lancet Countdown on health and
climate change: leveraging climate actions for healthy ageing. Lancet Public Health 7(12),
e1073–e1090 (2022). https://doi.org/10.1016/S2468-2667(22)00224-9 p
g
4. Liu, K., Nie, T., Liu, W.: A machine learning approach to predict outdoor thermal comfort
using local skin temperatures. Sustain. Cities Soc. 59, 102216 (2020). https://doi.org/10.1016/
j.scs.2020.102216 5. Shah,R.,Pandit,R.K.,Gaur,M.K.:Urbanphysicsandoutdoorthermalcomfortforsustainable
street canyons using ANN models for composite climate. Alex. Eng. J. 61(12), 10871–10896
(2022). https://doi.org/10.1016/j.aej.2022.04.024 6. Ravuri, S., Lenc, K., Willson, M.: Skilful precipitation nowcasting using deep generative
models of radar. Nature 597(7878), 672–677 (2021). https://doi.org/10.1038/s41586-021-
03854-z 7. Huang, C., Zhang, G., Yao, J.: Accelerated environmental performance-driven urban design
with generative adversarial network. Build. Environ. 224, 109575 (2022). https://doi.org/10. 1016/j.buildenv.2022.109575 8. Fiala, D., Havenith, G., Bröde, P.: UTCI-Fiala multi-node model of human heat transfer and
temperature regulation. Int. J. Biometeorol. 56(3), 429–441 (2012). https://doi.org/10.1007/
s00484-011-0424-7 9. Bła˙zejczyk, K., Jendritzky, G., Bröde, P.: An introduction to the universal thermal clim
index (UTCI). Geogr. Pol. 86(1), 5 (2013). https://doi.org/10.7163/GPol.2013.1 0. Havenith, G., Fiala, D., Błazejczyk, K.: The UTCI-clothing model. Int. J. Biometeorol. 56(3)
461–470 (2012). https://doi.org/10.1007/s00484-011-0451-4 11. Blazejczyk, K., Epstein, Y., Jendritzky, G.: Comparison of UTCI to selected thermal indices. Int. J. Biometeorol. 56(3), 515–535 (2012). https://doi.org/10.1007/s00484-011-0453-2 12. Jendritzky, G., de Dear, R., Havenith, G.: UTCI—Why another thermal index? Int. J. Biometeorol. 56(3), 421–428 (2012). https://doi.org/10.1007/s00484-011-0513-7 13. Zhang, S., Zhang, X., Niu, D.: Physiological equivalent temperature-based and universal ther-
mal climate index-based adaptive-rational outdoor thermal comfort models. Build. Environ. 228, 109900 (2023). https://doi.org/10.1016/j.buildenv.2022.109900 p
g
j
14. Exploration of applicability of UTCI and thermally comfortable sun and wind conditions
outdoors in a subtropical city of Hong Kong. Sustain. Cities Soc. 52, 101793. https://doi.org/
10.1016/j.scs.2019.101793 15. Liu, Y., Ma, H., Zhang, C.: Watering on porous pavement for improvement of environmental
human thermal comfort in an ecological community in arid area: a case study in Lanzhou,
China. Sustain. Cities Soc. 85, 104081 (2022). https://doi.org/10.1016/j.scs.2022.104081 16. Goodfellow,I.J.,Pouget-Abadie,J.,Mirza,M.:Generativeadversarialnetworks(2020). http://
arxiv.org/abs/1406.2661 17. Guo, X., Wang, Z., Yang, Q.: GAN-Based virtual-to-real image translation for urban scene
semantic segmentation. Neurocomputing 394, 127–135 (2020). https://doi.org/10.1016/j.neu
com.2019.01.115 18. Davidson, C., Jaganathan, V., Sivakumar, A.N.: NDVI/NDRE prediction from standard RGB
aerial imagery using deep learning. Comput. Electron. Agric. 203, 107396 (2022). References https://
doi.org/10.1016/j.compag.2022.107396 283 Modeling on Outdoor Thermal Comfort Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), 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. Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), 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 chapter are included in the chapter’s Creative
Commons license, unless indicated otherwise in a credit line to the material. If material is not
included in the chapter’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.
|
https://openalex.org/W2948121021
|
https://journals.assaf.org.za/index.php/tvl/article/download/6269/7614
|
English
| null |
The ghostly matter of asylum in Kivu Ruhorahoza’s A Tree Has Fallen (Europa)
|
Tydskrif vir letterkunde
| 2,019
|
cc-by-sa
| 8,058
|
The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) This paper applies Avery Gordon’s insights in Ghostly Matters to Kivu Ruhorahoza’s 2019 film, A Tree Has Fallen, and vice-versa. For Gordon, the ghost reveals visibility itself to be “a complex system of permissions and prohibitions.” The ghost is a case, as
Gordon puts it, of “visible invisibility,” of seeing that one is not there. In Ruhorahoza’s film, the protagonist, Simon, is an African
asylum seeker in the UK, now a ghost. Even before he becomes ghostly matter, Simon is already ghostly: he is held in limbo,
consistently denied, deemed threatening, highly visible yet rendered invisible, a figure whose claims to a past are deemed invalid
in official channels. For Gordon, the ghost is a liminal presence, “what appears dead, but is nevertheless powerfully alive.” In
Ruhorahoza’s film, the protagonist appears to be alive, but is nevertheless powerfully dead. Gordon notes the refusal of modern
social scientists to acknowledge, or to speak to ghosts: what happens when British subjects speak to its African ghosts, and vice
versa? This paper investigates what the ghostly relations in the film suggest about political subjectivity, visibility, and the politics
of asylum. Potentially, the essay offers a reading of what may no longer be visible in Ruhorahoza’s film, as the essay was written
before Ruhorahoza edited A Tree Has Fallen, transformed it, and re-titled it Europa. Keywords: Kivu Ruhorahoza, Avery Gordon,
asylum, African cinema, Ghostly Matters. For Zygmunt Bauman, our present, liquid life instils fear of an uncertain expiration
date in an environment of ubiquitous disposability. The haunting fear in liquid
life, Bauman asserts, is that one will not adapt adequately enough to modernity’s
fluctuating demands, that one will be left behind—that one will “outstay [one’s]
welcome” (Bauman, Liquid Life 3). To narrate this liquid modernity, Bauman asserts,
is not to tell the story of constantly emerging beginnings, but rather to “tell the story
of successive endings” (Liquid Life 3). The ghost resists an ending. The story of a ghost begins with an expiration of life,
an expiration of breath, but it resists its expiration date, even if the ghost is a case,
as Avery Gordon (42) puts it in Ghostly Matters, of seeing that one is not there. MaryEllen Higgins MaryEllen Higgins
MaryEllen (Ellie) Higgins is Associate Professor of English at the Pennsylvania State University. Her books
include The Western in the Global South, coedited with Rita Kerestezi and Dayna Oscherwitz, and Hollywood’s
Africa After 1994. Email: mxh68@psu.edu
ORCID: https://orcid.org/0000-0002-2119-1630 MaryEllen Higgins
MaryEllen (Ellie) Higgins is Associate Professor of English at the Pennsylvania State University. Her books
include The Western in the Global South, coedited with Rita Kerestezi and Dayna Oscherwitz, and Hollywood’s
Africa After 1994. Email: mxh68@psu.edu
ORCID: https://orcid.org/0000-0002-2119-1630 MaryEllen Higgins
MaryEllen (Ellie) Higgins is Associate Professor of English at the Pennsylvania State University. Her books
include The Western in the Global South, coedited with Rita Kerestezi and Dayna Oscherwitz, and Hollywood’s
Africa After 1994. Email: mxh68@psu.edu
ORCID: https://orcid.org/0000-0002-2119-1630 The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) For
Gordon, the ghost directs attention to, among other things, “what appears dead,
but is nevertheless powerfully alive.” As I watched Kivu Ruhorahoza’s 2018 A Tree
Has Fallen, a film about a ghost, I wondered what the filmmaker makes of that line. TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 24 Avery’s formula for the ghost—what appears dead, but is nevertheless powerfully
alive—takes on another dimension in Ruhorahoza’s film: its protagonist, Simon
Adefolake (Oris Erhuero), appears to be very much alive—he unlocks doors with his
keys, takes a shower, walks the streets, makes dinner, has sex with his lover, talks to
people who see, hear, and touch him—but he is nevertheless powerfully dead. The
British subjects whom Simon’s ghost haunts know that he is already dead; they are
aware that he is a ghost—the task of the film’s British subjects is then to speak with
the African ghost, to piece together the meanings of his hauntings. A Tree Has Fallen is a film, in the director’s parlance, about dead black men who
refuse to leave.1 Simon is a ghost who haunts politely, a ghost who is meant to be to
invisible, but who dares to reveal himself and who refuses to play dead. He evokes
Gordon’s interpretation of haunting, which occurs “when things are not in their as
signed places, when the cracks and rigging are exposed, when the people who are
meant to be invisible show up without any sign of leaving” (xvi). Yet even before he
becomes ghostly matter, even before he inhabits the ghost’s body, Simon is already
ghostly: he has barely survived civil conflict; he has lost his entire family in a mas
sacre; he seeks asylum in the UK. He is treated, as Jonathan Darling (74) notes of
asylum seekers, as a “liminal presenc[e] within the nation.” The asylum seeker is held
in limbo by the home office, a figure held precipitously at the edge of devastation,
facing oppression in the home country if deported. Simon is subject to the sovereign’s
expiration date, to the space of “failed” asylum. His claims are consistently denied,
and his appeal drags on for ten years. He is ultimately rejected, and is classified as
an illegal being. The zone of asylum is a ghostly zone. We see, in this film (and elsewhere), that
the recognition of the seeker’s political subjectivity in this zone is, to adapt Gordon,
not there. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) The language of asylum packages political subjectivity not as a given, but
as a thing given, a thing offered or denied by the sovereign who decides whether a
person’s fears and losses are valid, whether a person’s narratives are to be believed,
whether to send a person back into the realm of his or her oppressors. In Ruho
rahoza’s film, there is a flashback to Simon’s audition for the part of Shylock in a
play. He performs an improvisational scene set in an immigration appeals court
in which his character makes his final plea for asylum. He recites these lines: “The
home office have challenged my asylum claim. They have rejected all my appeals. They have expressed significant doubts about my story. Even my name, I’ve been
told, is possibly not mine. But your Honor, I am who I have always claimed to be.”
Simon then lifts his shirt to reveal the scars produced by three bullet wounds on his
stomach, and where each bullet entered his body—damaging his liver, perforating
his stomach, and nearly hitting his spine—suturing the performance to his own life. Simon is compelled, in a climate of suspicion and denial that circulates a “colonial-
style set of assumptions about applicants’ dishonesty,” as Frances Webber (40) puts TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 25 it, to assure his audience that his claim is not bogus: the wounds, he adds, “are not
self-inflicted.” Simon concludes with the lines, “Your honor, you can go ahead and
dismiss everything that I’ve said, or you can listen to little voice of humanity deep
inside of you, and give me back my life.” Political subjectivity and rights become
matters to be processed by the bureaucracy—cases that can be thrown out and
disposed, things subject to expiration dates. The “adjudication of asylum claims,”
Elizabeth Dauphinee writes, manifests itself in “the relationship of a technology to
its applications; it is the bits of paper that undergo judgment, and those that stand
behind those bits of paper are denied their own faces, their own voices, their own
skins. They appear before tribunals and are erased” (236); they are judged to be
“ineligible for life” (237). For Gordon, the ghost provides us with “fugitive knowledge;” the ghost reveals
what Foucault referred to as subjugated knowledge, or “what official knowledge
represses within its own terms, institutions, and archives” (Gordon xviii). The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) To express
subjugated knowledge is a performance of citizenship; following Jacques Rancière,
Engin F. Isin and Kim Rygiel argue that political subjectivity has always been enacted
by people who make claims to rights they do not currently possess. In this proposition,
despite being assigned to a zone that attempts to prevent him from acting as a politi
cal subject and citizen with economic, social, and political rights, Simon’s very act of
seeking rights is itself an act of political subjectivity and citizenship. Simon performs
what Darling (77) refers to, in his analysis of asylum in the UK, as the “part with no
part.” Simon’s protest against the subjugation of his narratives, of the suppression
of his past by a home office that qualifies and disqualifies at will, a home office that
can toss his claims and his rights in the dumpster, is an act of political subjectivity,
an act of playing a part. However, this action, this agency in the film, has the aura
of a ghostly performance, even if this scene of the audition in the film is a flashback
to a moment when Simon is still alive. Simon is reduced to a supplicant pleading in
a theatre for rights to be a part with a part, like an audition for existence. The theatricality of the asylum zone plays out in the streets. One of the early
figures to cross Simon’s path is a man who drapes himself in the flag of Saint George
(Dimitri Lambridis). The film visually notes the anti-immigrant, fortress identity
politics of Brexit in this scene. It recalls Stuart Hall’s description of a nation “haunted
by the fantasy of a late return to the flag” (18). The flag is the visual incarnation of a
discourse that makes particular claims to Britishness, and by extension, to resources
and rights that are assumed to belong exclusively to those with legitimate claims to
this version of Britishness. Simon gazes briefly at the flag-wrapped figure, who does
not make eye contact. He passes Simon by as if he is invisible, as if he is not there,
as if his ghost is not there. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) Yet Simon, and other postcolonial asylum seekers and
immigrants, are very much there; they are at the core of the discourse on what it
means to be British, of who is included and excluded, of who is welcome and where, TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 26 Figure 1: Man with flag of Saint George draped on his shoulders and who has, to return to Bauman, outstayed one’s welcome. While immigrants
from formerly colonized spaces are depicted as “strangers at our door” (Bauman,
Strangers at Our Door), they are not strangers to the histories of imperialism, to those
ghostly, unsettled, and unsettling histories in which the question of who is host
and who is guest, of who is foreign or strange, or who is allowed to make oneself at
home in the world takes on multiple, entangled dimensions.2 As Paul Gilroy (110)
writes, the postcolonial immigrant in melancholic Europe “is here because Britain,
Europe, was once out there.”3 And thus for Gilroy, the asylum seeker haunts, even
if unwittingly: “It is the infrahuman political body of the immigrant rather than the
body of the sovereign that comes to represent all the discomforting ambiguities of the
Empire’s painful and shameful but apparently nonetheless exhilarating history […]
the incomers may be unwanted and feared precisely because they are the unwitting
bearers of the imperial and colonial past” (110). When the flag-bearer crosses paths
with the ghost, it is not a moment of I see that you are not there, but a moment of “I
treat you as if you do not exist,” a moment of refusing to come to terms, as Gordon
(18) writes, the “with what modern history has rendered ghostly.” When Simon is alive, the zone of asylum is a zone of estrangement. To make a
living, Simon plays the role of the sexualizable black body available for consump
tion in a nightclub. His arms are bound and tied in glittery ribbons; his buttocks are
bare and strapped in. Through Simon’s voice-over, one learns that his family was
attacked and killed on a night when he was dancing. He remembers the night in a
flashback in which he performs an exotic dance as women drink, laugh, converse
with each other, and gaze at him in vertical lines. His gesticulating movements on
the floor are reminiscent of the opening scenes in Jean-Pierre Bekolo’s Les Saignantes. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 27 Yet this is not the “new anatomy” of Bekolo’s oeuvre (see Omelsky). The bound and
gyrating body is there, but it is not the decolonized, hyperbolized, and rebellious
sex-worker/bloodette thrusting her body in a harness above the patriarch in Les Saig
nantes (see Harrow). Rather, in Ruhorahoza’s rendering, Simon’s public dance evokes
an old, imprisoning mode of embodiment, one that simultaneously sexualizes and
binds the black body. It is an aestheticized evocation of a bound past to the beat of
techno-drumming, one that glosses over Simon’s bullet wounds, eclipses the traces
of his losses and injuries, and turns it into a performance choreographed for those
who pay to watch with pleasure. This scene is there not to aestheticize the bound
body, but to show how aestheticization is done, how the histories of Simon’s bullet
wounds and survivorhood are packaged in a spectacular ritual of pornographic,
effacing entertainment. Figure 2: Bound: dance club scene Fi
2 B
d d
l b Figure 2: Bound: dance club scene Yet this is not the “new anatomy” of Bekolo’s oeuvre (see Omelsky). The bound and
gyrating body is there, but it is not the decolonized, hyperbolized, and rebellious
sex-worker/bloodette thrusting her body in a harness above the patriarch in Les Saig
nantes (see Harrow). Rather, in Ruhorahoza’s rendering, Simon’s public dance evokes
an old, imprisoning mode of embodiment, one that simultaneously sexualizes and
binds the black body. It is an aestheticized evocation of a bound past to the beat of
techno-drumming, one that glosses over Simon’s bullet wounds, eclipses the traces
of his losses and injuries, and turns it into a performance choreographed for those
who pay to watch with pleasure. This scene is there not to aestheticize the bound
body, but to show how aestheticization is done, how the histories of Simon’s bullet
wounds and survivorhood are packaged in a spectacular ritual of pornographic,
effacing entertainment. Yet this is not the “new anatomy” of Bekolo’s oeuvre (see Omelsky). The bound and
gyrating body is there, but it is not the decolonized, hyperbolized, and rebellious
sex-worker/bloodette thrusting her body in a harness above the patriarch in Les Saig
nantes (see Harrow). Rather, in Ruhorahoza’s rendering, Simon’s public dance evokes
an old, imprisoning mode of embodiment, one that simultaneously sexualizes and
binds the black body. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) It is an aestheticized evocation of a bound past to the beat of
techno-drumming, one that glosses over Simon’s bullet wounds, eclipses the traces
of his losses and injuries, and turns it into a performance choreographed for those
who pay to watch with pleasure. This scene is there not to aestheticize the bound
body, but to show how aestheticization is done, how the histories of Simon’s bullet
wounds and survivorhood are packaged in a spectacular ritual of pornographic,
effacing entertainment. Let’s return, then, to Gordon’s (18) question: “How do we reckon with what
modern history has rendered ghostly?” Or, we might ask this question as Derrida
does before Gordon, in Spectres of Marx: how do we apostrophize the ghost, or
speak the language of the ghost? (12) In his ghostly form, Simon visits four people:
Brother Joshua, who preaches in the street (Paul Morris), Simon’s former landlady
Peggy (Jennie Lathan), his lover Anna (Lisa Moorish), and Bruce Warnock (Matt
Ray Brown), the co-director, with Anna, of the play for which Simon auditions. Bruce, who was once Anna’s lover, stalks Anna and Simon, even following Anna
to a poetry club after Simon’s death, where she reads a poem in Simon’s memory. Bruce spies on the ghost when he visits Anna; we see him gazing up to her apart
ment where they are having sex, and then lurking around a corner after they exit
her apartment. Bruce seems to represent those in liquid modernity who channel TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 28 fears of their own pending disposability on others: after the grant proposal for his
theatre project has been rejected by the Victoria Arts Fund, we see him kneel, and
what look like bits of shredded paper pour out from his mouth as the text of the
rejection letter scrolls on the screen. After the rejection, Bruce increasingly expresses
his disdain for “others.”
h
dd
h
f
h d
lk
b
Figure 3: Bruce’s surveillance Figure 3: Bruce’s surveillance Figure 3: Bruce’s surveillance fears of their own pending disposability on others: after the grant proposal for his
theatre project has been rejected by the Victoria Arts Fund, we see him kneel, and
what look like bits of shredded paper pour out from his mouth as the text of the
rejection letter scrolls on the screen. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) After the rejection, Bruce increasingly expresses
his disdain for “others.” fears of their own pending disposability on others: after the grant proposal for his
theatre project has been rejected by the Victoria Arts Fund, we see him kneel, and
what look like bits of shredded paper pour out from his mouth as the text of the
rejection letter scrolls on the screen. After the rejection, Bruce increasingly expresses
his disdain for “others.” fears of their own pending disposability on others: after the grant proposal for his
theatre project has been rejected by the Victoria Arts Fund, we see him kneel, and
what look like bits of shredded paper pour out from his mouth as the text of the
rejection letter scrolls on the screen. After the rejection, Bruce increasingly expresses
his disdain for “others.” When Bruce addresses the topic of migration, he does not talk about Brexit or
immigrants directly; instead, he talks about films. He dismisses Steven Frears’ Dirty
Pretty Things (2002), and particularly Nicolas Provost’s film L’envahisseur (2011) (The
Invader), as a waste of time. He tells Anna that he “hated that piece of shit” film,
The Invader (a film about the relations between a rich woman and an undocumented
African immigrant in Brussels). Bruce complains that the husband and female lead
represent “everything that is wrong with Europe:” it is “sluggish, undignified,
submissive.” In an implicit reference to the stories of undocumented immigrants in
Europe, he then asks how much of taxpayers’ money has been spent on “fucking
unbelievable,” “tear-jerking diversity stories.” As Bruce trivializes the plight of asylum
seekers and others, his characterization of Europe echoes Kerry Moore’s (349) analysis
of discourses of asylum in the UK that position the British state as “manipulated and
compromised, emasculated by the supposed ‘abuse’ of its immigration system and
its failure to deal with an ‘asylum crisis.’” Yet Bruce’s lament about what he sees as an emasculated Europe is less invested
in his status as a British national than it is in another insecure attachment: losing his
former lover Anna to Simon. The ghostly matter of asylum in Kivu
Ruhorahoza’s A Tree Has Fallen (Europa) Anna asks Bruce, “Why don’t you just be honest about
what it is that causes negativity and hostility toward everything that doesn’t look like
you?” The question remains open—Bruce may be displacing his frustration with his TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 29 insignificance in liquid modernity—but if neoliberalism posits the “free, possessive
individual” as its protagonist, as Stuart Hall (10) reminds us, Bruce is ultimately a
man clinging possessively to that which he cannot possess. Anna is not his—she
does not belong to anyone. Later in Ruhorahoza’s film, in his ghostly form, Simon
visits Bruce, who asks Simon what he wants from him, adding that the ghost has
some “nerve” showing up at the door. Simon assures Bruce that he wants nothing
from him, then speaks to him as if he is addressing the British sovereign about its
possessive compulsions. Simon tells him, You haven’t lost anything, Bruce. You are still the powerful one. And that is how it is
going to be for a very long, long time. All of this is yours. You are still the mighty one
[…] We are all just trying to survive in a world you created for us. You are still the
master of all of us—the master of dreams, beliefs, and narratives. If you think about
it, that is all that counts, right? Dreams, beliefs, and narratives. But enough of that. I
have come to forgive you […] for being a patriot, for reporting an immigration crime,
anonymously. Bruce then orders him: “Get out of my house.” Bruce then orders him: “Get out of my house.” “Get out of my house:” words that suture the film narrative to the world just
outside of the film, a world in which Prime Minister Teresa May and her support
ers attempt to discourage immigration through the active production of a “hostile
environment.” The appellation “environment” leaves out the human activity that
deliberately renders the social environment in the “home” toxic; the home policy is
one of decided unwelcoming, designed to reinforce separateness and detachment,
to bar people from having bank accounts or to work, to prevent the seeker from
making a living, to disallow free movement, and to generate an atmosphere of
rigorous estrangement. Susan Sontag, in Regarding the Pain of Others, imagined that
photographs of distant tragedies—those tragedies from which asylum seekers try to
escape—might animate in their viewers not mere sympathy, but a contemplation of
how their own histories and privileges are linked to the suffering of the afflicted. In
this case, when the afflicted are at the border, the regime of hostility aims to foster
disregard for the pain of ‘others’ by dismissing their suffering altogether, and by
disassociating the ‘home’ from politics ‘over there.’ In this theatre, ‘others’ are as
signed the roles of the characters who only arrive to take. In one scene, Simon gazes
at paintings in Anna’s art gallery. He lingers over two works. In both, individual human faces are distorted beyond
recognition. As Simon gazes at these works, one can perceive another painting, this
one of an elephant being lifted into the air in a harness. The space of Anna’s gallery
is a space of silent, visual recognition of the deliberate distortion of subjects, and in
the backdrop an irony, a haunting, a reminder of the British Empire’s extraction of
the resources of those distant lands, of an imperial past and postcolonial present in TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 30 which the right of the British subject to wander and to take is assumed as a given. These paintings, like the ghost, engage in a live haunting, a break with the illusion, as
Gilroy (2) describes it, that “Britain has been or can be disconnected from its imperial
past.” Simon studies, with a pained expression, a painting of a tortured face bound
at the neck, tongue hanging out. Bruce then orders him: “Get out of my house.” He gazes at another work in which paint has been
smeared over the eyes and mouth, wiped over with sweeping strokes of the brush. The subjects in these paintings exist, but parts of their faces have been swept away. Their faces in the frame are now estranged faces—rearranged ‘identities’—faces
distorted beyond recognition. There is an irony in Simon’s ghostly performance as
a live being—and those who say there is no need for him to continue acting as if he
were alive—for how does this powerfully dead, ghostly performance differ from
the performance of Simon’s living self, a self divested of his narratives? Indeed, as
Dauphinee (236) writes, “In the [fictive] logic of the sovereign ban, death does not
happen. The dead did not die if they were constituted as subjectless in the first place.” The effacement of subjectivity, however, is not the ending. Near the film’s con
clusion, Bruce enters Simon’s apartment when Simon and Anna are having sex and
he shoots Simon. It appears that Bruce has shot Simon’s ghost. The ghost bleeds,
but this is not the conclusion. Like the ghost, A Tree Has Fallen does not settle on an
ending. It does not settle on the ghost’s explanations of its final visits, or its reasons
for leaving and then returning, or its death. There is a resistance to the denouement,
to the successive ending. If there is a settling in this ghostly narrative—a settling
and not an ending—the narrative settles on the movement of ghosts. Whereas the
asylum seeker is hemmed in even as he or she makes profound claims to political
subjectivity, the ghost is, as Amos Tutuola’s work tells us, a wandering subject. In
Achille Mbembe’s (17) meditation on Tutuola, he asserts, “there is no body except
in and through movement. That is why there is no subject but a wandering one. The wandering subject moves from one place to another. Journey as such does not
need a precise destination: the wanderer can go about as he pleases.” Mbembe’s
(or Tutuola’s, if you accept the interpretation) wanderer recalls the foreign corre
spondents traipsing through the forests of Rwanda, who imagine themselves to be
explorers in Ruhorahoza’s 2015 film, Things of the Aimless Wanderer. Bruce then orders him: “Get out of my house.” The inequities
of wandering subjecthood in A Tree Has Fallen come to the fore: Simon’s ability to
wander is only possible after death.The very first words of the film invoke a world
inhabited by spirits without borders. Simon recites the poem in a voiceover, which
includes this stanza: The dead are never gone They are in the paling shadow They are in the thickening shadow The dead are not under the water They are in the rustling tree TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 31 They are in the groaning wood
They are in the water that runs
And the water that sleeps
They are in the hut
They are in the crowd
The dead are not dead This is Birago Diop’s “Souffles,” translated here by Ruhoroza himself for the film. The
title can be rendered as “Spirits,” and also “Breaths.” We hear Simon’s voice recite the
poem as Anna stands silently on a rooftop; her hair moves in the wind. The poem
evokes those wandering, breathing subjects, the ancestors. After his death, after
the expiration of breath, Simon has the liberty to choose where he will wander, and
while his remains are buried in his birthplace, he chooses to return to London. He is
a dead black man who has the right to come back and to refuse to leave. As a ghost
he is not only free to wander but to haunt, and as Gordon (xvi) writes, “Haunting
is one way in which abusive systems of power make themselves known and their
impacts felt in everyday life, especially when they are supposedly over and done
with […] or when their oppressive nature is denied.” Haunting is a matter of see
ing that which has been denied or reassigned out of consciousness, including the
recognition of the imperial past and its enduring denial of the dignity of the formerly
colonized. It is seeing that world, Simon asserts, created for us. Bruce then orders him: “Get out of my house.” In Ruhorahoza’s incorporation of the ghost, it is not only a matter of seeing the
ghost, or of following the ghost. Bruce follows the ghost in an attempt to police it. Simon the ghost, too, follows another ghostly figure on a bridge, whom he eventu
ally loses. There is the compelling matter of speaking to the ghost, and listening to
the ghost, but also of finally feeling the ghost, and of imagining how the ghost feels
and is felt. This engagement in feeling begins by welcoming Simon at the door, and
it is this feeling that disturbs Bruce the most. When Simon returns to Anna, she
meets him at the door of her apartment. In the next scene they are naked; they have and goings because it begins by coming back.” There is an interesting divergence from
what Derrida calls the specter’s visor effect, that is, “we do not see who looks at us”
(6). Derrida’s model of the ghost is Hamlet’s father whom we cannot see hidden
behind his armour. Simon, in contrast, wanders in plain view; Anna feels his body
against hers, he paints his former landlady’s nails. What Derrida calls the ghost’s
“supreme insignia of power: the power to see without being seen” is stripped away. I
R h
h
’ i
i
f h
h
i i
l
f
i
h p
g
p
p
g
pp
y
In Ruhorahoza’s incorporation of the ghost, it is not only a matter of seeing the
ghost, or of following the ghost. Bruce follows the ghost in an attempt to police it. Simon the ghost, too, follows another ghostly figure on a bridge, whom he eventu
ally loses. There is the compelling matter of speaking to the ghost, and listening to
the ghost, but also of finally feeling the ghost, and of imagining how the ghost feels
and is felt. This engagement in feeling begins by welcoming Simon at the door, and
it is this feeling that disturbs Bruce the most. When Simon returns to Anna, she
meets him at the door of her apartment. In the next scene they are naked; they have
sex and his hands cling to the skin on her back; he cries holding her. The camera
lingers on the skin of the lovers in an embrace. Bruce then orders him: “Get out of my house.” This is a world in
which, as Lucy Mayblin’s (24) work on asylum shows us, “displacements resulting
from colonialism and decolonisation in the past are left out of many accounts of the
history of asylum policy that precede analyses of the present.” In the discourse of
this world, narratives of colonial and neocolonial persecution become ghostly mat
ter: “it then also becomes logical to leave unmentioned the legacies of colonialism
for refugee-producing situations, for destination country choice and for ongoing
practices of border control” (24). For Simon, it is not only a matter of seeing how
one is there but rendered invisible, or seeing what is supposed to be invisible, but a
way of saying I have returned, I am everywhere: not only in the paling shadow, in
the rustling tree, in the groaning wood, in the water that runs over the skin, in the
crowd, in our intertwined histories, but at the front door, ringing the bell. Rancière wrote this about politics: “Politics is generally seen as the set of proce
dures whereby the aggregation and consent of collectivities is achieved, the organiza
tion of powers, the distribution of places and roles, and the systems of legitimizing
this distribution. I propose to give this system of distribution another name. I propose
to call it the police” (28). If there is an alternate model for coexistence in the film that
defies the social and spatial assignments managed by the police, it is the ancestors,
moving. The ghost is not to be managed by any home office: can one deport an illegal
ghost? Derrida (Spectres 11) says of the revenant, “One cannot control its comings TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 32 and goings because it begins by coming back.” There is an interesting divergence from
what Derrida calls the specter’s visor effect, that is, “we do not see who looks at us”
(6). Derrida’s model of the ghost is Hamlet’s father whom we cannot see hidden
behind his armour. Simon, in contrast, wanders in plain view; Anna feels his body
against hers, he paints his former landlady’s nails. What Derrida calls the ghost’s
“supreme insignia of power: the power to see without being seen” is stripped away. In Ruhorahoza’s incorporation of the ghost, it is not only a matter of seeing the
ghost, or of following the ghost. Bruce follows the ghost in an attempt to police it. Bruce then orders him: “Get out of my house.” Simon the ghost, too, follows another ghostly figure on a bridge, whom he eventu
ally loses. There is the compelling matter of speaking to the ghost, and listening to
the ghost, but also of finally feeling the ghost, and of imagining how the ghost feels
and is felt. This engagement in feeling begins by welcoming Simon at the door, and
it is this feeling that disturbs Bruce the most. When Simon returns to Anna, she
meets him at the door of her apartment. In the next scene they are naked; they have
sex and his hands cling to the skin on her back; he cries holding her. The camera
lingers on the skin of the lovers in an embrace. Skin is, as Marc Lafrance (9) writes,
an “instrument of interpersonal engagement and exchange.” During Simon’s visit
with Peggy, we do not see Peggy’s face, initially. The first image of their meeting is a
shot of their hands. Simon holds Peggy’s hand and gently runs his thumb over her
knuckles, over her aging skin. When we see her face she seems to hold back tears;
Simon strokes her forehead. Peggy has been alone; her family exists but does not
Figure 4: Simon at the door and goings because it begins by coming back.” There is an interesting divergence from
what Derrida calls the specter’s visor effect, that is, “we do not see who looks at us”
(6). Derrida’s model of the ghost is Hamlet’s father whom we cannot see hidden
behind his armour. Simon, in contrast, wanders in plain view; Anna feels his body
against hers, he paints his former landlady’s nails. What Derrida calls the ghost’s
“supreme insignia of power: the power to see without being seen” is stripped away. In Ruhorahoza’s incorporation of the ghost, it is not only a matter of seeing the
ghost, or of following the ghost. Bruce follows the ghost in an attempt to police it. Simon the ghost, too, follows another ghostly figure on a bridge, whom he eventu
ally loses. There is the compelling matter of speaking to the ghost, and listening to
the ghost, but also of finally feeling the ghost, and of imagining how the ghost feels
and is felt. This engagement in feeling begins by welcoming Simon at the door, and
it is this feeling that disturbs Bruce the most. Bruce then orders him: “Get out of my house.” When Simon returns to Anna, she
meets him at the door of her apartment. In the next scene they are naked; they have
sex and his hands cling to the skin on her back; he cries holding her. The camera
lingers on the skin of the lovers in an embrace. Skin is, as Marc Lafrance (9) writes,
an “instrument of interpersonal engagement and exchange.” During Simon’s visit
with Peggy, we do not see Peggy’s face, initially. The first image of their meeting is a
shot of their hands. Simon holds Peggy’s hand and gently runs his thumb over her
knuckles, over her aging skin. When we see her face she seems to hold back tears;
Simon strokes her forehead. Peggy has been alone; her family exists but does not
visit her. Simon prepares her a bath, soaps and runs water over her skin, washes
her hair, and massages lotion on her feet. The attendance to the needs of the body
and the care of the skin relay a welcoming, an intimate friendship, a way of sharing
space that is felt first in the skin. Close-up scenes of Simon’s tender care of Peggy’s
Figure 4: Simon at the door Figure 4: Simon at the door Figure 4: Simon at the door and goings because it begins by coming back.” There is an interesting divergence from
what Derrida calls the specter’s visor effect, that is, “we do not see who looks at us”
(6). Derrida’s model of the ghost is Hamlet’s father whom we cannot see hidden
behind his armour. Simon, in contrast, wanders in plain view; Anna feels his body
against hers, he paints his former landlady’s nails. What Derrida calls the ghost’s
“supreme insignia of power: the power to see without being seen” is stripped away. and goings because it begins by coming back.” There is an interesting divergence from
what Derrida calls the specter’s visor effect, that is, “we do not see who looks at us”
(6). Derrida’s model of the ghost is Hamlet’s father whom we cannot see hidden
behind his armour. Simon, in contrast, wanders in plain view; Anna feels his body
against hers, he paints his former landlady’s nails. What Derrida calls the ghost’s
“supreme insignia of power: the power to see without being seen” is stripped away. Bruce then orders him: “Get out of my house.” Skin is, as Marc Lafrance (9) writes,
an “instrument of interpersonal engagement and exchange.” During Simon’s visit
with Peggy, we do not see Peggy’s face, initially. The first image of their meeting is a
shot of their hands. Simon holds Peggy’s hand and gently runs his thumb over her
knuckles, over her aging skin. When we see her face she seems to hold back tears;
Simon strokes her forehead. Peggy has been alone; her family exists but does not
visit her. Simon prepares her a bath, soaps and runs water over her skin, washes
her hair, and massages lotion on her feet. The attendance to the needs of the body
and the care of the skin relay a welcoming, an intimate friendship, a way of sharing
space that is felt first in the skin. Close-up scenes of Simon’s tender care of Peggy’s TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 33 skin occur before they exchange any words. Peggy discloses that she has missed his
kindness and his home-cooked meals; Simon apologizes for leaving suddenly. She
tries to assure him that it is okay; he opens a window and magically creates an image
of light. They are not denied, to return to Dauphinee, their own skins. These scenes
lie in a stark contrast to the dance club scene in which Simon’s body and tattooed
skin are put on display. In the club there is no connection, no magic between Simon
and the anonymous women who smile at him during his pornographic performance,
no intimate connection when a woman briefly stops her conversation to touch his
skin as he dances suggestively. Also in contrast is Bruce, who protects his skin and
conceals its porousness: in the final scenes, he rides about the streets fully covered
in his motorcycle gear and helmet. The skin, Lafrance (9) writes, is not a mere “crude
container, a one-sided shield, or an impenetrable shield.” Rather, as Sara Ahmed and
Jackie Stacey write, it “has the potential to break down the dichotomous elabora
tions of inside/outside, surface/depth and self-other that often permeate accounts of
embodied subjectivity” (qtd in Lafrance 11). It is Simon’s affectionate attachments
to others, the welcome touches of the skin, that Bruce cannot manage, and tries
desperately to police. Bruce then orders him: “Get out of my house.” The model of the borderless world of the ancestors is likewise a stark contrast to
present zones of managed, policed existence: one’s belonging in the world of the
ancestors is uncontested. Yet the proposition of being free to wander only after death
is rather unsettling. Simon returns as a witness “from the inside of death,” to adapt
Dauphinee (231). He is free to wander where he pleases, but the living are still there
in their ghostly zones, where they can be reduced to the status of threats, problems,
frauds, financial burdens, crisis, overflow—where they can be returned to other
zones, as Dauphinee writes, where they may be imprisoned, tortured, or murdered
by those whom they fled in the first place. To expand upon Gordon’s question, how
do we reckon with the alienating zones that render living people ghostly? In the
film’s initial scenes after the recitation of Diop’s poem about spirits, we see Simon
sitting still on a train, fatigued, passing through high-rise apartment buildings, in a
seat that propels him backward. There are no sounds of voices, only the motion of
the train. He evokes Benjamin’s angel of history, his face gazing back at the world. But he is a ghost. We cannot yet know, at this point, that he is ghostly matter. Several claims are made upon ghosts; ghosts, too, have their assigned roles. In
Derrida, “the dead are dead […] Just because the dead no longer exist does not mean
that we are done with spectres” (Derrida and Stiegler 49). For Gordon (xix), the
ghost is a messenger: it reveals to us a “living inheritance.” When the ghost appears,
Gordon (xvi) asserts, “We are notified that what’s been concealed is very much alive
and present.” In the film, Simon explains that after a death, one does not say that
someone is dead. When one joins the other world, one says that a tree has fallen. In
a similar vein, at the ending of the film, one does not speak of an ending. We hear TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 34 Simon’s voice return to Diop’s “Souffles” as Anna is walking. Among the last lines are:
Figure 5: Simon on the train Simon’s voice return to Diop’s “Souffles” as Anna is walking. Among the last lines are: They are in the forest; They are in the house. The dead are not dead. Bruce then orders him: “Get out of my house.” Gordon (6) writes that “[c]ajoling is in the nature of the ghost, the very distinctions
between there and not there, past and present, force and shape.” The film itself is
ghostly. It disorients: in one moment Simon stoops to leave flowers near an apart
ment building, looks up to the rooftop, and there is a cut to a view of the rooftop
from below, then a cut to an image of Simon on a roof. We see him from the back,
in a different coat, looking down. Is this the roof the ghost has been looking at? Did
Simon jump from it in the time before? Answers are withheld. Then a cut to Anna,
on a rooftop, perhaps the same one but the view has changed. She stands where we
saw her before, at the beginning of the film, in the same coat. What else happens on
the roof is not spoken or seen. I am writing about a film that, at this moment, like a ghost, only a few have seen. I’ve seen the fifth cut. Further cuts might make some of these words on the page
appear as if I have seen something that is not there. But they existed. And perhaps
a new image will appear, and I will have missed it, until it appears before me. In
between the writing of this piece and my “proofs,” Ruhorahoza added several new TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 35 scenes to A Tree Has Fallen, transformed it, and re-titled it Europa. Europa is a film
altered and rearranged: the story about Simon is still there, alongside an artful mix
of ghostly fiction, quasi-ethnography, explication du texte, sociological documentary,
and an autobiographical essay about an African filmmaker making a film out of Africa
titled A Tree Has Fallen. Potentially, then, this essay offers a reading of what may no
longer be visible in Ruhorahoza’s film. In any event, “to write stories concerning
inclusions and exclusions,” Gordon (17) asserts, “is to write ghost stories.” A Tree Has
Fallen presents narratives, memories, suggestions, and omissions. A ghost story. It
displays the sounds and images of wind that rustles branches, of water caressing the
skin, of fires, spirits, and of breath. They are there and not there, past and present,
expiring and breathing—shape, image, sound, summoning. Notes 1. From an email correspondence with the director, 2018. 2. I make a reference here to Jacques Derrida’s Of Hospitality: Anne Dufourmantelle Invites Jacques Derrida
to Respond (2000). My reading of Derrida in this instance is inspired by Joshua Mills-Knutsen’s ques
tions about hosts, guests, and foreigners in his article, “Becoming Stranger”. 2. I make a reference here to Jacques Derrida’s Of Hospitality: Anne Dufourmantelle Invites Jacques Derrida
to Respond (2000). My reading of Derrida in this instance is inspired by Joshua Mills-Knutsen’s ques
tions about hosts, guests, and foreigners in his article, “Becoming Stranger”. 3. The phrase “We are here because you were there” was also articulated by Ambalavaner Sivanandan
(see Younge). Acknowledgments I would like to thank Kivu Ruhorahoza for sharing the various versions of his films
with me and for providing the film stills that accompany this essay. 1. From an email correspondence with the director, 2018. Works Cited Bauman, Zygmunt. Liquid Life. Polity, 2005. _____. Strangers at Our Door. Polity, 2016. Bauman, Zygmunt. Liquid Life. Polity, 2005. __. Strangers at Our Door. Polity, 2016. Darling, Jonathan. “Asylum and the Post-Political: Domopolitics, Depoliticisation and Acts of Citizenship:
Asylum and the Post-Political.” Antipode vol. 46, no. 1, 2014, pp. 72–91. DOI: https://doi.org/10.1111/
anti.12026. Dauphinee, Elizabeth. “Living, Dying, Surviving II.” The Logics of Biopower and the War on Terror. Eds. Elizabeth Dauphinee & Cristina Masters. Palgrave Macmilllan, 2007. Derrida, Jacques. Of Hospitality: Anne Dufourmantelle Invites Jacques Derrida to Respond. Trans. Rachel
Bowlby. Stanford U P, 2000. _____. Spectres of Marx. Trans. Peggy Kamuf. Routledge, 1994. Derrida, Jacques & Bernard Stiegler. “Spectographies.” The Spectralities Reader: Ghosts and Haunting in
temporary Cultural Theory. Eds. María del Pilar Blanco & Esther Peeren. Bloomsbury, 2013, pp. 38 Gilroy, Paul. After Empire: Melancholia or Convivial Culture? Routledge, 2004. Gordon, Avery F. Ghostly Matters: Haunting and the Sociological Imagination. U of Minnesota P, 200 Hall, Stuart. “The Neoliberal Revolution.” Soundings no. 48, 2011, pp. 9–27. https://www.lwbooks.co.uk/
soundings/48/neoliberal-revolution. Accessed 19 Feb. 2019. g
Harrow, Ken. “‘Let Me Tell You about Bekolo’s Latest Film, Les Saignantes, but First …’” Exit: Endings and
New Beginnings in Literature and Life. Eds. Stefan Helgesson, et al. Rodopi, 2011. Henderson, Ailsa, et al. “How Brexit Was Made in England.” The British Journal of Politics and International
Relations vol. 19, no. 4, 2017, pp. 631–46. DOI: https://doi.org/10.1177/1369148117730542. Isin, Engin F. & Kim Rygiel. “Abject Spaces: Frontiers, Zones, Camps.” The Logics of Biopower and the War
on Terror. Eds. Elizabeth Dauphinee & Cristina Masters. Palgrave Macmillan, 2007. 36 TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 Lafrance, Marc. “Skin Studies: Past, Present, and Future.” Body and Society vol. 24, no. 1–2, 2018, pp. 3–32. DOI:
https://doi.org/10.1177/1357034X18763065. Mayblin, Lucy. Asylum After Empire: Colonial Legacies in the Politics of Asylum Seeking. Rowman & Littlefield,
2017. Mbembe, Achille. “Life, Sovereignty, and Terror in the Fiction of Amos Tutuola.” Research in African Literatures
vol. 34, no. 4, 2003, pp. 1–26. https://www.jstor.org/stable/4618325. Accessed 19 Feb. 2019. Mills-Knutsen, Joshua. “Becoming Stranger: Defending the Ethics of Absolute Hospitality in a Potentially Hostile
World.” Religion and the Arts vol. 14, 2010, pp. 522–33. DOI: https://doi.org/10.1163/156852910X529304. Moore, Kerry. “‘Asylum Shopping’ in the Neoliberal Social Imaginary.” Media, Culture & Society vol. 35, no. 3,
2013, pp. 348–65. DOI: https://doi.org/10.1177/0163443712472090. Omelsky, Matthew. “Jean-Pierre Bekolo’s African Cyborgian Thought.” Nka: Journal of Contemporary African
Art vol. 31, no. Works Cited 1, 2012, pp. 6–21. DOI: https://doi.org/10.1215/10757163-1586445. Rancière, Jacques. Disagreement: Politics and Philosophy, trans. Julie Rose. London: Verso, 1999. Ruhorahoza, Kivu & Antonio Ruberio. Sundance Interview: The Story of “Things of the Aimless Wanderer.” https://
www.youtube.com/watch?v=Hc_X6PkEC1M. Accessed 19 Feb. 2019. Webber, Frances. “Borderline Justice.” Race & Class vol. 54, no. 2, 2012, pp. 39–54. DOI: https://doi. org/10.1177/0306396812454988. Younge, Gary. “Ambalavaner Sivanandan Obituary: Director of the Institute of Race Relations Who Helped
Change the Way Britain Thought About Race.” The Guardian. 7 Feb. 2018. https://www.theguardian.com/
world/2018/feb/07/ambalavaner-sivanandan. Accessed 11 Jul. 2018. TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 TYDSKRIF VIR LETTERKUNDE • 56 (1) • 2019 37
|
https://openalex.org/W1724717467
|
https://journal.binus.ac.id/index.php/commit/article/download/487/465
|
Indonesian
| null |
PERANCANGAN APLIKASI e-SCM PADA PT CAHAYA BUANA FURINDOTAMA
|
CommIT (Communication and Information Technology) Journal/Commit Journal
| 2,008
|
cc-by-sa
| 5,556
|
Pendahuluan dengan keinginan dari pihak manajemen, sehingga terjadi
keterlambatan data inventory yang menghambat pengadaan
bahan baku dan proses produksi. Selain itu, antar divisi dalam
perusahaan juga belum terintegrasi dengan baik. Pada masa sekarang ini, perusahaan di dunia terus-
menerus berkembang dari hari ke hari dan berusaha untuk
meningkatkan kinerjanya dengan kecanggihan teknologi. Ketatnya persaingan, baik secara lokal maupun global
membuat perusahaan terfokus pada bagaimana meningkatkan
proses bisnis dan dapat berkompetisi dengan para pesaing. ABSTRACT PT Cahaya Buana Furindotama is a company incorporated in the Olympic Group. This company is a manufacturing
company which produces furniture and household-wares made from plastic material. Problems en countered were raw
material shortage and production delay, though the company already had a partnership with several suppliers because of
the un-integrated data and there was a “human error” in data entry. Results of the problem were the company could lose
market share and threatened with competitors’ position. Based on the problems which arose, it was proposed to use an e-SCM
application system at the company because e-SCM application system could help the company to manage flow of raw material
better and strengthen the company’s relationship between suppliers and distributors. The methods used were Porter’s Five
Forces to analyze the company’s position in market and SWOT Method to determine a strategy could be used by the company. Based on the internal matrix (IFE), the result obtained was 3:08. Whereas, the external matrix (EFE), the result obtained was
3:34. Based on both results, it can be concluded that the company is in a strong position in matrix IE. Whereas, the strategy
used is Strengths-Opportunity (SO) which is in SWOT matrix. It is expected that the SO strategy and e-SCM application
system, the company can be more strength its position in the market. Keywords: e-SCM, SWOT, internal matrix, external matrix Keywords: e-SCM, SWOT, internal matrix, external matrix ABSTRAK PT Cahaya Buana Furindotama adalah salah satu perusahaan yang tergabung dalam Olympic Group. Perusahaan
ini adalah perusahaan manufaktur yang memproduksi furniture dan peralatan rumah tangga berbahan baku plastik. Masalah
yang sering terjadi antara lain kekurangan bahan baku dan keterlambatan produksi, walaupun perusahaan memiliki hubungan
kerja sama dengan beberapa supplier karena tidak terintegrasinya data dan adanya “human error” dalam pemasukan data. Akibatnya perusahaan dapat kehilangan pangsa pasar dan terancam dengan posisi para pesaing. Berdasarkan masalah yang
muncul, maka diusulkan untuk menggunakan sistem aplikasi e-SCM karena sistem dapat membantu mengelola aliran bahan
baku perusahaan secara lebih baik dan mempererat hubungan perusahaan antara supplier dan distributor. Metode yang
diterapkan adalah Porter’s Five Forces untuk menganalisis posisi perusahaan di pasar dan Metode SWOT untuk menentukan
strategi yang dapat digunakan oleh perusahaan. Berdasarkan matrikss internal (IFE), didapatkan hasil 3.08. Sedangkan
matrikss eksternal (EFE) didapatkan hasil 3.34. Jadi, berdasarkan kedua hasil tersebut, perusahaan berada di posisi yang
kuat dalam matrikss IE..Sedangkan strategi yang digunakan adalah Strengths-Opportunity (SO) yang ada dalam matrikss
SWOT. Diharapkan dengan strategi SO dan sistem aplikasi e-SCM, perusahaan dapat lebih memperkuat posisinya di dalam
pasar. Kata kunci: e-SCM, SWOT, matrikss internal, matrikss eksternal Keywords: e-SCM, SWOT, internal matrix, external matrix Perancangan Aplikasi E-SCM... (Honni; dkk) PERANCANGAN APLIKASI e-SCM PADA
PT CAHAYA BUANA FURINDOTAMA Honni1, Robertus Tang Herman2, Erick Christanto3 1, 2, 3Jurusan Sistem Informasi, Fakultas Ilmu Komputer, Universitas Bina Nusantara,
Jln. K.H. Syahdan No.9, Palmerah, Jakarta Barat 11480
honni@binus.edu Ruang Lingkup Ruang lingkup penulisan ini mencakup perancangan
aplikasi e-SCM berbasis web yang dapat membantu
perusahaan meningkatkan kinerjanya; dan fasilitas-fasilitas
yang disediakan, antara lain: pendataan sumber daya di
gudang, proses hubungan dengan supplier yang mencakup
pemesanan sumber daya, proses aliran sumber daya yang
digunakan dalam produksi perusahaan, pemberian data, dan
informasi dalam membantu pihak manjemen menghasilkan
solusi bagi perusahaan, dan layanan yang diberikan kepada
pelanggan. p
p
p
g
p
p
g
PT Cahaya Buana Furindotama yang berlokasi
di Bogor, fokus pada pengolahan produk-produk rumah
tangga dan furniture berbahan baku plastik. Hubungan yang
terjalin antara perusahaan dengan supplier kurang terjalin,
sehingga menyebabkan berbagai kendala manajemen. Pertukaran dokumen-dokumen dalam perusahaan ataupun
antara perusahaan dengan supplier terjadi secara manual. Hal ini kurang efektif dalam menghadapi persaingan bisnis
di era globalisasi ini. Inventory juga sering tidak sejalan 17 Perancangan Aplikasi E-SCM... (Honni; dkk) Tujuan dan Manfaat purchasing dan pengiriman. Kedua adalah internal supply
chain segment. Segmen ini meliputi keseluruhan proses yang
dilakukan oleh perusahaan dalam mentransformasi bahan
baku yang dikirim oleh supplier menjadi barang jadi. Ketiga
adalah downstream supply chain segment. Segmen ini meliputi
seluruh proses yang melibatkan distribusi dan pengiriman
barang akhir atau barang jadi ke konsumen tingkat akhir. Tujuan dari penulisan ini adalah untuk mengetahui
proses-proses bisnis dan operasional dari perusahaan, sehingga
dapat mengidentifikasi secara lengkap kendala manajemen
yang dihadapi perusahaan; memperbaiki hubungan yang
dibina antara perusahaan dengan konsumen, ataupun antara
perusahaan dengan supplier; menganalisis dan merancang
aplikasi e-SCM yang dapat diajukan sebagai solusi pemecahan
masalah di perusahaan dan berguna bagi peningkatan
proses bisnis perusahaan; serta mereduksi penggunaan cara
konvensional di perusahaan seperti pemakaian kertas untuk
dokumen, sehingga lebih terotomatisasi dan menambah
efisiensi perusahaan. SCM Planning dan Execution Menurut Kalakota (2001: 283), SCM sangat penting
demi keberhasilan strategi e-business. SCM adalah kerangka
kerja bisnis yang terdiri dari berbagai aplikasi yang dapat dibagi
menjadi 2 kelompok. Pertama adalah Supply Chain Planning
(SCP). Ini adalah aplikasi yang mengintegrasikan fungsi-
fungsi planning, seperti peramalan permintaan, simulasi
persediaan, distribusi, transportasi, serta perencanaan dan
penjadwalan produksi. Kualitasi software perencanaan akan
meningkatkan ketepatan peramalan, penjadwalan produksi
yang optimal, mengurangi persediaan dan biaya transportasi,
serta meningkatkan pelayanan konsumen. Kedua adalah
Supply Chain Execution (SCE). Mengintegrasikan fungsi-
fungsi eksekusi seperti procurement, manufacturing, dan
distribusi produk melalui rantai nilai. Aplikasi Supply Chain
Execution mengatur aliran produk melalui pusat distribusi dan
gudang, serta membantu memastikan bahwa produk dikirim
ke lokasi yang benar, menggunakan alternatif transportasi
terbaik yang disediakan. i
p
Manfaat dari penulisan ini adalah. Pertama,
bagi perusahaan. Manfaatnya adalah perusahaan dapat
mengatur dan merencanakan proses bahan baku produksi
perusahaan agar lebih efisien; memberikan masukan bagi
pihak manajemen dalam mengatasi masalah yang terjadi
dan merencanakan strategi perusahaan yang lebih baik dan
terintegrasi dengan sistem; serta membina hubungan yang
baik antara perusahaan dengan supplier dan antara perusahaan
dengan pelanggan. Kedua, bagi pelanggan. Manfaatnya adalah
memudahkan pelanggan dalam mendapatkan produk-produk
perusahaan dan mendapatkan pelayanan yang memuaskan
dari perusahaan. Ketiga, bagi supplier. Manfaatnya adalah
untuk meningkatkan kepercayaan dan hubungan dengan
perusahaan; serta pesanan dari perusahaan akan lebih
terorganisir dan memudahkan supplier dalam mengontrol
arus barang. PEMBAHASAN Menurut Iwan Purwanto (2007: 131), matrikss
SWOT merupakan matching tool yang membantu para
manajer mengembangkan 4 tipe strategi, yaitu: Strategi SO
(Strengths-Opportunity) adalah strategi yang digunakan
perusahaan dengan memanfaatkan atau mengoptimalkan
kekuatan/Strengths (S) yang dimiliki untuk memanfaatkan
berbagai peluang/Opportunity (O); strategi WO (Weakness-
Opportunity) adalah strategi yang digunakan perusahaan,
dengan seoptimal mungkin meminimalisir kelemahan/
Weakness (W) yang ada untuk memanfaatkan berbagai
peluang/Opportunity (O); strategi ST (Strengths-Threats)
adalah
strategi
yang
digunakan
perusahaan
dengan
memanfaatkan atau mengoptimalkan kekuatan/Strengths
(S) untuk mengurangi berbagai ancaman/Threats (T) yang
mungkin melingkupi perusahaan; serta strategi WT (Weakness-
Threats) adalah strategi yang digunakan untuk mengurangi
kelemahan/Weakness (W) dalam rangka meminimalisir atau
menghindari ancaman/Threats (T). Proses Supply Chain Management Menurut Kalakota (2001: 274), supply chain sebuah
perusahaan mencakup fasilitas di mana bahan mentah,
produk setengah jadi, dan barang jadi diperoleh, dipindahkan,
disimpan, dan dijual. External Factor Evaluation (EFE) Matrix Sedangkan menurut James A. O’Brien (2006),
manajemen rantai pasokan adalah sistem antar perusahaan
lintas fungsi, yang menggunakan teknologi informasi untuk
membantu mendukung, serta mengelola berbagai hubungan
antara beberapa proses bisnis utama perusahaan dan dengan
pemasok, pelanggan, dan para mitra bisnis. Menurut Fred David (Iwan Purwanto, 2007: 113), ada 5
tahapan dalam pembuatan EFE matrikss. Pertama adalah buat
critical success factors seperti yang diidentifikasikan dalam
faktor-faktor lingkungan eksternal yang menjadi peluang
(opportunities) maupun ancaman (threats). Kedua adalah
menentukan bobot atau timbangan critical success factors,
dimulai dari 0,0 untuk faktor yang sangat tidak penting sampai
1,0 untuk faktor yang sangat penting. Ketiga adalah untuk
setiap faktor yang telah diberi bobot, juga diberi peringkat
mulai dari angka 1 sampai 4. Nilai 4 (respon sangat bagus)
artinya jika respon perusahaan terhadap lingkungan eksternal
sangat baik dan optimal dibanding dengan perusahaan lain
dalam industri. Keempat adalah setiap bobot pada langkah
kedua dikalikan dengan peringkat yang telah ditentukan
pada langkah tiga untuk mendapatkan nilai timbangannya. Pertama adalah jumlah nilai tertimbang untuk setiap variabel
yang digunakan merupakan total nilai tertimbang perusahaan
tersebut. Pengertian Supply Chain Management Lee & Whang (Lina Anatan dan Lena Ellitan, 2000)
mendefinisikan manajemen rantai pasokan sebagai integrasi
proses bisnis dari pengguna akhir melalui pemasok yang
memberikan produk, jasa, informasi, dan bahkan peningkatan
nilai untuk konsumen dan karyawan. Supply Chain Management (SCM) Pengertian Supply Chain Management Internet Internet menurut Turban (2001: 208) adalah sebuah
interkoneksi jaringan yang besar dari jaringan-jaringan
komputer dan komputer-komputer di seluruh penjuru dunia,
melalui saluran telepon, satelit, dan sistem komunikasi
lainnya, guna melakukan pertukaran informasi. Permasalahan yang Dihadapi Melalui pembahasan supply chain planning dan
supply chain execution, maka ditemukan bahwa dalam
melakukan proses bisnisnya PT Cahaya Buana Furindotama
menghadapi beberapa permasalahan, antara lain: lamanya
waktu yang dibutuhkan untuk memproses pemesanan
pelanggan; data bahan baku seringkali tidak lengkap atau
tidak akurat, dapat terjadi juga kekosongan bahan baku pada
saat jadwal produksi akan dibuat; terjadi ”human error” di
mana karyawan salah memasukkan data untuk nota atau
laporan; terjadi kesalahan komunikasi dengan retail dan
supplier karena tidak terintegrasinya data perusahaan dengan
data yang dimiliki oleh supplier dan retail; pihak retail yang
ingin memesan seringkali ragu-ragu karena tidak mengetahui
kapasitas produk yang bisa disediakan oleh perusahaan pada
saat retail tersebut membutuhkan. Object
Oriented
Analysis
and
Design
(OOA&D) Object Menurut Lars Mathiassen (2000: 51), object adalah
suatu entitas yang memiliki identitas, status, dan sifat. Untuk menentukan sesuatu sebagai object, harus dapat
mendeskripsikan sebuah entitas terlebih dahulu. Identitas
object adalah bagian object yang terpisah dari bagian-bagian
object lainnya. Status object terdiri dari semua bagian object
yang statis dan dynamic. Sifat object adalah rangkaian
peristiwa yang terjadi, baik secara aktif atau pasif dalam
kehidupan. Ancaman Masuknya Pendatang Baru (Threat of New
Entrants) Ancaman Masuknya Pendatang Baru (Threat of New
Entrants) Use-case Diagram Menurut Whitten, Bentley, dan Dittman (2004: 418),
Use-case Diagram merupakan diagram yang menggambarkan
interaksi antara sistem, sistem eksternal, dan pengguna. Dengan kata lain, secara grafis mendeskripsikan siapa yang
akan menggunakan sistem dan dalam cara apa pengguna
mengharapkan interaksi dengan sistem tersebut. Ancaman Produk Subtitusi (Threat of Subtitute Products
or Services) System Definition Salah satu pesaing baru yang cukup memiliki kelebihan
untuk mengancam keberadaan PT Cahaya Buana Furindotama
adalah PT Green Leaf Indonesia karena perusahaan ini
disokong oleh perusahaan plastik ternama dari Jepang. PT
Green Leaf Indonesia memiliki berbagai ragam produk
yang menarik para pelanggan dan segi kualitas produk yang
cukup baik. Sedangkan pesaing-pesaing lainnya sulit untuk
berkembang karena terdapat beberapa faktor hambatan. Menurut Lars Mathiassen (2000: 24), system definition
adalah deskripsi singkat sistem komputerisasi yang dijelaskan
dengan bahasa yang sederhana dan mudah dimengerti. System definition menerangkan bagian fundamental dalam
penggunaan dan pengembangan sistem. Internal Factors Evaluation (IFE) Matrix yang dipahami atau dimengerti oleh pelukis (Gambar 1). Rich Pictures fokus kepada aspek dominan yang mencuri
perhatian dari pelukis. Dengan Rich Pictures, pemirsa diajak
untuk memahami dan merasakan kepentingan dari aspek
tersebut. Rich Pictures digunakan dalam seleksi sistem
untuk menunjukkan semua persepsi yang dihadapi dalam
pengembangan sistem. Langkah membuat IFE matrikss sama dengan
membuat EFE matrikss. Hanya saja, jika pada EFE matrikss
yang didata adalah faktor-faktor eksternal (peluang dan
ancaman), sedangkan pada IFE matrikss yang didata adalah
faktor-faktor internal (kekuatan dan kelemahan). Sistem Aplikasi
e-SCM
PT. Cahaya Buana Furindotama
Logistic Manager
Supplier 1
Karyawan
1. Login
Supplier 2
1. Login
1 Login
Database
2. Username
& Password
3. Konfirmasi
4. Tender
5. Database
Tender
6. Status
Tender
6. Status
Tender
7. Pengajuan
tender
7. Pengajuan
tender
8. Status
Tender
9. Pilih
Pemenang
Tender
10. Konfirmasi
Pemenang
Logistic
11. Bahan Baku
12. MRV
P
a
y
to
$
Finance
13. MRV
P
a
y
t
o
$
10. Konfirmasi
Tender
14. Pembayaran
$
$
Gambar 1 Rich Pictures Proses Tender Bahan Baku Ancaman Produk Subtitusi (Threat of Subtitute Products
or Services) Kenaikan harga jual produk menyebabkan timbulnya
barang subtitusi atau barang pengganti yang dapat
mempengaruhi penjualan produk utama. Meskipun masih
ada konsumen yang membeli produk utama dengan kualitas
yang lebih baik, tetapi tidak menutup kemungkinan bahwa
pangsa pasarnya dapat direbut karena adanya konsumen
yang membeli produk kelas dua yang kualitas produknya
tidak sebaik produk utama, akan tetapi untuk PT Cahaya Porter’s Five Forces Menurut Iwan Purwanto (2007: 88), strategi dan
tujuan perusahaan dipengaruhi oleh daya saing industri untuk
menjalankan bisnis dan posisi sektor industri tersebut. Suatu
industri dapat digambarkan sebagai serangkaian perusahaan
yang bersaing satu sama lain untuk meraih pangsa pasar yang
tinggi dalam mencapai skala ekonomi dan strategi yang telah
ditentukan. Intensitas persaingan dalam suatu industri atau
perusahaan bukanlah masalah kebetulan atau nasib buruk. Keadaan persaingan dalam suatu industri tergantung pada 5
kekuatan pesaing pokok. Lima kekuatan yang mempengaruhi
manajemen strategi, yaitu: sektor pelanggan. Perencana
strategi yang efektif menaruh perhatian pada jenis konsumen,
serta kebutuhan dan keinginan konsumen. para perencana
strategi berkepentingan dengan siapa dan di mana calon
konsumen berada dan kecenderungan di masa depan yang
dapat mengakibatkan perubahan pola beli konsumen. Sektor
ini membahas 3 faktor, antara lain: identitas pembeli, faktor
demografi, dan faktor geografi; sektor pemasok; sektor pesaing
Industri; sektor produk pengganti; serta sektor pendatang
baru. Pendatang baru pada suatu industri membawa kapasitas
baru, keinginan merebut bagian pasar, dan seringkali jumlah
sumber daya yang besar. Akibatnya harga dapat menjadi turun
atau biaya membengkak, sehingga mengurangi laba. Gambar 1 Rich Pictures Proses Tender Bahan Baku Komponen Utama Supply Chain Menurut Turban (2003: 320), supply chain terdiri dari
3 segmen utama sebagai berikut. Pertama adalah upstream
supply chain segment. Ini merupakan supply chain dari
sisi supplier dan organisasinya. Aktivitas utamanya adalah 18 CommIT, Vol. 2 No. 1 Mei 2008, hlm. 17 - 24 Matriks IE PT Cahaya Buana Furindotama y
p
p
Berdasarkan Porter’s Five Forces PT Cahaya Buana
Furindotama, diketahui bahwa posisi perusahaan dalam
industri sudah cukup kuat. Perusahaan mempunyai persaingan
yang cukup tinggi dengan perusahaan yang memiliki modal
kuat atau adanya dukungan dari perusahaan luar negeri. Perusahaan dapat menambah strategi yang dapat diterapkan
dalam menghadapi perusahaan-perusahaan pesaing yang
bermodal kuat. Berdasarkan matriks IFE dan EFE, didapatkan nilai
rata-rata tertimbang untuk EFE adalah 3.34 (Gambar 2). Sedangkan untuk IFE, didapatkan nilai rata-rata tertimbang
sebesar 3.08. Dari kedua nilai tertimbang rata-rata tersebut,
diketahui bahwa PT Cahaya Buana Furindotama termasuk
dalam bagian I dalam matriks IE. Kelemahan dan ancaman yang ada dapat ditutupi
oleh kekuatan dan peluang perusahaan, sehingga perusahaan
sangat berpotensi untuk berkembang menjadi lebih baik lagi. Dengan menerapkan beberapa solusi bisnis dan pemakaian
teknologi, akan mendukung proses bisnis perusahaan. Persaingan Diantara Sesama Perusahaan yang Sejenis
(Rivalry Among Existing Competitors) Persaingan Diantara Sesama Perusahaan yang Sejenis
(Rivalry Among Existing Competitors) Matriks IFE berikut ini berupa tabel, merupakan tools
dalam strategic management untuk mengevaluasi kekuatan
(strengths) dan kelemahan (weakness) dalam area fungsional
bisnis PT Cahaya Buana Furindotama (Tabel 2). Pada dasarnya, dalam setiap bisnis pasti terdapat
persaingan. Setiap perusahaan pasti ingin mencapai posisi
yang paling tinggi, dibandingkan dengan pesaingnya dalam
bidang yang sama. Sifat persaingan yang terjadi di antara para
pebisnis tersebut berbeda-beda; dari persaingan yang halus
sampai pada tingkat persaingan yang saling menjatuhkan,
tergantung pada seberapa agresif perusahaan-perusahaan
melakukan tindakan-tindakan yang mengancam perolehan
laba pesaingnya, serta seberapa diperhatikannya etika dalam
berbisnis oleh perusahaan-perusahaan yang bersaing dalam
industri sejenis. PT Cahaya Buana Furindotama memiliki
beberapa pesaing yang bergerak dalam bisnis furniture plastik,
yaitu: Owl Plast, Maspion Group, dan Lion Star Plastic. y
(
)
Dari tabel di atas, diketahui bahwa kekuatan terbesar
perusahaan adalah dari segi kualitas SDM dan memiliki
banyak distributor yang menopang proses bisnis perusahaan. Sedangkan dari sisi kelemahan yang tertinggi adalah variasi
produk. Variasi produk perusahaan tidak sebanyak variasi
produk pesaingnya, sehingga posisi perusahaan lebih lemah
dibandingkan dengan pesaingnya. Rich Pictures Matriks IFE PT Cahaya Buana Furindotama Rich Pictures Menurut Lars Mathiassen (2000: 26), Rich Pictures
adalah gambaran informal yang menggambarkan situasi 19 Perancangan Aplikasi E-SCM... (Honni; dkk) Pada tabel tersebut, terlihat pada variabel internet
sebagai suatu kebutuhan sehari-hari, diketahui bahwa
internet dapat memberikan peluang yang sangat tinggi bagi
perusahaan dengan nilai sebesar 0.32. Sedangkan dari sisi
ancaman, perusahaan mendapatkan ancaman terbesar dari
keberadaan produk-produk palsu yang beredar di pasar
dengan nilai sebesar 0.64. Buana Furindotama dengan produk Napolly tidak memiliki
barang subtitusi dari segi tingkat harga yang lebih rendah. Sedangkan dari segi kualitas produk, perusahaan memiliki
pesaing-pesaing yang memproduksi produk berbahan baku
kayu dan besi. Pesaing-pesaing tersebut antara lain adalah:
PT Cofemo furniture, PT Max Havelaar Furniture Tbk, dan
PT Chitose Furniture. Kekuatan Tawar Menawar Supplier (The Bargaining
Power of Suppliers) Tabel 1 Matriks EFE PT Cahaya Buana Furindotama Variabel
Bobot
Rating
Nilai
Peluang (Opportunities)
Tingkat harga
1. 0.10
3
0.30
Kepercayaaan Pelaku
2. Bisnis
0.25
4
1
Internet sebagai suatu
3. kebutuhan sehari-hari
0.08
4
0.32
Peluang menuju pasar
4. internasional
0.03
3
0.09
Tingkat perkembangan
5. industri yang cukup baik
0.04
2
0.08
Ancaman (Threatness)
Tingkat persaingan
•
0.08
2
0.16
Resesi global
•
0.10
3
0.30
Ancaman perusahaan
•
asing
0.07
4
0.21
Produk palsu
•
0.16
4
0.64
Perubahan kebutuhan
•
dan kepuasan konsumen
0.08
3
0.24
Total
1
3.34
Tabel 1 Matriks EFE PT Cahaya Buana Furindotama
Sumber: PT Cahaya Buana Furindotama PT Cahaya Buana Furindotama mengutamakan
kualitas dan mutu produk yang baik. Hal ini merupakan
salah satu cara agar mencegah para pelanggan berpindah ke
perusahaan lain. Penawaran produk yang berkualitas rendah
dapat memberikan dampak negatif bagi perusahaan, sehingga
dapat mempengaruhi tingkat penjualan perusahaan, yang
pada akhirnya perusahaan tidak dapat bersaing dengan baik di
dalam pasar industri yang ada. PT Cahaya Buana Furindotama
memiliki satu supplier besar yang memiliki kontrak kerja,
yaitu PT Tri Polyta Indonesia Tbk. dan beberapa supplier
skala kecil yang memasok bahan baku pada perusahaan-
perusahaan cabang. Kekuatan Tawar Menawar Pembeli (The Bargaining
Power of Customers) Kekuatan tawar menawar pembeli memiliki pengaruh
yang sangat besar terhadap volume penjualan perusahaan. Daya tawar pembeli terus menerus mengalami perubahan,
mengingat jumlah populasi produk yang semakin tinggi. Untuk mengantisipasi kendala itu, maka PT Cahaya Buana
Furindotama terus menerus melakukan pemantauan terhadap
pengembangan mutu dan kualitas produk yang beraneka
ragam, agar dapat menyesuaikan produk-produk yang dijual
terhadap daya tawar menawar konsumen. PT Cahaya Buana
Furindotama tidak pernah menjual produknya langsung
kepada end-user. Saran Adapun saran-saran yang dapat dilakukan adalah
sebagai berikut. Pertama, melakukan tahap implementasi
sistem yang telah dibangun. Kedua, melakukan pelatihan
mengenai sistem yang dibangun untuk para karyawan,
sehingga karyawan dapat mengoperasikan sistem secara
optimal. Ketiga, melakukan penambahan fitur-fitur yang
lebih lengkap, sehingga sistem aplikasi e-SCM ini dapat
diintegrasikan sebagai suatu sistem Entreprise Resource
Planning (ERP) secara keseluruhan. Keempat, dapat
menggabungkan sistem aplikasi e-SCM dengan sistem
pembayaran secara online, sehingga pembayaran dapat
dilakukan secara lebih efisien dan tanpa harus menunggu
waktu lebih lama. Kelima, menambahkan fungsi-fungsi
Knowledge Management, sehingga karyawan perusahaan
dengan supplier dan distributor dapat melakukan pertukaran
pengetahuan. User Interface Layar Home Karyawan Halaman ini hanya dapat diakses oleh karyawan yang
sudah melakukan login. Setiap username yang terdaftar
memiliki status tersendiri, sehingga dapat dibedakan apakah
yang melakukan login adalah user dengan status karyawan,
supplier, ataupun distributor (Gambar 5). Strategy Decision Setelah melihat latar belakang perusahaan, struktur
organisasi, permasalahan, dan peluang yang ada, maka PT
Cahaya Buana Furindotama dapat mengembangkan strategi
bisnisnya dengan menerapkan sistem aplikasi e-SCM. Diharapkan sistem aplikasi e-SCM ini dapat memberikan
perusahaan beberapa keuntungan, yaitu: otomatisasi proses
bisnis perusahaan; menghemat waktu yang dibutuhkan karena
mempercepat aliran informasi antara supplier, perusahaan,
dan customer; serta mengurangi biaya operasional perusahaan
seperti biaya pemakaian kertas, biaya telepon, biaya
pengiriman surat-surat kepada supplier, dan lain lain. Use Case Diagram Aplikasi e-SCM Upstream Tabel 2 Matriks IFE PT Cahaya Buana Furindotama Tabel 2 Matriks IFE PT Cahaya Buana Furindotama Variabel
Bobot
Rating
Nilai
Kekuatan (Strengths)
Kualitas SDM
1. 0.21
4
0.84
Memiliki banyak
2. distributor
0.21
4
0.84
Tingkat teknologi yang
3. tinggi
0.10
4
0.40
Letak perusahaan yang
4. strategis
0.06
3
0.18
Memiliki hubungan
5. dengan supplier besar
0.09
3
0.27
Kelemahan (Weakness)
Kurang pengetahuan
•
tentang pelanggan. 0.06
1
0.06
Variasi produk kurang
•
0.10
2
0.2
Kurangnya iklan
•
mengenai produk
0.07
2
0.14
Kurang kepercayaan
•
terhadap pemakaian
internet
0.05
1
0.05
Pemakaian kertas yang
•
berlebih. 0.05
2
0.10
Total
1
3.08
Sumber: PT Cahaya Buana Furindotama Ada 2 actor yang terlibat dalam use case diagram
aplikasi e-SCM Upstream, yaitu karyawan dan supplier, serta
terdiri dari 16 use case yaitu: login, mengubah profil, melihat
daftar bahan baku, membuat PO, melihat daftar PO, membuat
tender, melihat daftar tender, mengajukan penawaran tender,
melihat inbox, membalas message, memberikan kritik dan
saran, melihat status pembayaran, membuat surat jalan,
melihat daftar surat jalan, menambah daftar supplier, dan
melihat daftar user (Gambar 4). Kesimpulan Berdasarkan analisis dan perancangan yang telah
dilakukan pada PT Cahaya Buana Furindotama, dapat ditarik
kesimpulan sebagai berikut. Pertama, proses bisnis PT Cahaya
Buana Furindotama sudah tertata dengan baik dan berpotensi
untuk dilakukan implementasi sistem aplikasi e-SCM, agar
proses bisnis perusahaan dapat lebih berkembang. Kedua,
posisi perusahaan di dalam industri sudah kuat, berdasarkan
analisis manajemen dan dapat memberikan masukan dalam
perancangan sistem aplikasi e-SCM. Ketiga, sistem aplikasi
e-SCM yang dibangun diharapkan dapat meningkatkan
efisiensi dan kecepatan arus informasi maupun bahan baku
dari perusahaan dengan supplier dan distributor. Keempat,
sistem yang dibangun selain untuk mempercepat arus
informasi dan bahan baku, juga untuk mengintegrasikan data
antara data perusahaan dengan data supplier dan distributor. Kelima, dengan menggunakan sistem aplikasi e-SCM,
perusahaan diharapkan dapat menambah daya saing, guna
lebih memperkokoh posisi perusahaan di pasar. Keenam,
sistem aplikasi e-SCM memberikan fungsi-fungsi tambahan
pada proses bisnis perusahaan, antara lain: pemesanan produk
oleh distributor secara online dan adanya tender online untuk
pengadaan bahan baku dapat meningkatkan hubungan bisnis
dengan para supplier. Sumber: PT Cahaya Buana Furindotama dalam mengidentifikasi kekuatan, kelemahan, peluang, dan
ancaman pada PT Cahaya Buana Furindotama. Strategi-
strategi yang akan dilakukan, dijelaskan dan dapat dilihat
pada Tabel 3. p
Dapat diketahui berdasarkan matriks kekuatan internal
dan eksternal perusahaan, bahwa perusahaan memiliki posisi
yang kuat dan memiliki kekuatan yang mendukung posisi
perusahaan tersebut. Oleh karena itu, diusulkan perusahaan
menggunakan strategi SO pada matriks SWOT, di mana
perusahaan dapat menerapkan strategi yang mengoptimalkan
kekuatan-kekuatan yang dimiliki oleh perusahaan, untuk
mencapai peluang-peluang yang ada, sehingga perusahaan
dapat terus berkembang dan bersaing dengan para pesaing
yang sudah terlebih dahulu menguasai pasar seperti Lion
Star. Analisis Strengths, Weakness, Opportunities,
dan Threats (SWOT) Pada Tabel 1, terlihat matrikss EFE PT Cahaya Buana
Furindotama berupa beberapa critical success factors yang
ada dalam faktor-faktor lingkungan eksternal sebagai peluang
(opportunities) maupun ancaman (threatness). Analisis SWOT dapat digunakan sebagai tolok ukur 20 CommIT, Vol. 2 No. 1 Mei 2008, hlm. 17 - 24 Class Diagram Aplikasi e-SCM DAFTAR PUSTAKA
Anatan, Lina., Ellitan, Lena. (2008). Supply Chain
Management, Cetakan pertama. Bandung: Alfabeta. DAFTAR PUSTAKA
Anatan, Lina., Ellitan, Lena. (2008). Supply Chain
Management, Cetakan pertama. Bandung: Alfabeta. APPENDIX I
II
III
IV
V
VI
VII
VIII
IX
Total Rata-Rata Tertimbang IFE
Kuat 3.00-400 Sedang 2.00-2.99
Lemah
1.00-1.99
Tinggi 3.00-4.00
Sedang 2.00-2.99
Lemah 1.00-1.99
Total Rata-Rata Tertimbang EFE
Gambar 2 Matriks IE PT Cahaya Buana Furindotama Gambar 2 Matriks IE PT Cahaya Buana Furindotama Gambar 2 Matriks IE PT Cahaya Buana Furindotama y
+Login()
+Memproses Pemesanan()
+Memberikan saran dan kritik()
+Memproses Pembayaran()
+Menerima Surat Jalan()
+LogOut()
Pelanggan
-Kode Customer
-Nama Toko
-Contact Person
-Alamat
-Email
-No Telp
-No CP
+Dibuat()
+Dikirim()
+Diterima()
Sales Order
-Kode SO
-Kode Customer
-Kode Produk
-Jumlah
-Harga
+Login()
+Memproses SO()
+Memproses SPBB()
+Memproses PO()
+Memproses MRV()
+Memproses Jadwal Produksi()
+Memproses Produk()
+Memproses SHP()
+Memproses Bahan Baku()
+Memproses Pembayaran()
+Memproses Surat Jalan()
+Memproses Perawatan Mesin()
+LogOut()
Karyawan
-Kode Karyawan
-Divisi
-Nama
-Posisi
-Alamat
-Email
-No Telp
+Memilih Produk()
+Memesan Produk()
+Membuat Produk()
+Mengirimkan Produk()
Produk
-Kode produk
-Kode Bahan Baku
-Jenis
-Harga
1..*
1..1
0..1
1..1
1..*
1..1
+Dicek()
+Dikirim()
+Diterima()
+Diproduksi()
Bahan Baku
-Kode Bahan Baku
-Jenis
-Jumlah
1..1
1..*
+Login()
+Memproses PO()
+Memproses Bahan Baku()
+Memproses Pembayaran()
+LogOut()
Supplier
-Kode Supplier
-Nama Perusahaan
-Contact Person
-No Telp
-No CP
-Email
+Dibuat()
+Dikirim()
+Diterima()
MRV
-Kode MRV
-Kode Supplier
-Kode Bahan Baku
+Dibuat()
+Dikirim()
+Diteima()
Purchase Order
-Kode PO
-Kode Bahan Baku
-Kode Supplier
-Total Harga
+Dibuat()
+Dikirim()
+Diterima()
Surat Jalan
-Kode Surat Jalan
-Kode Customer
-Kode Produk
+Membuat PO()
+Menerima PO()
Detil _PO
-Kode PO
-Kode Bahan Baku
-Kode Supplier
-Jumlah
-Harga
-Tgl Permintaan
-Tgl realisasi
+Dibuat()
+Melihat Jadwal Produksi()
Jadwal Produksi
-Kode Jadwal Produksi
-Kode Produk
-Kode Bahan Baku
-Tgl Mulai
-Tgl Selesai
-Pengawas
-Jumlah
+Dibuat()
+Dikirim()
+Diterima()
SPBB
-kode SPBB
-Kode Bahan Baku
-Kode Jadwal Produksi
+Melakukan Pembayaran()
+Memeriksa Status Pembayaran()
+menerima Pembayaran()
Pembayaran
-Kode Pembayaran
-Kode SO
-Kode PO
-Kode User
-Jumlah
-Total Pembayaran
+Memproses Perawatan Mesin()
Perawatan Mesin
-Kode Perawatan
-Tgl Perawatan
-Tgl Perawatan Akan Datang
-Mesin
-Keterangan
+Dibuat()
+Dikirim()
+Diterima()
SHP
-Kode SHP
-Kode Jadwal Produksi
1..*
1..1
1
0..1
1..1
1..*
0..1
1..*
0..1
1..1
1..1
1..*
0..1
1..1
1..1
0..1
1..*
0..1
1..*
0..1
1..1
1..*
1..1
0..1
+Dibuat()
+Dikirim()
+Diterima()
Detil SHP
-Kode SHP
-Kode Jadwal Produksi
-Jumlah
1..1
1..*
1
0..1
1..1
1..1
1..1
1..*
1..1
1..*
+Dibuat()
+Dikirim()
+Diterima()
Detil SPBB
-Kode SPBB
-Kode Bahan Baku
-Kode Jadwal Produksi
-Jumlah
-Tanggal
+Dibuat()
+Dikirim()
+Diterima()
Detil MRV
-Kode MRV
-Kode Bahan Baku
-Kode Supplier
-Jumlah
-Gudang
1..1
0..1
1..*
1..1
1
0..1
1
0..1
1..1
1..1
Gambar 3 Class Diagram Aplikasi e-SCM PT Cahaya Buana Furindotama Gambar 3 Class Diagram Aplikasi e-SCM PT Cahaya Buana Furindotama 22 CommIT, Vol. APPENDIX Purwanto, Iwan. (2008). Manajemen Strategi, Cetakan kedua. DAFTAR PUSTAKA Aplikasi e-SCM PT Cahaya Buana Furindotama
melibatkan beberapa object yang saling berhubungan. Hubungan antar object ini digambarkan melalui class
diagram (Gambar 3). Anatan, Lina., Ellitan, Lena. (2008). Supply Chain
Management, Cetakan pertama. Bandung: Alfabeta. 21 Perancangan Aplikasi E-SCM... (Honni; dkk) Kalakota, R., dan Marcia Robinson. (2001). E-Business 2.0
Roadmap for Success, 2nd edition. Massachusetts, USA:
Addison Welsey. CV. Bandung: Yrama Widya. Robbins, Stephen P., dan Mary Coulter. (2002). Manajemen,
Edisi ketujuh. Jakarta: Indeks. Mathiassen, L. et al. (2000). Object-Oriented Analysis and
Design. Denmark: Marko Publishing, Aps. Tunggal, Amin Widjaja. (2009). Manajemen Logistik dan
Supply Chain Management, Cetakan pertama. Jakarta:
Havarindo. McLeod Jr., Raymond, dan George P. Schell. (2004). Management Information Systems. New Jersey: Pearson
Education, Inc. Turban, Efraim, R. Kelly Rainer Jr., dan Richard E. Potter. (2005). Introduction to Information Technology, 3rd
edition. New York: John Wiley and Sons, Inc. O’Brien, James A. (2003). Introduction to Information
System, 11th edition. New York: McGraw-Hill. 2. Meningkatkan
kemampuan
daya
saing
perusahaan dengan mengaplikasi software-
software pendukung pengambilan keputusan
(W4,W5,T1,T4) 1. Melakukan evaluasi dengan melakukan
riset pendapat masyarakat mengenai produk
perusahaan (W1,W3,O1,O5) APPENDIX •
Peluang (Opportunities)
Tingkat harga
1. Kepercayaaan Pelaku Bisnis
2. Internet sebagai suatu kebutuhan
3. sehari-hari. Peluang menuju pasar internasional
4. Tingkat perkembangan industri
5. yang cukup baik. Strategi SO
1. Penggunaan
teknologi
dapat
mendongkrak kinerja dan efisiensi
perusahaan, selain itu dapat digunakan
internet sebagai salah satu proses
bisnis perusahaan karena tingkat
penggunaan internet yang tinggi
dalam masyarakat dan internet dapat
merambah seluruh dunia bukan hanya
terbatas pada suatu wilayah (S3,O3,
O4)
2. Mempertahankan kepercayaan dari
pelaku bisnis dengan peningkatan
kinerja perusahaan dengan melakukan
pelatihan-pelatihan karyawan yang
dibarengi
dengan
penggunaan
teknologi (S1, S3, O2)
3. Meningkatkan
layanan
kepada
masyarakat
dengan
berinteraksi
lanrgsung
melalui
internet,
memberikan layanan secara online ,
memberikan pengetahuan mengenai
produk perusahaan kepada konsumen-
konsumen ditempat yang jauh secara
langsung dan memberikan laporan
kepada investor guna menambah
kepercayaan
mereka. (S1,S2,
S3,O2,O3,O4,O5)
Strategi WO
1. Menambah jumlah variasi produk sesuai
dengan permintaan pasar, yang dilakukan
dengan startegi riset dan pengembangan
produk. Sasaran yang dituju bukan hanya
pada pasar lokal tetapi juga mulai merambah
pasar internasional. (O1,O4,O5,W1,W2)
2. Meningkatkan efektifitas produk iklan atas
produk dengan merencanakan lebih matang
pemasangan iklan yang strategis yang
diharapkan dapat meningkatkan pengenalan
dan
kepercayaan
kepada
perusahaan
(O2,W3)
3. Mengurangi penggunaan kertas dengan
mengadopsi serta mengembangkan strategi
manajemen
perusahaan
asing
karena
sudah banyak perusahaan asing umumnya
menghindari pemakaian kertas yang berlebih,
selain itu hal ini merupakan salah satu upaya
mengenali situasi perusahaan internasional
(O4, W5)
Ancaman (Threatness)
1. Tingkat persaingan
2. Resesi global
3. Ancaman perusahaan asing
4. Produk palsu
5. Perubahan kebutuhan dan kepuasan
konsumen
Strategi ST
1. Terus
mengikuti
perkembangan
teknologi yang dapat menambah
kualitas
produk
dan
berusaha
mengaplikasi
teknologi
tersebut
dalam perusahaan karena tingginya
tingkat persaingan, maka perusahaan
harus dapat berinovasi baik dalam
produk maupun kinerja perusahaan
(S1,S3,T1,T3,T5). 2. Memberikan jasa bagi pelanggan
yang membeli produk perusahaan
agar produk yang dihasilkan tetap
menjadi pilihan utama pelanggan. Menempatkan
karyawan-karyawan
yang berkualitas dalam posisi tersebut
(S1,S2,S3,T1,T3,T4). 3. Perusahaan
dapat
menugaskan
karyawan yang berpotensi untuk
menarik investor-investor baru untuk
mengatasi dampak resesi global. Perusahaan harus dapat menunjukkan
kemampuan
dalam
menghadapi
pesaing dan ancaman yang ada untuk
menarik Investor(S1,S3,T2,T1). Strategi WT
1. Melakukan evaluasi dengan melakukan
riset pendapat masyarakat mengenai produk
perusahaan (W1,W3,O1,O5)
2. Meningkatkan
kemampuan
daya
saing
perusahaan dengan mengaplikasi software-
software pendukung pengambilan keputusan
(W4,W5,T1,T4)
3. Melakukan pendekatan kepada konsumen
dan memberikan pengenalan produk dengan
penggunaan iklan sehingga masyarakat
tidak keliru dengan produk-produk palsu,
hal ini membantu pencitraan perusahaan
sehingga
dapat
mengalahkan
pesaing
(W2,W3,T1,T3,T4) 2. APPENDIX 2 No. 1 Mei 2008, hlm. 17 - 24 Kekuatan (Strengths)
Kualitas SDM
•
Memiliki banyak distributor
•
Tingkat teknologi yang tinggi
•
Letak perusahaan yang strategis
•
Memiliki hubungan dengan supplier
•
besar
Kelem
K
•
V
•
K
•
K
•
in
pe
•
Peluang (Opportunities)
Tingkat harga
1. Kepercayaaan Pelaku Bisnis
2. Internet sebagai suatu kebutuhan
3. sehari-hari. Peluang menuju pasar internasional
4. Tingkat perkembangan industri
5. yang cukup baik. Strategi SO
1. Penggunaan
teknologi
dapat
mendongkrak kinerja dan efisiensi
perusahaan, selain itu dapat digunakan
internet sebagai salah satu proses
bisnis perusahaan karena tingkat
penggunaan internet yang tinggi
dalam masyarakat dan internet dapat
merambah seluruh dunia bukan hanya
terbatas pada suatu wilayah (S3,O3,
O4)
2. Mempertahankan kepercayaan dari
pelaku bisnis dengan peningkatan
kinerja perusahaan dengan melakukan
pelatihan-pelatihan karyawan yang
dibarengi
dengan
penggunaan
teknologi (S1, S3, O2)
3. Meningkatkan
layanan
kepada
masyarakat
dengan
berinteraksi
lanrgsung
melalui
internet,
memberikan layanan secara online ,
memberikan pengetahuan mengenai
produk perusahaan kepada konsumen-
konsumen ditempat yang jauh secara
langsung dan memberikan laporan
kepada investor guna menambah
kepercayaan
mereka. (S1,S2,
S3,O2,O3,O4,O5)
Strateg
1. M
de
de
pr
pa
pa
2. M
pr
pe
dih
da
(O
3. M
me
ma
su
me
se
me
(O
Ancaman (Threatness)
1. Tingkat persaingan
2. Resesi global
3. Ancaman perusahaan asing
4. Produk palsu
5. Perubahan kebutuhan dan kepuasan
konsumen
Strategi ST
1. Terus
mengikuti
perkembangan
teknologi yang dapat menambah
kualitas
produk
dan
berusaha
mengaplikasi
teknologi
tersebut
dalam perusahaan karena tingginya
tingkat persaingan, maka perusahaan
harus dapat berinovasi baik dalam
produk maupun kinerja perusahaan
(S1,S3,T1,T3,T5). 2. Memberikan jasa bagi pelanggan
yang membeli produk perusahaan
agar produk yang dihasilkan tetap
menjadi pilihan utama pelanggan. Menempatkan
karyawan-karyawan
yang berkualitas dalam posisi tersebut
(S1,S2,S3,T1,T3,T4). 3. Perusahaan
dapat
menugaskan
karyawan yang berpotensi untuk
menarik investor-investor baru untuk
mengatasi dampak resesi global. Perusahaan harus dapat menunjukkan
kemampuan
dalam
menghadapi
pesaing dan ancaman yang ada untuk
menarik Investor(S1,S3,T2,T1). Strateg
1. M
ris
pe
2. M
pe
so
(W
3. M
da
pe
tid
ha
seh
(W
Tabel 3 Matriks SWOT PT Cahaya Buana Furindot Tabel 3 Matriks SWOT PT Cahaya Buana Furindotama Kekuatan (Strengths)
Kualitas SDM
•
Memiliki banyak distributor
•
Tingkat teknologi yang tinggi
•
Letak perusahaan yang strategis
•
Memiliki hubungan dengan supplier
•
besar
Kelemahan (Weakness)
Kurang pengetahuan tentang pelanggan. •
Variasi produk kurang
•
Kurangnya iklan mengenai produk
•
Kurang kepercayaan terhadap pemakaian
•
internet
pemakaian kertas yang berlebih. 3. Melakukan pendekatan kepada konsumen
dan memberikan pengenalan produk dengan
penggunaan iklan sehingga masyarakat
tidak keliru dengan produk-produk palsu,
hal ini membantu pencitraan perusahaan
sehingga
dapat
mengalahkan
pesaing
(W2,W3,T1,T3,T4) APPENDIX Meningkatkan efektifitas produk iklan atas
produk dengan merencanakan lebih matang
pemasangan iklan yang strategis yang
diharapkan dapat meningkatkan pengenalan
dan
kepercayaan
kepada
perusahaan
(O2,W3) 2. Mempertahankan kepercayaan dari
pelaku bisnis dengan peningkatan
kinerja perusahaan dengan melakukan
pelatihan-pelatihan karyawan yang
dibarengi
dengan
penggunaan
teknologi (S1, S3, O2) 3. Mengurangi penggunaan kertas dengan
mengadopsi serta mengembangkan strategi
manajemen
perusahaan
asing
karena
sudah banyak perusahaan asing umumnya
menghindari pemakaian kertas yang berlebih,
selain itu hal ini merupakan salah satu upaya
mengenali situasi perusahaan internasional
(O4, W5) 3. Mengurangi penggunaan kertas dengan
mengadopsi serta mengembangkan strategi
manajemen
perusahaan
asing
karena
sudah banyak perusahaan asing umumnya
menghindari pemakaian kertas yang berlebih,
selain itu hal ini merupakan salah satu upaya
mengenali situasi perusahaan internasional
(O4, W5) 3. Meningkatkan
layanan
kepada
masyarakat
dengan
berinteraksi
lanrgsung
melalui
internet,
memberikan layanan secara online ,
memberikan pengetahuan mengenai
produk perusahaan kepada konsumen-
konsumen ditempat yang jauh secara
langsung dan memberikan laporan
kepada investor guna menambah
kepercayaan
mereka. (S1,S2,
S3,O2,O3,O4,O5) 1. Terus
mengikuti
perkembangan
teknologi yang dapat menambah
kualitas
produk
dan
berusaha
mengaplikasi
teknologi
tersebut
dalam perusahaan karena tingginya
tingkat persaingan, maka perusahaan
harus dapat berinovasi baik dalam
produk maupun kinerja perusahaan
(S1,S3,T1,T3,T5). 2. Memberikan jasa bagi pelanggan
yang membeli produk perusahaan
agar produk yang dihasilkan tetap
menjadi pilihan utama pelanggan. Menempatkan
karyawan-karyawan
yang berkualitas dalam posisi tersebut
(S1,S2,S3,T1,T3,T4). 3. Perusahaan
dapat
menugaskan
karyawan yang berpotensi untuk
menarik investor-investor baru untuk
mengatasi dampak resesi global. Perusahaan harus dapat menunjukkan
kemampuan
dalam
menghadapi
pesaing dan ancaman yang ada untuk
menarik Investor(S1,S3,T2,T1). 23 Perancangan Aplikasi E-SCM... (Honni; dkk) an
Sistem Informasi
PT. Cahaya Buana Furindotama
Login
Melihat Daftar PO
Membuat PO
Supplier
Melihat Daftar
Bahan Baku
Melihat Daftar
Tender
Mengajukan
Penawaran Tender
Membuat Tender
Melihat Inbox
Membalas Message
Mengubah Profil
Memberikan Kritik
dan Saran
Melihat Status
Pembayaran
Membuat Surat Jalan
Melihat Daftar
Surat Jalan
Menambah Daftar
Supplier
Melihat Daftar User
Gambar 4 Use Case Aplikasi e-SCM Upstream
PT Cahaya Buana Furindotama
Gambar 5 User Interface Layar Home Karyawan Gambar 4 Use Case Aplikasi e-SCM Upstream
PT Cahaya Buana Furindotama Gambar 5 User Interface Layar Home Karyawan Gambar 5 User Interface Layar Home Karyawan 24 CommIT, Vol. 2 No. 1 Mei 2008, hlm. 17 - 24
|
https://openalex.org/W2112988638
|
https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1003257&type=printable
|
English
| null |
Human Cytomegalovirus Infection Elicits New Decidual Natural Killer Cell Effector Functions
|
PLOS pathogens
| 2,013
|
cc-by
| 15,016
|
Received April 5, 2012; Accepted February 5, 2013; Published April 4, 2013 Received April 5, 2012; Accepted February 5, 2013; Published April 4, 2013 Copyright: 2013 Siewiera et al. This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. wiera et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: JS is supported by a PhD Fellowship from the French Ministe`re de l’Education Nationale de la Recherche et de la Technologie. Financial support was
provided by INSERM, CNRS and Toulouse University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: nabila.jabrane-ferrat@inserm.fr Johan Siewiera1,2,3, Hicham El Costa1,2,3, Julie Tabiasco1,2,3, Alain Berrebi4, Ge´raldine Cartron4,
Philippe Bouteiller1,2,3, Nabila Jabrane-Ferrat1,2,3* 1 Institut National de la Sante´ et de la Recherche Me´dicale; UMR 1043, Toulouse, France, 2 Centre National Recherche Scientifique; UMR 5282, Toulouse, France,
3 Universite´ Toulouse III Paul Sabatier, Toulouse, France, 4 Service de Gyne´cologie-Obste´trique, Centre Hospitalo-Universitaire de Toulouse, Hoˆpital Paule de Viguier,
Toulouse, France PLOS Pathogens | www.plospathogens.org Introduction dNK cells during pregnancy is not yet fully understood. Their
contribution to successful placentation versus their potential ability
to exert cytotoxicity remains a major paradox [8,9]. By secreting a
unique profile of cytokines/chemokines and angiogenic factors,
dNK cells are thought to be crucial for successful placentation and
materno-fetal
immune tolerance
[9–15]. dNK
cells exhibit
different phenotypic and functional characteristics from other
peripheral blood NK cells (pNK). The majority of dNK cells are
CD56brightCD16neg and they express a repertoire of activating and
inhibitory receptors (NKRs) that resembles that of early differen-
tiation stages of pNK cells [9,16–19]. The lack of dNK cell
cytotoxicity has been attributed to defects in the formation of the
immunological synapse and/or failure of 2B4 receptor to convey
activating signals [8,20,21]. Human cytomegalovirus (HCMV) infection is mostly asymp-
tomatic in healthy adults and results in the establishment of long
term latency. On the contrary, life threatening diseases may occur
in immunocompromised patients after viral reactivation or
primary HCMV infections. HCMV is the most common cause
of intra-uterine viral infections and a leading cause of congenital
infection [1,2]. Even though maternal-fetal transmission is not
systematic [3], the prevalence of HCMV transmission is about
30% in the first trimester of pregnancy and can reach up to 72%
in the third trimester [4]. It is believed that the first steps of
infection and amplification take place in the decidua where both
maternal and fetal cells are in close contact [5]. Human placentation is associated with a large increase of
decidual NK cells (dNK). During the first trimester of pregnancy,
dNK cells are the major population of maternal immune cells as
they count for 70% of total immune cells present in the decidua in
the first trimester of pregnancy [6,7], whereas other immune cells,
macrophages, T cells (including CD8, CD4 and cd T cells) and
dendritic cells count for 20, 10 and 2% respectively. The role of In contrast to the clearly defined role of human and mouse pNK
cells in controlling viral infections [22–31], little is known about
the ability of dNK cells to control viral infections during
pregnancy [17,32,33]. Abstract During the first trimester of pregnancy the uterus is massively infiltrated by decidual natural killer cells (dNK). These cells are
not killers, but they rather provide a microenvironment that is propitious to healthy placentation. Human cytomegalovirus
(HCMV) is the most common cause of intrauterine viral infections and a known cause of severe birth defects or fetal death. The rate of HCMV congenital infection is often low in the first trimester of pregnancy. The mechanisms controlling HCMV
spreading during pregnancy are not yet fully revealed, but evidence indicating that the innate immune system plays a role
in controlling HCMV infection in healthy adults exists. In this study, we investigated whether dNK cells could be involved in
controlling viral spreading and in protecting the fetus against congenital HCMV infection. We found that freshly isolated
dNK cells acquire major functional and phenotypic changes when they are exposed to HCMV-infected decidual autologous
fibroblasts. Functional studies revealed that dNK cells, which are mainly cytokines and chemokines producers during normal
pregnancy, become cytotoxic effectors upon their exposure to HCMV-infected autologous decidual fibroblasts. Both the
NKG2D and the CD94/NKG2C or 2E activating receptors are involved in the acquired cytotoxic function. Moreover, we
demonstrate that CD56pos dNK cells are able to infiltrate HCMV-infected trophoblast organ culture ex-vivo and to co-localize
with infected cells in situ in HCMV-infected placenta. Taken together, our results present the first evidence suggesting the
involvement of dNK cells in controlling HCMV intrauterine infection and provide insights into the mechanisms through
which these cells may operate to limit the spreading of viral infection to fetal tissues. Citation: Siewiera J, El Costa H, Tabiasco J, Berrebi A, Cartron G, et al. (2013) Human Cytomegalovirus Infection Elicits New Decidual Natural Killer Cell Effector
Functions. PLoS Pathog 9(4): e1003257. doi:10.1371/journal.ppat.1003257 Editor: Stipan Jonjic, University of Rijeka, Croatia Received April 5, 2012; Accepted February 5, 2013; Published April 4, 2013 Author Summary Human cytomegalovirus (HCMV) is a herpes virus that can
establish persisting infection in immunocompetent hosts. HCMV primary infection during pregnancy is devastating;
it can result in up to 75% of congenital infections and it is a
known cause of fetal death. The immune system and
particularly natural killer cells (NK) are known to play a key
role in the clearance of several viruses in healthy adults. Whether decidual NK cells (dNK), present in the pregnant
uterus, have a role during HCMV infection is not known. We analyze changes in dNK cell function and phenotype in
the presence of HCMV-infected targets in an autologous
setting. We demonstrate the acquisition of cytotoxic
profile which is associated with changes in dNK cell
receptor repertoire and cytokine production. Finally, we
find that dNK cells are able to sense HCMV infection,
migrate and infiltrate infected tissues both in tissular organ
culture and in situ in infected placenta. Together our
results present the first report demonstrating the involve-
ment of dNK cells in controlling HCMV infection. To further confirm the cytotoxic function of dNK cells, we next
investigated lytic capacities of dNK cells in an MHC mismatched
(heterologous) setting (Figure S2D–E). dNK cells were purified
from one decidua basalis and their killing activity was tested against
either
uninfected
or
HCMV-infected
heterologous
decidual
fibroblasts. While very little killing was observed after 4 h of
contact (Figure S2D), up to 60% of uninfected and HCMV-
infected heterologous fibroblasts were killed after 18 h of contact
(Figure S2E). To exclude any external bias that could be
responsible for initiating dNK cell cytotoxicity against heterolo-
gous fibroblasts, we tested the ability of dNK cells to kill K562
classical NK cell targets (Figure S2F). In agreement with previous
studies [21], very little lysis was observed in the presence of dNK
cells while pNK cells killed up to 75% of K562 cells (Figure S2F). Further analyses demonstrate that while dNK cells killed more
than 55% of HCMV-infected autologous fibroblasts after 18 h of
contact, they did not kill semi-allogeneic fetal trophoblasts (Figure
S2G). In the same manner pNK cells did not kill semi-allogeneic
trophoblasts (data not shown). involved in limiting HCMV viral spreading to fetal tissues. To test
this possibility, we have conducted detailed analysis of functional
and phenotypic changes of first trimester of pregnancy dNK cells
after their exposure to infected target autologous fibroblasts. Author Summary We
found that dNK cells acquire cytotoxic effector function that is
associated with phenotypic alterations in their receptor repertoire
expression and involves key receptor-ligand pairs. Furthermore,
we found that dNK cells were able to sense HCMV infection,
migrate and infiltrate placental tissues both in tissue organ culture
and in situ in HCMV-infected placenta. These results suggest that
dNK cells control HCMV spreading across mucosal tissues
probably through the acquisition of cytotoxic profile. These observations suggest that dNK cells that are tolerant both
in vivo and in vitro to semi-allogeneic fetal trophoblasts become
activated when there is a danger signal such as HCMV-infection. Results NK cells achieve target cell killing either through delivery of
soluble mediators or by triggering death receptor-ligand pathways
such as Fas ligand (FasL) or the tumor necrosis factor-related
apoptosis-inducing ligand (TRAIL). To provide insights into the
mechanisms involved in dNK cell killing of HCMV-infected
fibroblasts, we investigated the involvement of the death receptor-
ligand pathway (Figure 1D–E). We used neutralizing antibodies to
either TRAIL or FasL that are expressed on dNK cells, to block
their interaction with cognate death receptors expressed on target
cells. After 18 h of co-culture, the blockade of either FasL
(Figure 1D) or TRAIL (Figure 1E) did not affect dNK cell
cytotoxicity against HCMV-infected autologous fibroblasts. The
blocking ability of both mAbs was confirmed since they prevented
TRAIL- or FasL-induced killing of Jurkat cell line (see Figure
S2H). These data strongly suggest that dNK cell killing of HCMV-
infected fibroblasts proceeds through mechanisms independent of
the death receptor-ligand pathways. Decidual NK cells efficiently kill HCMV-infected
autologous target cells Our previous study provided evidence indicating that cytolytic
function of dNK cells during normal pregnancy is partially
controlled by negative signals that involve NKG2A receptor [9]
suggesting that such function might be modulated upon viral
infections. Therefore, to test the possible involvement of dNK cells
in controlling HCMV infection we examined their cytotoxic
effector function against HCMV-infected autologous decidual
fibroblasts. dNK cells and decidual fibroblasts (Figure S1A) were
purified from the same decidua basalis. High purity fibroblasts
(Vimentinpos and Cytokeratin-7neg, Figure S1A) were infected with
two strains of HCMV; the VHLE clinical isolate and the
laboratory strain AD169. Decidual fibroblasts were efficiently
infected by both strains as evidenced by staining for HCMV-IE
nuclear protein (Figure S1B and data not shown) where more than
60%63 (mean 6 S.D.) of cells were infected after 48 h (Figure
S1C). Uterine NK Cell Response to HCMV Infection targets. Significant increases were also observed in pNK cell lysis
of HCMV-infected autologous fibroblasts after 18 h of contact
(Figure S2C). Given that no major differences were observed
between VHLE or AD169 strains, we extended the analysis of
dNK cell cytotoxicity to a cohort of 10 decidua basalis and
confirmed that dNK cells can specifically kill AD169-infected
fibroblasts, although with some variable efficiency (Figure 1C). Taken together, these data suggest that under HCMV infection
dNK cells become cytotoxic against infected autologous fibro-
blasts. PLOS Pathogens | www.plospathogens.org Introduction dNK cells represent the major decidual
lymphoid population in the first trimester of pregnancy [7,6] and
vertical transmission of HCMV to the fetus is quite low during this
trimester, therefore it is conceivable that dNK cells might be April 2013 | Volume 9 | Issue 4 | e1003257 1 PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection dNK cells engage immune synapse with HCMV-infected
autologous fibroblasts and polarize their lytic machinery
towards HCMV-infected targets Through co-cultures in autologous settings, we then investigated
the cytotoxicity of dNK cells by conventional chromium release
assay. Neither dNK cells nor pNK cells killed efficiently HCMV-
infected decidual fibroblasts after 4 h of contact (Figure S2A & B). However, after 18 h dNK cells efficiently killed VHLE- or AD169-
infected fibroblasts (Figure 1A & B). With both strains up to 75%
of killing was reached at the effector to target ratio of 50 and no
killing of autologous uninfected fibroblasts was observed indicating
the specificity of the cytotoxic function against HCMV-infected The delivery of perforin/granzyme lethal hits is a highly
regulated multistep mechanism that involves the formation of a
dynamic structure, namely immunological synapse (IS), between
NK cell and its target [34]. We undertook a stepwise approach to
dissect the involvement of perforin-induced killing mechanisms. First, we analyzed the capacity of dNK cells to form IS with
autologous targets. Conjugates formation between dNK cells and April 2013 | Volume 9 | Issue 4 | e1003257 2 Figure 1. dNK cells are cytotoxic against HCMV-infected autologous fibroblasts. Decidual fibroblasts were kept uninfected or inf
48 h with HCMV. dNK cell cytotoxicity was determined by 51Cr-release assay after 18 hours of contact at different E/T ratios. (A) Fibrobla
infected with VHLE clinical isolate (n = 3), (B) cells infected with AD169 laboratory strain of HCMV (n = 5). (C) Analysis of dNK cell cytotoxici
cohort of 10 decidua samples at the 50 to 1 ratio. (D) dNK cells were pre-incubated with anti-FasL or (E) anti-TRAIL blocking mAbs at
concentration of 10 mg/ml for 20 min and cytotoxicity was monitored after 18 h. Control (CTRL), lysis performed in the presence of IgG con
data point is calculated as the mean lysis 6 S.D. from at least five independent experiments done in replicate tissue culture wells. S
comparisons of mean lysis of uninfected versus HCMV-infected were performed using two-way ANOVA test. ***, p,0.001; **, p,0.01
significant, p.0.05. doi:10.1371/journal.ppat.1003257.g001
Uterine NK Cell Response to HCMV Uterine NK Cell Response to HCMV Infection Figure 1. dNK cells are cytotoxic against HCMV-infected autologous fibroblasts. Decidual fibroblasts were kept uninfected or infected for
48 h with HCMV. dNK cell cytotoxicity was determined by 51Cr-release assay after 18 hours of contact at different E/T ratios. (A) Fibroblasts were
infected with VHLE clinical isolate (n = 3), (B) cells infected with AD169 laboratory strain of HCMV (n = 5). Uterine NK Cell Response to HCMV Infection MTOC, the secretory machinery and clustering of CD2 activating
receptor at the intercellular contact zone. uninfected/HCMV-infected autologous decidual fibroblasts was
analyzed after 20 min of interaction by monitoring F-actin
remodeling
and
confocal
microscopy. Although
dNK
cells
recognized both uninfected and HCMV-infected target cells, as
evidenced by their actin-enriched flattened shape (Figure S3A),
only 17% uninfected cells were engaged in conjugates with dNK
cells while more than 55% AD169-infected fibroblasts were
recognized by dNK cells (Figure S3B). Thus, dNK cells form
conjugates preferentially with HCMV-infected fibroblasts and
reorganize their F-actin cytoskeleton at 20 min. uninfected/HCMV-infected autologous decidual fibroblasts was
analyzed after 20 min of interaction by monitoring F-actin
remodeling
and
confocal
microscopy. Although
dNK
cells
recognized both uninfected and HCMV-infected target cells, as
evidenced by their actin-enriched flattened shape (Figure S3A),
only 17% uninfected cells were engaged in conjugates with dNK
cells while more than 55% AD169-infected fibroblasts were
recognized by dNK cells (Figure S3B). Thus, dNK cells form
conjugates preferentially with HCMV-infected fibroblasts and
reorganize their F-actin cytoskeleton at 20 min. We next examined whether dNK cells were able to degranulate
upon recognition of HMCV-infected fibroblasts by analyzing the
cell surface expression of the Lysosomal-associated membrane
protein 1 (LAMP1/CD107a) (Figure 2D). The kinetics of CD107a
cell surface expression by dNK cells in contact with HCMV-
infected autologous fibroblasts was carried out for 8 hours. Very
little variations were observed within the first four hours of contact. After six hours of contact, a significant increase of CD107a
expression was observed in dNK cells that are in contact with
HCMV-infected
autologous
fibroblasts. The
degranulation
reached maximal level by 8 hours of contact (Figure 2D). The
significant increase in CD107a cell surface expression indicates
that IS formation is accompanied by efficient release of lytic
granules and that dNK cells cytotoxicity is perforin-dependent but
only after six hours of contact. Being critical for the trafficking and delivery of lytic granules to
the IS in NK cells [35,36], we then analyzed the microtubule
organizing center (MTOC) (Figure 2A) and the Golgi apparatus
polarization (Figure S3A) in fixed conjugates after 20 min of
interaction. dNK cells in contact with uninfected cells displayed a
random localization of the MTOC (Figure 2A). In contrast, the
majority of conjugates formed with HCMV-infected targets
displayed a reoriented dNK cell MTOC towards the immune
synapse (Figure 2A). HCMV infection modulates dNK cell receptor repertoire HCMV infection modulates dNK cell receptor repertoire
The repertoire of NK activating and inhibitory receptors
(NKRs) plays a critical role in cytotoxic activity of pNK cells
and modulation of NKRs expression by these cells is often
associated with their response to HCMV [30]. Thus, to provide
further insights into the mechanisms involved in dNK cell cytolytic
activity against HCMV, we analyzed whether these cells modulate
their NKRs repertoire upon recognition of infected fibroblasts
(Figure 3). Similar to freshly isolated dNK cells (data not shown),
more than 76.365% (mean 6 S.D.) of dNK cells co-cultured with
uninfected autologous fibroblasts were CD56bright (Figure 3). Exposure to HCMV-infected fibroblasts significantly decreased
the percentage of CD56bright dNK cells (4866.3%), but signifi-
cantly increased the percentage of CD56dim cells (4064%). The
dampening down of CD56 expression was observed even after
18 hours of contact (Figure S4B) consistent with the acquisition of
the cytotoxic profile. The changes in CD56 expression profile is
always associated with the acquisition of CD16 expression (41%
compared to 4.3%). There was a slight decrease in the mean
fluorescence intensity of CD69 but the absolute number of
CD69pos dNK cells (8565%) did not vary after contact with
HCMV-infected fibroblasts. Although optimal changes were
reached by 48 h, our data demonstrate that HCMV infection
orchestrate dampening of CD56 and increase of CD16 expression
observed as early as 18 h of contact (Figure S4B) which is
consistent with acquisition of a cytotoxic profile. We next analyzed the distribution of lytic granules containing
perforin after 20 min of conjugation (Figure 2). Similar to the
MTOC, perforin containing granules were localized in a random
manner, but upon recognition of HCMV-infected cells, dNK cells
polarized their perforin containing granules with the MTOC close
to the contact zone (Figure 2A). Quantification of perforin
polarization in a large number of immune synapses, demonstrated
that while the majority of dNK cells that formed immune synapse
with AD169-infected fibroblasts showed polarization of their lytic
granules (8364%), only 28% of dNK cells showed polarization
towards uninfected targets (Figure 2C). Interestingly, when using a
mixture of infected and non infected cells (one to one ratio), dNK
cells polarize their MTOC and secretory machinery preferentially
towards HCMV-infected fibroblasts (data not shown). dNK cells engage immune synapse with HCMV-infected
autologous fibroblasts and polarize their lytic machinery
towards HCMV-infected targets (C) Analysis of dNK cell cytotoxicity from a
cohort of 10 decidua samples at the 50 to 1 ratio. (D) dNK cells were pre-incubated with anti-FasL or (E) anti-TRAIL blocking mAbs at the final
concentration of 10 mg/ml for 20 min and cytotoxicity was monitored after 18 h. Control (CTRL), lysis performed in the presence of IgG control. Each
data point is calculated as the mean lysis 6 S.D. from at least five independent experiments done in replicate tissue culture wells. Statistical
comparisons of mean lysis of uninfected versus HCMV-infected were performed using two-way ANOVA test. ***, p,0.001; **, p,0.01; ns, not
significant, p.0.05. doi:10.1371/journal.ppat.1003257.g001 April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org 3 Uterine NK Cell Response to HCMV Infection PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection We then finely defined the MTOC
reorientation by measuring the distance between dNK cell MTOC
and the center of the IS for each conjugate (defined as the center of
the interaction zone dNK cell-target, see scheme Figure 2B). The
distance between the MTOC and the center of IS showed a quite
compact distribution in dNK cells that contacted AD169-infected
fibroblasts with a mean distance of 4.661.25 mm (mean 6 S.D.)
(Figure 2B). In contrast, the distance from the MTOC to the
center of the contact zone was very variable in dNK cell that
formed conjugates with uninfected cells (Figure 2B) with a mean
distance of 9.163.4 mm (mean 6 S.D.). Collectively, these findings indicate that dNK display cytotoxic
activity towards HCMV-infected autologous decidual fibroblasts
but also emphasize the unique properties of dNK cells cytotoxicity. Even if dNK cells can form mature IS within normal range of time
they do need extended time frame in order to release their lytic
granules and perform efficient killing of HCMV-infected autolo-
gous fibroblasts. HCMV infection modulates dNK cell receptor repertoire By contrast to NKp30L, ligands
for NKp44, and NKG2D were highly expressed in uninfected
decidual fibroblasts (Figure 4A, S5A). Both HCMV strain induced
significant decreases in the expression of NKp44L and NKG2DL
(Figure 4A, S5A). decreased after HCMV infection. By contrast to NKp30L, ligands
for NKp44, and NKG2D were highly expressed in uninfected
decidual fibroblasts (Figure 4A, S5A). Both HCMV strain induced
significant decreases in the expression of NKp44L and NKG2DL
(Figure 4A, S5A). We then investigated whether HCMV infection affected the
expression level of HLA-E cell surface molecules. As shown in
figure 4A (and S5A), decidual fibroblasts expressed both the
nonclassical HLA-E and the classical HLA-A,-B,-C molecules at
their surface. While infection with HCMV resulted in a significant
decrease in HLA-E expression, only small effect was observed for
the expression of classical HLA-A,-B,-C. This striking observation
of HLA-E downregulation by HCMV prompted us to perform
further analyses comparing the impact of HCMV infection in
additional decidual fibroblasts and in other cells (Figure S5B). Consistently, we observed downregulation of cell surface expres-
sion of molecules HLA-E in HCMV-infected decidual fibroblasts
(Figure 4, S5A, S5B). Consistent with previous studies using HFFF
cells [42,43] and in contrast to decidual fibroblasts, HCMV
resulted in upregulation of cell surface HLA-E in MRC-5
fibroblasts and in HEK293T cells (Figure S5B and data not
shown). We also observed a small decrease in the level of HLA-A,-
B,-C in these cell lines (Figure S5B and data not shown). Western
blot analyses of total amount of HLA-E molecules demonstrated
that HCMV-infection did not affect total amount of HLA-E
proteins in decidual fibroblasts while increased levels were
observed in MRC-5 cells expression (Figure S5C). The CD94/
NKG2X (-A, -C or -E) family members recognize HLA-E
molecule but these receptors can transmit opposing signals
[23,44,45]. The differences between the two systems imply that
HCMV infection of decidual fibroblasts might trigger their
recognition and promote their killing through engagement of
CD94/NKG2C/E activating receptors. This is in line with
observed up-regulation of NKG2C on dNK upon their recogni-
tion of infected decidual fibroblast (Figure 3) and the high levels of
NKG2E on dNK cells [12]. It has been suggested that de novo expression of MHC-II by NK
cells and their acquisition of an APC-like phenotype could regulate
the activation of numbering immune cells in particular T cells. HCMV infection modulates dNK cell receptor repertoire Consistent
with the MTOC and lytic granules, the Golgi apparatus was also
distributed in clusters close to the MTOC only in dNK cells that
formed immune synapses with AD169-infected fibroblasts (Figures
S3A), but not in those that formed conjugates with uninfected
targets (Figure S3A see right enlargement panels). One of the critical step in the NK-IS formation includes the
clustering of specific receptors that contribute to NK cell activation
[34]. Despite the fact that CD9 would have been a better choice as
it is mainly expressed by dNK cells but not pNK, decidual
fibroblasts (data not shown) and other human fibroblasts express
substantial amounts of this receptor [37,38] we choose to analyze
the localization of CD2 receptor for two main reasons. CD2 is
expressed on the majority of dNK [9,21] and it has been shown to
rapidly cluster at the NK-IS [34,39,40]. Confocal analyses
revealed that CD2 receptor microclusters were concentrated at
the intercellular contact zone only in dNK cells that formed
conjugates with infected fibroblasts (Figure S3C). We did not
observe any changes in CD56 localization (data not shown). Thus,
dNK cells engage mature immune synapse with HCMV-infected
autologous fibroblasts that is characterized by polarization of the To further characterize phenotypic changes in dNK cell
receptor repertoire, we analyzed the expression of natural
cytotoxicity receptors (NCRs) (NKp30, NKp44, and NKp46),
NKG2D that recognize viral or stress induced ligands and
NKG2A or C receptors that are expressed by a large fraction of
dNK cells and recognize HLA-E molecules (Figure 3). The
frequency of dNK cells expressing NKp44 activating receptor was
significantly increased in dNK cells that were exposed to HCMV-
infected fibroblasts as compared to those exposed to uninfected
fibroblasts (90% versus 46%). Furthermore, co-culture with
infected cells also induced major changes in the expression of
NKp46 receptor. A significant shift in the fluorescence intensity
towards an NKp46low profile with a complete loss of the bimodal April 2013 | Volume 9 | Issue 4 | e1003257 4 Uterine NK Cell Response to HCMV Infection April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection Figure 2. Polarization of the MTOC and lytic granules to the immune synapse formed with HCMV-infected fibroblasts. Uninfected
(AD1692) or HCMV-infected (AD169+) decidual fibroblasts (F) plated on glass coverslips were incubated with autologous dNK cells (dNK) for 20 min at
37uC. HCMV infection modulates dNK cell receptor repertoire Therefore, to further examine the modulation in dNK cells
properties and phenotype upon exposure to HCMV-infected
fibroblasts we examined the expression of HLA-DR in dNK cells
co-cultured with infected and non-infected cells (Figure 3). A great
fraction of dNK cells exposed to HCMV-infected fibroblasts, but
not uninfected cells, acquired significant de novo expression of
MHC-II DR at their cell surface (48%) displaying a bimodal
distribution of fluorescence with a prevalence of positive cells
expressing intermediate levels of these cell surface molecules. The
acquisition of HLA-DR expression was effective even after
18 hours of contact (Figure S4B). Increases of HLA-DR expression
were also observed in pNK cells that were in contact with HCMV-
infected autologous fibroblasts (Figure S4C). Taken together, these data show that exposure to HCMV-
infected fibroblasts not only modulates dNK cell receptor
repertoire but also increases the expression of key elements of
adaptive response (HLA-DR). Using Fc-chimeric proteins to block specific receptor/ligand
interactions, we found that neither blockade of NKp30 (Figure 4B)
nor of NKp46 (Figure 4C), both modulated upon HCMV
infection, interaction with their putative ligand(s) had an effect
on dNK cell killing of autologous HCMV-infected fibroblasts. Blocking the interaction of NKp44 activating receptor with its
ligand resulted in 50% increased killing of infected autologous
fibroblasts (Figure 4D). In contrast, interference with NKG2D
receptor ligation induced a significant decrease in dNK cell
cytotoxicity; the mean lysis of HCMV-infected fibroblasts was
50% whereas only 20% of infected cells were lysed in the presence
of NKG2D-Fc chimeric protein (Figure 4E). The decrease in
cytotoxicity after blockade of NKG2D ligation to its cognate PLOS Pathogens | www.plospathogens.org HCMV infection modulates dNK cell receptor repertoire (A) Representative images of maximum intensity projection. Microtubules (a-tubulin in green), lytic granules containing Perforin (red), HCMV-IE
antigen (blue). Arrowhead points to the MTOC polarization (aster). Bar represent 20 mm. (B) Left cartoon shows schematic representation of the
immunological synapse (IS), D (distance in mm). The center of IS was defined as the center of the contact zone between dNK and target cell (see
cartoon, red line). Zero on the Y axis (mm) represents synaptic area; blue dot represents the microtubule organizing center (MTOC) and microtubules
are in green. The MTOC polarization (Right graph) defined by the distance between the MTOC and the center of IS formed with uninfected (AD1692)
and HCMV-infected (AD169+) fibroblasts. Distances were calculated for 50 conjugates from five independent experiments. Statistical analysis was
performed using unpaired Student’s t-test. ***, p,0.001. (C) Percentage of conjugates showing polarized perforin containing granules to the NKIS. Results from 5 independent conjugations were averaged, values represent means and S.D.s. At least 300 conjugates were analyzed. Statistical analysis
was performed using unpaired Student’s t-test. *** p = 0.0002. (D) Kinetic of CD107a cell surface expression was analyzed by flow cytometry on dNK
cells that were in contact with uninfected or AD169-infected autologous fibroblasts. Values presented in the bar graphs are mean values calculated
from three independent experiments done in triplicates at the ratio 1 to 1. Error bars are SEM. Statistical comparisons were performed using unpaired
Student’s t-test, ** p,0.01. d i 10 1371/j
l
1003257 002 doi:10.1371/journal.ppat.1003257.g002 NKp46hi and NKp46low expression pattern was observed when
dNK cells were exposed to HCMV-infected cells. More than 80%
of dNK cells become NKG2C+ after their exposure to HCMV-
infected fibroblasts, while only minor yet reproducible decreases in
the percentage of NKG2A+ cells was observed. Exposure to
HCMV-infected cells induced significant decrease in the percent-
age of cells expressing KIR2DL1, KIR2DL4 and ILT-2, while no
changes were observed with those expressing KIR2DL2/3 (Figure
S4A). Some of the changes in the expression of dNK cell repertoire
were observed after 18 hours of contact (Figure S4B) while only
discrete changes were observed for pNK cells (Figure S4C) further
highlighting the originality of dNK cells and stretching their
differences compared to pNK cells. Altogether, our data indicate
that HCMV infection induces major changes in dNK cell receptor
repertoire with increases in NKp44, NKG2C and decreases in
NKp46, KIR2DL1, KIR2DL4 and ILT2 expression. decreased after HCMV infection. NKG2D and CD94/NKG2 activating receptors modulate
dNK cell responsiveness to HCMV-infected fibroblasts Cytotoxic function of NK cells could involve several NKRs. To
provide insights to their possible involvement in dNK cell
cytotoxicity against HCMV-infected fibroblasts, we took advan-
tage of Fc-chimeras to analyze NKR ligands expression in
uninfected,
AD169-infected
(Figure
4A),
or
VHLE-infected
(Figure S5A) decidual fibroblasts. Uninfected fibroblasts expressed
low levels of NKp30L. Similar to human fetal foreskin fibroblasts
(HFFF) [41], HCMV infection led to an increase in NKp30L
expression by decidual fibroblasts (Figure 4A, S5A). Decidual
fibroblasts expressed low levels of NKp46L that was further April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org 6 Uterine NK Cell Response to HCMV Infection ligands expressed on HCMV-infected fibroblasts underscored a
Since HLA-E is a ligand for both inhibitory CD94/NKG2A
Figure 3. Exposure to infected cells modulates dNK cell receptor repertoire expression. dNK cells were co-cultured with autologous
fibroblasts that were either uninfected or infected with HCMV-AD169 for 48 h. dNK cells were stained for surface expression of the indicated receptor
using fluorochrome-conjugated antibodies and analyzed by flow cytometry. Representative FACS histograms gated on CD56pos CD3neg dNK cells are
shown (n = 5). Specific receptors are indicated by the arrow. dNK cells in contact with uninfected fibroblasts are represented by black line, dNK cells in
contact with HCMV-infected fibroblasts are represented by shaded gray. Dotted gray line represents isotype-matched control Ig. One representative
histogram out of five independent experiments is shown. doi:10.1371/journal.ppat.1003257.g003
Uterine NK Cell Response to HCMV Infection Figure 3. Exposure to infected cells modulates dNK cell receptor repertoire expression. dNK cells were co-cultured with autologous
fibroblasts that were either uninfected or infected with HCMV-AD169 for 48 h. dNK cells were stained for surface expression of the indicated receptor
using fluorochrome-conjugated antibodies and analyzed by flow cytometry. Representative FACS histograms gated on CD56pos CD3neg dNK cells are
shown (n = 5). Specific receptors are indicated by the arrow. dNK cells in contact with uninfected fibroblasts are represented by black line, dNK cells in
contact with HCMV-infected fibroblasts are represented by shaded gray. Dotted gray line represents isotype-matched control Ig. One representative
histogram out of five independent experiments is shown. doi:10 1371/journal ppat 1003257 g003 Since HLA-E is a ligand for both inhibitory CD94/NKG2A
and activating CD94/NKG2C/E receptors, we explored its
involvement in dNK cell cytotoxic response against HCMV-
infected fibroblasts. To this end, we performed lysis assay in the
presence of an anti-HLA-E blocking monoclonal antibody. Uterine NK Cell Response to HCMV Infection Figure 4. Functional analysis dNK cells specific receptors. (A) HCMV-infection modulates the expression of NKR ligands on decidual
fibroblasts. The binding of human NKp30-Fc, NKp46-Fc, NKp44-Fc, and NKG2D-Fc chimera was used to evaluate the cell surface expression of specific
receptor ligands. The expression of HLA-E and HLA-A,-B,-C molecules was evaluated using specific mAb. Uninfected fibroblasts are represented by
black line, HCMV-infected fibroblasts (shaded gray). Dotted gray line represents represent negative control or isotype-matched control Ig. One
representative FACS histogram out of five independent experiments is shown. (B–E) Decidual fibroblasts uninfected (AD1692) or infected (AD169+)
were incubated with soluble receptor-Fc fusion protein at the concentration of 1 mg/ml and dNK cell cytotoxicity was analyzed by chromium release
assay after 18 h of co-culture. Control lysis was performed in the presence of CD99-Fc chimera (CTRL). Lysis analyzed in the presence of (B) NKp30-Fc,
(C) NKp46-Fc, (D) NKp44-Fc, (E) NKG2D-Fc. (F) Analysis of NKG2A and NKG2C function was performed in the presence of blocking antibody against
HLA-E molecules (a-HLA-E). Control lysis performed in the presence of isotype match control Ig (CTRL). Data sets represent mean lysis 6 S.D. from five
independent experiments done in replicate. Statistical comparisons were performed using two-way ANOVA test. ***, p,0.001; **, p,0.01. doi:10.1371/journal.ppat.1003257.g004 compared to 75% for IgG1 isotype control) (Figure 4F). This
inhibitory effect suggests that in our system model, HLA-E on
infected-fibroblasts binds to the CD94/NKG2C or -E activating
receptors rather than to CD94/NKG2A inhibitory receptor and
such binding could mediate the cytotoxic effect of dNK. (Figure 5C). On the other hand, the production of MIP-1b, IL-8,
IP10 (Figure 5B), GM-CSF, RANTES, MIP-1a (Figure 5C) was
significantly decreased after stimulation with HCMV-infected
cells. Finally, all other cytokines and chemokines tested were either
below cut-off levels (IFN-c, IFN-v, TGF-a, TNF-a/b, IL-1b, IL-
2, IL2RA, IL-4, IL-5, IL-10, IL-12, IL-15, IL-17A/F, EGF, E-
Selectin and Leptin) or did not vary after exposure to HCMV-
infected fibroblasts (basic FGF, IFN-a2, IFN-b, IL-1a, IL-1RA,
IL-22, SDF-1, sFas, sFasL, TRAIL, Eotaxin-3/CCL26, Fractalk-
ine/CX3CL1) (data not shown). Overall, these data demonstrate
that HCMV-infection modulates the secretory profile of dNK
cells, with increased production of cytotoxic factors that may
constitute virus-specific immune response. dNK cells infiltrate HCMV-infected trophoblast The maternal decidua is the main fetal-maternal interface where
maternal dNK cells are in close contact with invasive fetal
trophoblast. HCMV virions are believed to disseminate from
decidual cells to the invasive trophoblasts and in floating and
anchoring villous trophoblasts [5]. To support the relevance of our
results, we developed an organ culture model of trophoblastic villi
explants to assess the ability of dNK cells to infiltrate infected
tissues. Villous explants were isolated, infected (48 h) or not and
cultured for 2 h with autologous dNK cells that were labeled with
CellTraker Red. As shown in Figure 6A and supplementary
movies very few dNK cells were able to establish contact with
autologous uninfected trophoblast. However, large number of
dNK cells was able to infiltrate and establish close cellular contacts
within HCMV-infected organ explants. We were able to analyze
organ culture over 250 mm deep section and demonstrate that
dNK cells were able to formed synapse like structures with infected
cells throughout the section (see 3D-reconstitution movie). These
data demonstrate that dNK cells are able to sense and migrate
within the infected tissues. Exposure to infected fibroblasts modulates dNK cells
cytokine and chemokine secretion In normal pregnancy, dNK cells are known to secrete great
amount of soluble factors that play a key role in trophoblast
attraction and vasculature remodeling. Since some of dNK cell
soluble factors have also been found in HCMV secretome [46], we
first analyzed the secretion profile of uninfected and HCMV-
infected decidual fibroblasts, using a 42-multiplexed cytokine/
chemokine/growth factor Luminex assay (Figure S7). Decidual
fibroblasts produced GRO-a/CXCL-1, sICAM-1, IL-6, IL-8,
IP10, MCP-1, MIP1b, MIP1b, and VEGF-A. After HCMV-
infection, mild variations were observed for IL-8, MIP-1b and
VEGF-A without reaching statistical significance and only IL-6
secretion was significantly increased in AD169-infected decidual
fibroblasts (1.7 fold increase) (Figure S7). To examine whether
HCMV infection modulates dNK cell secretion profile, we
analyzed specific dNK cell secretion in co-cultures either with
uninfected or HCMV-infected autologous decidual fibroblasts
(Figure 5). Although large variations were observed amongst the
donors, only a limited number of secreted cytokines, chemokines
and growth factors varied after 24 h of co-culture with HCMV-
infected autologous targets (Figure 5). Similar to freshly isolated
dNK cells ([9] and data not shown), dNK cells that were in contact
with autologous uninfected decidual fibroblasts produced VEGF-
A, sICAM-1, GRO-a/CXCL-1, IL-6, Granzyme B (GZ-B)
(Figure 5A), MIP-1b/CCL4, IL-8/CXCL8 and IP-10/CXCL10
(Figure 5B). They also produced substantial amounts of GM-CSF,
RANTES/CCL5, MIP-1a/CCL3 and low amounts of MCP-1/
CCL2 (Figure 5C). Stimulation of dNK cells with HCMV-infected
fibroblasts led to significant increased secretion of VEGF-A (1.6-
fold), sICAM-1 (1.7-fold), GRO-a/CXCL-1 (2-fold), IL-6 (1.5-
fold), GZ-B (2.1-fold) (Figure 5A) and MCP-1/CCL-2 (3.5-fold) We then investigated the ability of dNK cells to interact with
infected tissues in vivo; we analyze biopsies of placental samples
from 24–26 weeks HCMV+ termination of pregnancy (Figure 6B). Thin sections of placental samples were analyzed by IHC for the
presence of NK cells using anti-CD56 marker and anti-CMV-IE
antibodies. Analysis of infected placenta showed that CD56pos cells
were present at the vicinity of infected HCMV positive cells
(Figure 6B) while no CD56pos cells were present in the HCMV
negative tissue. Together these results clearly demonstrate that
dNK (CD56pos) cells are able to infiltrate HCMV-infected tissue
both in vitro in organ culture model and in situ within HCMV+
placentas, providing thus solid evidence for the implication of
dNK cells in controlling HCMV infection and spreading. Uterine NK Cell Response to HCMV Infection Examination of pNK cell cytotoxicity shows that even though
some minor changes were constantly observed with NKp30,
NKp46 and NKp44 receptors (Figure S6B–D), only the NKG2D
receptor played a major role in the killing of HCMV-infected
autologous decidual fibroblasts (Figure S6E) as its blockade
resulted in significant decrease in pNK cell cytotoxicity against
autologous fibroblasts. The blockade of HLA-E did not impair
pNK cell cytotoxicity (Figure S6F). Taken together, our data uncover a crucial role of NKG2D and
CD94/NKG2C or -E activating receptors in dNK cell cytotoxic
response
against
HCMV-infected
fibroblasts,
while
neither
NKp30 nor NKp46 are implicated in dNK cell response. By
contrast to its activating role in peripheral blood NK [24], NKp44
have an inhibitory effect in the control of dNK cell cytotoxic
function. NKG2D and CD94/NKG2 activating receptors modulate
dNK cell responsiveness to HCMV-infected fibroblasts Blockade of HLA-E ligation with its cognate receptor resulted in
a two-fold decrease of the sensitivity to dNK cell lysis (36% ligands expressed on HCMV-infected fibroblasts underscored a
role for NKG2D receptor in dNK cell cytotoxicity. Since neither
NKp30-Fc nor NKp46-Fc had an effect on dNK cells lysis, we
tested the ability of these chimeras to block pNK cell cytotoxicity. The binding of either chimera significantly decreased the killing of
K562 cell line by pNK cells (Figure S6A) indicating that both
chimeras are functionally active. April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 7 Uterine NK Cell Response to HCMV Infection PLOS Pathogens | www.plospathogens.org April 2013 | Volume 9 | Issue 4 | e1003257 8 Uterine NK Cell Response to HCMV Infection Discussion Despite their importance in maintaining healthy pregnancy, the
control of maternal HCMV infection and spreading by dNK cells
is not yet fully understood. Our study is the first to assign a critical April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org 9 Uterine NK Cell Response to HCMV Infection Figure 5. HCMV infection modulates dNK cells cytokine/chemokine production. dNK cells were stimulated with uninfected (AD1692, gray)
or AD169-infected (AD169+, black) autologous decidual fibroblasts for 24 h. Cytokines were quantified in the supernatants using a 42-multi-plexed
cytokine assay. Representative histograms from selected cytokines-chemokines that showed significant differences are presented. (A & B) Soluble
factors that are produced at high levels by dNK cells. (C) Soluble factors that are produced at low levels by dNK cells. Concentrations are given as
differences between secretions of dNK cell in presence of uninfected or infected fibroblasts and the corresponding uninfected or infected fibroblasts. Normalized data points are given as mean 6 S.D. calculated as from four independent experiments. Statistical comparisons were performed using
Mann & Whitney test. **, p,0.01; *, p,0.05. doi:10.1371/journal.ppat.1003257.g005
Uterine NK Cell Response to HCMV Infection Figure 5. HCMV infection modulates dNK cells cytokine/chemokine production. dNK cells were stimulated with uninfected (AD1692, gray
or AD169-infected (AD169+, black) autologous decidual fibroblasts for 24 h. Cytokines were quantified in the supernatants using a 42-multi-plexe
cytokine assay. Representative histograms from selected cytokines-chemokines that showed significant differences are presented. (A & B) Solubl
factors that are produced at high levels by dNK cells. (C) Soluble factors that are produced at low levels by dNK cells. Concentrations are given a
differences between secretions of dNK cell in presence of uninfected or infected fibroblasts and the corresponding uninfected or infected fibroblast Figure 5. HCMV infection modulates dNK cells cytokine/chemokine production. dNK cells were stimulated with uninfected (AD1692, gray)
or AD169-infected (AD169+, black) autologous decidual fibroblasts for 24 h. Cytokines were quantified in the supernatants using a 42-multi-plexed
cytokine assay. Representative histograms from selected cytokines-chemokines that showed significant differences are presented. (A & B) Soluble
factors that are produced at high levels by dNK cells. (C) Soluble factors that are produced at low levels by dNK cells. Concentrations are given as
differences between secretions of dNK cell in presence of uninfected or infected fibroblasts and the corresponding uninfected or infected fibroblasts. Normalized data points are given as mean 6 S.D. Discussion dNK cells infiltrate infected placental tissues. (A) Two-color 3D-images o
trimester trophoblast either uninfected (AD1692) or HCMV-infected (AD169+). Nuclei we
and side panels show orthogonal XZ and YZ slices, respectively. Images are from two-ph
color IHC of 6-mm-thick sections from paraffin-embedded whole placental biopsies of
sections are presented. Representative immunostaining of HCMV-IE (blue, alkaline pho
staining) (n = 2). doi:10.1371/journal.ppat.1003257.g006 Figure 6. dNK cells infiltrate infected placental tissues. (A) Two-color 3D-images o
trimester trophoblast either uninfected (AD1692) or HCMV-infected (AD169+). Nuclei we
and side panels show orthogonal XZ and YZ slices, respectively. Images are from two-ph
color IHC of 6-mm-thick sections from paraffin-embedded whole placental biopsies of
sections are presented. Representative immunostaining of HCMV-IE (blue, alkaline pho
staining) (n = 2). doi:10.1371/journal.ppat.1003257.g006 Figure 6. dNK cells infiltrate infected placental tissues. (A) Two-color 3D-images of chorionic villi explants organ cultures established from first
trimester trophoblast either uninfected (AD1692) or HCMV-infected (AD169+). Nuclei were stained with dapi (cyan). Infiltrating dNK cells (red). Lower
and side panels show orthogonal XZ and YZ slices, respectively. Images are from two-photon Z-stack (total of 200 mm). Scale bars = 100 mm. (B) Two-
color IHC of 6-mm-thick sections from paraffin-embedded whole placental biopsies of HCMV+ pregnancy termination. HCMV+ and HCMV2 tissue
sections are presented. Representative immunostaining of HCMV-IE (blue, alkaline phosphatase staining) and CD56+ NK cells (brown, peroxidase
staining) (n = 2). doi:10 1371/journal ppat 1003257 g006 g
doi:10.1371/journal.ppat.1003257.g006 doi:10.1371/journal.ppat.1003257.g006 emphasizes the intrinsic ability of these cells to kill when they
are exposed to the right activating signals. Our observation that
dNK cells did not kill semi-allogeneic trophoblasts but killed
HCMV-infected autologous fibroblasts highlights their plasticity
and their specific ability to respond to HCMV infection. healthy conditions dNK cells are tolerant to semi-allogeneic fetal
trophoblasts. Although mechanisms that control cytotoxicity are
not well established, they may include strong interactions of
inhibitory receptors with their cognate ligands expressed by fetal
trophoblast, production of VEGF-C by dNK cells and/or
expression of anti-apoptotic proteins (XIAP) by target cells
[51,52]. The lack of dNK cell cytotoxicity can be reversed, at
least in vitro, after exposure to cytokines such as IL-5 and IL-18 or
upon engagement of specific activating receptors [9,21]. Here we
show that HCMV infection provides the necessary activating
signals to trigger dNK cell cytotoxicity. The fact that dNK cells
killed
heterologous
targets
from
a
different
donor
further healthy conditions dNK cells are tolerant to semi-allogeneic fetal
trophoblasts. Discussion calculated as from four independent experiments. Statistical comparisons were performed using
Mann & Whitney test. **, p,0.01; *, p,0.05. doi:10.1371/journal.ppat.1003257.g005 granules to the IS [34,47–50]. We show that although dNK cells
recognize and engage IS with HCMV-infected cells very rapidly,
they require longer exposure time in order to degranulate and
exert the cytotoxic effector function. The delay to unleash dNK
cell cytotoxic effector function might correspond to the time
necessary for dNK cells to mature and acquire necessary
functional changes to exert cytotoxicity. However, we cannot
exclude that HCMV-infected fibroblasts provide weak signal to
induce fast degranulation or that decidual fibroblasts have an
inherent resistance to cytotoxic granule mediated cell death. role to dNK cells in controlling maternal HCMV infection and in
limiting its spreading to fetal tissues through their capacity to
acquire potent cytotoxic activity when in contact with infected
decidual cells. During normal pregnancy, the majority of dNK
cells are CD56brightCD16neg. They secrete a large panel of
cytokines and chemokines that are necessary for placental
development. We demonstrate that dNK cells undergo phenotypic
and cellular changes that allow them to recognize and kill
autologous
HCMV-infected
cells in a
FasL- and TRAIL-
independent manner. Mechanisms that prevent dNK cell cytotoxicity are not
completely understood. Even though dNK and pNK cells exhibit
similar expression levels of cytotoxicity encoding genes [12], under Immunological synapse formation is a crucial step for the
delivery of lethal hits by effector cells. Rapid re-localization of the
MTOC is needed for the trafficking and the polarization of lytic PLOS Pathogens | www.plospathogens.org April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org April 2013 | Volume 9 | Issue 4 | e1003257 10 Uterine NK Cell Response to HCMV Infection Figure 6. dNK cells infiltrate infected placental tissues. (A) Two-color 3D-images of chorionic villi explants organ cultures established from first
trimester trophoblast either uninfected (AD1692) or HCMV-infected (AD169+). Nuclei were stained with dapi (cyan). Infiltrating dNK cells (red). Lower
and side panels show orthogonal XZ and YZ slices, respectively. Images are from two-photon Z-stack (total of 200 mm). Scale bars = 100 mm. (B) Two-
color IHC of 6-mm-thick sections from paraffin-embedded whole placental biopsies of HCMV+ pregnancy termination. HCMV+ and HCMV2 tissue
sections are presented. Representative immunostaining of HCMV-IE (blue, alkaline phosphatase staining) and CD56+ NK cells (brown, peroxidase
staining) (n = 2). doi:10.1371/journal.ppat.1003257.g006 Figure 6. April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection It has been clearly demonstrated that HCMV maintains
an inhibitory status either by preventing the cell-surface expression
of NKG2D activating ligands [55,56] or by UL40-mediated up-
regulation of HLA-E or MHC-I like surrogates molecules
expression. Although, there are some discrepancies between our
two observations, namely decreases of NKG2DL and acquired
cytotoxicity through NKG2D receptor, it is possible that decreases
in NKG2DL are selective resulting in the expression of high
affinity ligands. Alternatively, co-engagement of other activating
receptors is sufficient even if there is less NKG2D ligands. Further studies are needed to identify NKG2DL that are
expressed on decidual cells. Discovery of such ligand and the
characterization
of
specific
receptor-ligand
interactions
that
mediates dNK cellular cytotoxicity will help uncover potential
therapeutic target that, when activated in vivo, can limit viral
spreading and/or prevent congenital disease. We demonstrate that during HCMV infection, there is a bias of
the inflammatory environment in the decidua basalis. dNK cells
seem to lose their ‘‘decidual status’’ and become killers in order to
limit viral infection. Exposure to HCMV infection can imprint
dNK cell receptor repertoire towards killer activity. We demon-
strate that NKG2D, NKG2C/E activating receptors play a crucial
role in dNK cell cytotoxic response against HCMV-infected
fibroblasts. The fact that dNK cells are able to infiltrate HCMV-
infected tissue in vitro and engage immunological synapse-like
structures within the infected placentas in situ strongly suggest that
dNK cells are key players in controlling HCMV infection and
spreading during pregnancy. To our knowledge, we provide for
the first time evidence for the involvement of dNK cells in clearing
HCMV infection. In fact, we clearly show that dNK cells that are
present only in the decidua basalis during healthy pregnancy are in
contact with HCMV-infected fetal tissue in vivo. It is possible that
upon activation there is an increased dynamic of dNK cells Previous investigations demonstrated that both classical and
non-classical MHC-I molecules have been targeted by HCMV
evasion strategies. By contrast to human fetal foreskin fibroblasts
and fibroblastic cell lines [42,43], HCMV-infection resulted in
decreased cell surface HLA-E molecules without affecting the total
amount of proteins in decidual fibroblasts. The difference between
the two cellular systems might reside in the fact that decidual
fibroblasts express substantial amounts of HLA-E at the steady
state. In decidual fibroblasts, HCMV might interfere with the
stability of cell surface HLA-E molecules by impairing rapid
protein export or by increasing intra-cellular retention. Uterine NK Cell Response to HCMV Infection Although some variations were observed amongst different decidua
basalis, we found that decidual fibroblasts constitutively express
ligands for NKp44 and NKG2D while they barely express ligands
for NKp30 or NKp46. HCMV infection induced NKp30L and
resulted in significant decreases of NKp44L and NKG2DL but did
not affect the expression of NKp46L. These findings suggest that
HCMV infection interferes with the expression level of activating
receptor ligands even if some of them are of cellular rather than
virally induced. It is very intriguing that only few cytokines and chemokines
varied in the presence of HCMV infected fibroblasts. HCMV
infection induces IL-6 secretion most probably through the
expression of the viral-encoded chemokine receptor US28 and
the activation of the IL6/STAT3 signaling pathway [60]. Interestingly, IL-6 was further increased when dNK cells were in
contact with HCMV-infected fibroblasts, most probably through a
paracrine effect on dNK cells. sICAM-1 was also increased under
HCMV conditions. Previous reports suggested that IL-6 down-
regulates the production of several soluble factors [61], while
sICAM-1 increases have been correlated to HCMV reactivation
[62]. Both IL-8 and IP-10 are necessary for trophoblast migration
as these cells express a panel of receptors allowing them to respond
to these chemokines [10]. By lowering the level of IL-8 and IP-10,
dNK cells might reduce trophoblast invasion and prevent viral
spreading from decidual stroma to fetal tissue or be partially
responsible for fetal damages. Remarkably and in sharp contrast
with pNK cell response to viral infection [63,64], there were no
changes in secretion levels of cytokines such IL-12, IL15, type I
IFN, TNFa or IFN-c that are all known to regulate NK cell
function. Moreover, it is possible that changes in dNK cell
secretome create the necessary inflammatory environment that
will favor the recruitment and the initiation of anti-HCMV
adaptive immune response. Using chimeric proteins, we demonstrated that NKp44 receptor
plays an inhibitory function in dNK cell cytotoxicity. dNK cells
might express an inhibitory isoform of NKp44 receptor as a result
of NCR2 alternative splicing as it has been recently demonstrated
for NCR3 (NKp30) [54]. Alternatively, NKp44L expressed on
decidual fibroblasts might participate to uncoupling of activating
adaptor molecules thus promoting an inhibitory profile. However,
the expression of an inhibitory isoform is the most likely
explanation since dNK cells constitutively express the NKp44
receptor. Uterine NK Cell Response to HCMV Infection Uterine NK Cell Response to HCMV Infection infected fibroblasts further support the involvement of CD94/
NKG2C or possibly CD94/NKG2E activating receptors, both
greatly expressed by dNK cells [22–31]. In this context, HCMV
peptides might play a critical role in promoting the recognition of
HLA-E by activating members of CD94/NKG2C and CD94/
NKG2E receptors thus increasing susceptibility of decidual
fibroblasts to dNK cell cytotoxicity at early times of infection as
it has been shown previously for pNK cells [57]. It will be very
interesting to investigate whether late HCMV infection is
responsible for similar changes and whether specific HCMV
peptides play roles in the sequential changes in dNK cell function. Several NKRs have been involved in pNK cell cytotoxicity [53]. For instance, efficient control of HCMV infection involves
NKG2D receptor and can be associated with the emergence of
NKG2C+ subset that contribute to long term protective immune
response [30]. Exposure of dNK cells to HCMV-infected
fibroblasts resulted in an increased NKG2C+ expression without
major changes in NKG2A expression. The role of other receptors
in NK cell response to HCMV is not completely understood. HCMV is able to decrease a plethora of key receptor-ligand
interactions that are involved in NK-cell response. By contrast to
changes in pNK cell repertoire [22], opposite effects were
observed for NKp44 and NKp46 receptors while no changes
were observed for NKp30 receptor. These observations further
highlight differences between dNK and pNK cells modi operandi
during HCMV infection. In parallel to these changes in NK cell receptor, dNK cells
acquire de novo expression of MHC-II DR molecules. This
potential acquisition of an APC-like phenotype during the course
of HCMV immune response might play a crucial role in initiating
a cross-talk with neighboring immune cells, including CD4+ T
cells. Indeed, within the fetal-maternal interface, dNK cells are in
close proximity with decidual CD4+ T cells. Expression of MHC-
II DR antigens might be necessary for dNK cell activation and for
shaping up the adaptive immunity [58,59]. However, further
investigations are needed to demonstrate whether the expression
of MHC-II molecules is associated with the acquisition of APC
capabilities and HCMV antigen presentation. Since the nature of HCMV-induced cellular ligands is not
known, we took advantage of NKR-Fc chimeric receptors to
analyze the expression of NKR ligands on decidual fibroblasts. PLOS Pathogens | www.plospathogens.org Discussion Although mechanisms that control cytotoxicity are
not well established, they may include strong interactions of
inhibitory receptors with their cognate ligands expressed by fetal
trophoblast, production of VEGF-C by dNK cells and/or
expression of anti-apoptotic proteins (XIAP) by target cells
[51,52]. The lack of dNK cell cytotoxicity can be reversed, at
least in vitro, after exposure to cytokines such as IL-5 and IL-18 or
upon engagement of specific activating receptors [9,21]. Here we
show that HCMV infection provides the necessary activating
signals to trigger dNK cell cytotoxicity. The fact that dNK cells
killed
heterologous
targets
from
a
different
donor
further In contrast to pNK cells, very little is known about dNK cell
cytotoxicity as these cells are mainly cytokine and chemokine
producers [9,10,13,21]. We demonstrated that under HCMV-
infectious conditions, a significant fraction of dNK cells that are
CD56bright and CD16neg rapidly dampened down their CD56
expression level and acquired CD16 expression. These changes
are most probably due to the acquisition of cytotoxic function. April 2013 | Volume 9 | Issue 4 | e1003257 April 2013 | Volume 9 | Issue 4 | e1003257 11 PLOS Pathogens | www.plospathogens.org Flow cytometry Fibroblasts were cultured with dNK cells at 1:1 ratio. After 48 h
of co-culture, conjugates were disrupted mechanically by repeated
pipeting and dNK cells were collected and washed twice in PBS. dNK cell suspension were then liquoted in 100 ml containing
16105 cells and labeled with fluorophore-conjugated antibodies. The following mAbs were used: CD56-APC, CD3-PE-Cy7,
CD16-PE,
CD69-PE,
CD2-PE,
NKG2D-PE,
NKG2A-PE,
HLA-DR-FITC, KIR2DL1-PE and 2B4-PE (BD Pharmingen,
France); NKp30-PE, NKp44-PE, NKp46-PE, KIR2DL2/3-PE
(Beckman Coulter, France); NKG2C-PE, KIR2DL4 clone 181703
(R&D Systems, France); ILT2-PE (Biolegend); CD107a-PE, anti-
human HLA-I (HLA-A,-B,-C BC)-PE (BD Pharmingen) and
matched isotype controls. Histograms shown were obtained by
applying a gate on CD56pos CD3neg dNK cells. In conclusion, our data shed new light on the plasticity of dNK
cells and provide evidence for a correlation between phenotypic
changes and functional anti-viral response. We have demonstrated
the ability of dNK cells to exert anti-viral effector functions in vitro
and to infiltrate HCMV infected tissues both ex-vivo and form
immune synapse like-structures in vivo. Careful investigations of
dNK cell status in vivo in larger cohorts of HCMV+ termination of
pregnancy will be required to see whether this predicts clinical
outcome. Understanding mechanisms that regulate switch in dNK
cell immune tolerance will help us discover key factors/pathways
that are involved in the immunopathology of HCMV infection
during pregnancy and design strategies to limit congenital
infection. Fibroblasts were detached using 0.05% trypsin-EDTA, washed
twice in buffer containing 1% FCS. Cells (56105 to 106) were
resuspended in 100 ml of FCS containing buffer and incubated
either with primary specific Ab or isotype matched control
followed by mouse anti-human IgG1 FITC coupled Ab. The
expression of NCR-ligands on fibroblasts was analyzed by binding
of NCR-Fc chimera followed immunostaining with FITC-coupled
mouse anti-human IgG1 secondary Ab (Southern Biotec). The
following chimeras were used: NKp30-Fc, NKp44-Fc, NKp46-Fc
and NKG2D-Fc, CD99-Fc (R&D Systems, France). Non specific
binding was blocked by preincubating the cells for 30 min in 2%
FCS and 1% BSA containing buffer. Data were analyzed using
CellQuest (Becton Dickinson). Cell purification, cell lines dNK cells were purified from first-trimester decidua basalis (8–
12 wk of pregnancy) obtained after elective pregnancy termina-
tions as previously described [9]. Briefly, decidua samples were
minced, collagenase IV treated. Cell suspension is then allowed to
adhere in tissue culture plates. dNK cells were purified from the
non adherent cell fraction using MACS negative selection kits
according to the manufacturer procedure (Miltenyi Biotech). dNK
cells were kept at 4uC in conditioned media containing 20% heat-
inactivated fetal calf serum (FCS). Autologous fibroblasts were
purified from the adherent mononuclear cell fraction by successive
round of mild trypsin treatment. Materials and Methods This study was approved by the Research Ethical Comity
Haute-Garonne. All patients signed the informed consent before
samples were taken, Agence de la Biome´decine, PFS08-022. Uterine NK Cell Response to HCMV Infection The
inhibitory profile observed upon blockade of HLA-E in HCMV- April 2013 | Volume 9 | Issue 4 | e1003257 April 2013 | Volume 9 | Issue 4 | e1003257 12 PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection 72 hours. Trophoblastic villous explants were infected under the
same conditions. allowing them to rich fetal site, which is normally devoid of
maternal immune cells, and kill HCMV-infected cells. Recent
reports have clearly linked the ability of NK cells in controlling
HCV replication and liver fibrosis to specific soluble factor
secretion and/or specific activating receptor expression [65,66]. Future studies, with large cohort of placentas from medical
termination of pregnancy due to congenital HCMV infection, will
be necessary to clarify the dynamic of dNK cell activation in vivo as
well as the pivotal role of soluble factor secretion in mounting
proper anti-HCMV responses and limiting virus spreading. Immunofluorescence, Conjugation and Confocal
microscopy For conjugation, fibroblasts were seeded onto 24-well plates
containing glass coverslips. After 16 h adhesion, dNK cells were
added at a 1 to 2 ratio and incubated at 37uC. Cells were washed
briefly with PBS and fixed with 4% paraformaldehyde for 20 min
and washed in PBS. Intracellular staining was in the presence of
0.5% Saponin. Cells were incubated in PBS containing 1% heat-
inactivated calf serum for 30 min and stained with primary
antibodies followed by incubation with Alexa fluor conjugated
secondary antibody (Invitrogen) as previously described [68]. Filamentous actin cytoskeleton was visualized with Alexa fluor
conjugated phalloidin. After extensive washing, coverslips were
mounted with vectashield mounting medium (DAKO). Fluores-
cence was analyzed using Zeiss LSM710 confocal microscope
using a 63x oil objective (Carl Zeiss AG, Jena, Germany). The purity of both dNK cells and decidual fibroblasts was
assessed using a cocktail of antibodies. dNK cells were CD3neg and
CD56pos. Decidual fibroblasts purity was confirmed by immuno-
staining with an anti-cytokeratin and anti-vimentin antibodies,
fibroblasts are cytokeratin 7neg (NM_001047870) and Vimentinpos
(NM_003380). Cell morphology was analyzed by examining the phalloidin-
stained conjugates as an indicator of F-actin distribution. Images
correspond to maximum intensity projection along the z-axis (Zen
software). PLOS Pathogens | www.plospathogens.org Explant organ culture preparation and dNK cell tissue
invasion y
y
y
Target cells (16106cells) were labeled with 100 mCi 51Chromi-
um (Sodium chromate, 1 mCi/ml, Perkin Elmer, Courtaboeuf,
France). After 1 h incubation at 37uC, cells were washed 3 times in
HEPES-buffered RPMI. dNK effector cells were added to 51Cr
labeled target cells (56103) in replicate at various effector to target
ratios in a total volume of 200 ml RPMI containing 5% FCS per
well of 96-well round-bottomed microtiter plates. Microtiter plates
were centrifuged at 1200 rpm for 5 min and incubated at 37uC. After 4 h or 18 h of culture, 50 ml cell free supernatants were
transferred to Lumaplate (Perkin-Elmer) and the radioactivity was
measured on a TopCount (Perkin-Elmer). The specific cytotoxicity
was calculated. Spontaneous release was determined from wells
containing target cells alone. Maximum release was determined
from wells containing target cells lysed in 1% Triton X-100. The
data were expressed as follows:% specific cytotoxicity = 1006[-
Sample mean (cpm) - Spontaneous mean (cpm)/(Maximum mean
(cpm) 2 Spontaneous mean (cpm)]. To block cell lysis due to the
engagement of specific activating receptor engagement or specific
pathway, 51Cr-labeled target cells were incubated for 20 min on
ice with various soluble receptor-Fc IgG1 chimeric protein
(0.2 mg/ml), anti-HLA-E mAb (clone MEM-E/08, Exbio) or an
isotype match control (mouse IgG1) at the final concentration of
1.0 mg/ml then included as targets in cytotoxicity assay with dNK
effector cells. dNK cells were incubated with an anti-TRAIL and -
FasL antibodies (10 mg/ml) (R&D Systems, France) prior to
cytotoxicity assay. Recombinant TRAIL and FasL proteins (gifts
from A. Quillet-Mary, Toulouse, France) were used at the final
concentration of 10 mg/ml. Trophoblastic villous explants established from first trimester
elective termination of pregnancy samples. Tissue was minced (1
to 2 mm) and placed in 24 well tissue culture plates in complete
tissue culture media (PromoCell, France). After four hours of
culture at 37uC and two changes of culture media, explants were
at either left uninfected or infected with HCMV AD169 for two
days. For tissue invasion, trophoblast organ culture nuclei were
stained with 4 pM DAPI for 5 min, autologous dNK cells were
labeled with 1 mM Cell Tracker Red (Invitrogen) for 15 min. All
staining procedures were performed at 37uC and quenched with
10 ml of tissue culture media containing 10% FCS. Each explant
(1–2 mm) was incubated with 56105 dNK cells at 37uC. Mutiplex cytokine and chemokine arrays Mutiplex cytokine and chemokine arrays
dNK cells were co-cultured with uninfected or HCMV-infected
autologous decidual fibroblasts in complete medium in 96
microtiter plate. Controls experiments were performed using
dNK cells, uninfected fibroblasts, HCMV-infected autologous
fibroblasts that were cultured alone in the same conditions. Cleared supernatants replicates from 4 different experiments were
collected after 24 hours of culture and stored at 280uC. Cytokines, chemokines and growth factors levels were measured
using a 42-multiplexed Affymetrix cytokine assay according to the
manufacturer protocol (Procarta/Ozyme). The following cytokines
and chemokines were analyzed: IL-1a (NM_000575), IL-1RA
(NM_000577), IL-1b (NM_000576), IL-2 (NM_000586), IL-2RA
(NM_000417), IL-4 (NM_000589), IL-5 (NM_000879), IL-6
(NM_000600),
IL-8/CXCL8
(NM_0005843),
IL-10
(NM_000572), IL-12 (NM_002187), IL-15 (NM_000585), IL-
17A (NM_002190), IL-17F (NM_052872), IL-22 (NM_020525.4),
IP10/CXCL10 (NM_001565), Basic-FGF/FGF2 (NM_002006),
EGF (NM_005429.2), Eotaxin-3/CCL26 (NM_006072), E-selec-
tin/CD62E
(NM_000450),
sFas
(NM_000043),
sFasL
(NM_000639), Fractalkine/CX3CL1 (NM_002996), GM-CSF
(NM_000758), Granzyme B (NM_004131), GROa/CXCL-1
(NM_001511), sICAM-1 (NM_000201), Leptin (NM_000230),
IFN-a2
(NM_000605),
IFN-b
(NM_002176),
IFN-c
(NM_000619),
IFN-v
(NM_002177),
MCP-1/CCL2
(NM_002982), MIP-1a/CCL3 (NM_002983), MIP-1b/CCL4 HCMV+ whole placental biopsies were obtained from two
pathological termination of pregnancy (24.5 weeks and 25 weeks
of pregnancy). Tissues were fixed in 10% formalin, embedded in
paraffin and processed for IHC as previously described [69]
Briefly, 6-mm-thick sections of paraffin-embedded samples were
immunostained with an anti-CD56 mAb (1B6 clone) and an anti-
HCMV-IE mAb (Argene). Photographs were taken with 40X
objective of Leica microscope. p
y
y
dNK cells were co-cultured with uninfected or HCMV-infected
autologous decidual fibroblasts in complete medium in 96
microtiter plate. Controls experiments were performed using
dNK cells, uninfected fibroblasts, HCMV-infected autologous
fibroblasts that were cultured alone in the same conditions. Cleared supernatants replicates from 4 different experiments were
collected after 24 hours of culture and stored at 280uC. Cytokines, chemokines and growth factors levels were measured
using a 42-multiplexed Affymetrix cytokine assay according to the
manufacturer protocol (Procarta/Ozyme). The following cytokines
and chemokines were analyzed: IL-1a (NM_000575), IL-1RA
(NM 000577), IL-1b (NM 000576), IL-2 (NM 000586), IL-2RA Explant organ culture preparation and dNK cell tissue
invasion After two
hours of contact, organ explants were gently washed with excess of
complete media (4 washes), fixed in 4% paraformaldehyde for
20 min, washed twice in PBS and mounted for two-photon
microscopy analysis. Virus production and cellular infection Decidual fibroblasts were maintained in RPMI-1640 medium
(GIBCO) supplemented with 10% (v/v) FCS and penicillin-
streptomycin 100 U/ml each, under a 5% CO2 atmosphere at
37uC. The distance between the MTOC and the center of the IS was
measured from single plane of unprocessed images using the single
line function of the Imaris (Biplane Scientific Software). Two HCMV strains were used, AD169 laboratory strain
(ATCC strain, a gift from S. Michelson, Paris, France), and
VHLE clinical isolate (a gift from C. Sinzger, Tubingen,
Germany). Viral stocks were prepared from cell-released virions,
using MRC-5 cells as previously described [67]. High titer virus
stocks were stored in single use aliquots for up to six months at
280uC. The following antibodies were used to analyze microtubules and
MTOC, perforin, CMV infection: anti-human alpha tubulin
polyclonal Ab (Sigma-Aldrich, UK), anti-human golgin-97 (In-
vitrogen),
anti-human
perforin
and
anti-human
CD2
(BD
Pharmingen), anti-HCMV-IE (Argene), anti-human vimentin
and anti-cytokeratin 7 (Dako). The F-actin cytoskeleton was
analyzed using phalloidin coupled to either to Alexa fluor 488 or
Alexa fluor 747 (Invitrogen). Adherent cell monolayers of decidual fibroblasts or MRC-5 cell
line were infected with HCMV particles (MOI 3–5) for 48– April 2013 | Volume 9 | Issue 4 | e1003257 13 Uterine NK Cell Response to HCMV Infection (NM_002984), RANTES/CCL5 (NM_002985), SDF-1/CXCL12
(NM_000609), TGF-a (NM_001099691), TRAIL (NM_003810),
TNF-a
(NM_000594),
TNF-b
(NM_001159740),
VEGF-A
(NM_001025366). Measurement and analysis were performed
using the BioRad Bio-Plex System (BioRad, France). Data points
are expressed as follows: Specific dNK cell cytokine-chemokine
secretion = [Total concentration of dNK cells cultured in the
presence of fibroblasts - Fibroblasts secretion]. Degranulation assay, CD107a expression For
degranulation
assay,
fibroblasts
were
harvested
and
incubated at 37uC with dNK cells at a 1 to 1 ratio for different
time points. Reactions were stopped on ice, cells were stained with
fluorochrome-conjugated anti-human CD107a (BD Pharmingen)
or isotype matched control Ab in staining buffer containing 1%
FCS. Fluorescence was analyzed by Flow Cytometry. Two-photon microscopy Images were taken using Zeiss two-photon microscopy at
900 nm laser excitation. Fluorescence emission was collected using
dichroic mirrors to split fluorescence into three channels (blue,
green and red). Z stacks were taken at 10 mm slice intervals using
Zeiss Zen software. Imaris software was used to analyze the
acquired data. PLOS Pathogens | www.plospathogens.org Supporting Information The purity was analyzed using
anti-vimentin (green, fibroblasts) and anti-cytokeratin-7 (red,
cytotrophoblast) staining. Nuclei were stained with dapi (cyan). (B) Fibroblasts were infected with HCMV (AD169) for 48 h. Nuclei are stained with dapi (cyan) and HCMV-IE (red). Fibroblasts were stained for vimentin (green), a-tubulin (blue). Bar represent 20 mm. (C) Kinetics of fibroblasts infection was
quantified over three days. (TIF) Figure S2
Specificity of effector cell cytotoxicity. (A) dNK
cell cytotoxicity was analyzed against uninfected or AD169-
infected autologous decidual fibroblasts after 4 h of contact. (B &
C) pNK cell cytotoxicity was analyzed against autologous decidual
fibroblasts after 4 h (B) or 18 h (C) assay, mean specific lysis is
calculated from triplicates within the same experiment out of four. (D & E) dNK cell cytotoxicity against heterologous decidual
fibroblasts analyzed after 4 h (D) or 18 h (E) of contact. Data on
the graphs are from one representative experiment out of three. (F)
dNK and pNK cell cytotoxicity against K562 classical target cell
line after 4 h of contact. (G) dNK cell cytotoxicity towards semi-
allogeneic trophoblasts was evaluated in three different decidual
samples (Tropho_1, _2 and _3) and compared to lysis of
autologous infected decidual fibroblasts. (H) Recombinant FasL
and TRAIL induce lysis of Jurkat cell line. Jurkat cells were
incubated with recombinant TRAIL (rTRAIL) or FasL (rFasL). Specific lysis was performed in the absence or the presence of
blocking antibodies against TRAIL (a-TRAIL) or FasL (a-FasL). (TIF) Figure S5
HCMV infection down-modulates cell surface
expression of HLA-E without affecting the total amounts
of HLA-E. (A) HCMV-VHLE infection modulates the expression
of NKR ligands on decidual fibroblasts. The binding of human
NKp30-Fc, NKp46-Fc, NKp44-Fc, NKG2D-Fc and CD99-Fc
chimera was used to evaluate the cell surface expression of specific
receptor ligands. HLA-E cell surface expression evaluated using
MEM-E/08 mAb. One representative FACS histogram out of
three independent experiments is shown. Uninfected (black line),
VHLE-infected
(shaded
dark
gray). (B)
HLA-E
expression
evaluated in additional three decidual fibroblasts from three
independent deciduas (dFibro_1, _2, _3) or in MRC-5 cell line. HLA-A,-B,-C expression by MRC-5 cells was analyzed by specific
mAb. For MRC-5 cells, FACS histograms are representative of
three independent experiments. Uninfected (black line), HCMV-
infected (shaded dark gray). Light gray (line or shaded) histogram
represent isotype-matched control Ig. (C) HLA-E detected by
western blot in MRC-5 cells or decidual fibroblast from three
different deciduas. Supporting Information autologous fibroblasts that were either uninfected or infected with
HCMV-AD169 for 18 h. dNK cells were stained for surface
expression of the indicated receptor using fluorochrome-conjugat-
ed antibodies and analyzed by flow cytometry as indicated above. Representative FACS histograms gated on CD56pos CD3neg dNK
cells are shown (n = 5). dNK cells in contact with uninfected
fibroblasts are represented by black line, dNK cells in contact with
HCMV-infected fibroblasts are represented by shaded gray. Dotted gray line represents isotype-matched control Ig. One
representative histogram out of five independent experiments is
shown. (C) pNK cells were co-cultured with autologous decidual
fibroblasts that were either uninfected or infected with HCMV-
VHLE for 18 h. pNK cells were stained for surface expression of
the indicated receptor using fluorochrome-conjugated antibodies
and analyzed by flow cytometry as indicated above. Representa-
tive FACS histograms gated on CD56pos CD3neg pNK cells are
shown (n = 3). Cells in contact with uninfected fibroblasts are
represented by black line, with VHLE-infected fibroblasts are
represented by shaded dark gray. Light gray histograms represent
isotype-matched control Ig. One representative histogram out of
three independent experiments is shown. (TIF) autologous fibroblasts that were either uninfected or infected with
HCMV-AD169 for 18 h. dNK cells were stained for surface
expression of the indicated receptor using fluorochrome-conjugat-
ed antibodies and analyzed by flow cytometry as indicated above. Representative FACS histograms gated on CD56pos CD3neg dNK
cells are shown (n = 5). dNK cells in contact with uninfected
fibroblasts are represented by black line, dNK cells in contact with
HCMV-infected fibroblasts are represented by shaded gray. Dotted gray line represents isotype-matched control Ig. One
representative histogram out of five independent experiments is
shown. (C) pNK cells were co-cultured with autologous decidual
fibroblasts that were either uninfected or infected with HCMV-
VHLE for 18 h. pNK cells were stained for surface expression of
the indicated receptor using fluorochrome-conjugated antibodies
and analyzed by flow cytometry as indicated above. Representa-
tive FACS histograms gated on CD56pos CD3neg pNK cells are
shown (n = 3). Cells in contact with uninfected fibroblasts are
represented by black line, with VHLE-infected fibroblasts are
represented by shaded dark gray. Light gray histograms represent
isotype-matched control Ig. One representative histogram out of
three independent experiments is shown. (TIF) Figure S1
Characterization and HCMV-AD169 infectiv-
ity of decidual fibroblasts. (A) Decidual fibroblasts were
purified as described in M&M. Supporting Information Cells were HCMV-infected for 48 h. HLA-E
detected by MEM-E/06 (top gel) and anti-b-actin (bottom gel). The size of protein ladder is given in kDa. (TIF) Figure S3
MTOC polarization and Golgi relocalization
to the immune synapse. Uninfected (AD1692) or HCMV-
infected (AD169+) decidual fibroblasts (F) plated on glass coverslips
were incubated with autologous dNK cells (dNK) for 20 min at
37uC. (A) Formed conjugates were fixed and permeabilized for
intracellular staining of F-actin (blue), a-tubulin microtubules
(green) and Golgin (red) simultaneously. Scale bar represent
20 mm. Enlargement of the synaptic area of conjugates presented
in the right panels. Asterisks indicate the MTOC. Arrowheads
point to the Golgi apparatus. Scale bar represent 5 mm. (B) Bar
graphs show the frequency of conjugates formation between dNK
cells and autologous fibroblasts that were either kept uninfected
(AD1692) or HCMV-infected (AD169+). More than 500 fibro-
blasts (white graphs) and at least 50 conjugates (black graphs) were
scored
in
each
experiment
(n = 5). Statistical
analysis
was
performed using unpaired Student’s t-test. ***, p,0.001. (C)
Immunostaining for F-actin (phalloidin in green), HCMV-IE1
(pink), CD2 (red). Scale bar, 20 mm. (TIF) Figure S6
HCMV infection regulates NKR ligand ex-
pression in decidual fibroblasts: Role on pNK cell
cytotoxicity. (A) K562 cell line were incubated with CD99-Fc
(CTRL), NKp46-Fc, NKp30-Fc chimera and used as target cells
to evaluate pNK cell cytotoxicity in a 4 h chromium release assay. (B–E) pNK cells cytotoxicity against uninfected (gray plots) or
VHLE-infected autologous decidual fibroblasts (black plots) after
18 h of contact. (B) NKp30-Fc, (C) NKp46-Fc, (D) NKp44-Fc, (E)
NKG2D-Fc chimeric receptors were used to block the corre-
sponding specific ligands. CD99-Fc soluble receptor was used as
control (CTRL). (F) Analysis of NKG2A and NKG2C/E function
was performed in the presence of blocking antibody against HLA-
E molecules (a-HLA-E) or isotype matched control. Data sets
represent mean lysis 6 S.D. from three independent experiments
done in replicate. Statistical comparisons were performed using
two-way ANOVA test. ***, p,0.001. (TIF) Figure S4
Analysis of NK cell receptor repertoire during
HCMV
infection. (A)
dNK
cells
were
co-cultured
with
autologous fibroblasts that were either kept uninfected or infected
with HCMV AD169 for 48 h. dNK cells were stained for surface
expression of the indicated receptor using fluorochrome-conjugat-
ed antibodies and analyzed by FACS. Representative FACS
histograms gated on CD56pos CD3neg dNK cells are shown (n = 5). Specific receptors are indicated below each panel. Statistical analysis Unpaired Student t test was calculated using GraphPad Prism
4.0 (GraphPad Software). Unless otherwise indicated, data
represent the mean 6 S.D. from at least three independent
experiments. Gene accession numbers
CD69
(NM_001781),
NKp30/NCR3
(NM_001145466),
NKp44/NCR2
(NM_001199509),
NKp46/NCR1
(NM_001145457), NKG2D/KLRK1 (NM_007360), KIR2DL1/
CD158A (NM_014218), KIR2DL2/CD158B1 (NM_014219),
KIR2DL3/CD158B2
(NM_015868),
KIR2DL4/CD158D
(NM_001080770),
ILT2/LILRB1/CD85j
(NM_001081637),
NKG2C/KLRC2
(NM_002260),
HLA-A,-B,-C
(NM_001242758,
NM_005514,
NM_001243042),
HLA-E
(NM_005516), HLA-DR (NM_002124). April 2013 | Volume 9 | Issue 4 | e1003257 April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org 14 Uterine NK Cell Response to HCMV Infection PLOS Pathogens | www.plospathogens.org References Whitelaw PF, Croy BA (1996) Granulated lymphocytes of pregnancy. Placenta
17: 533–543. the control of HIV-1 infection. J Intern Med 265: 29–42. 8. Vacca P, Pietra G, Falco M, Romeo E, Bottino C, et al. (2006) Analysis of
natural killer cells isolated from human decidua: Evidence that 2B4 (CD244)
functions as an inhibitory receptor and blocks NK-cell function. Blood 108:
4078–4085. 27. Orr MT, Murphy WJ, Lanier LL (2010) ‘Unlicensed’ natural killer cells
dominate the response to cytomegalovirus infection. Nat Immunol 11: 321–327. p
y
g
28. Alter G, Heckerman D, Schneidewind A, Fadda L, Kadie CM, et al. (2011)
HIV-1 adaptation to NK-cell-mediated immune pressure. Nature 476: 96–100. 9. El Costa H, Casemayou A, Aguerre-Girr M, Rabot M, Berrebi A, et al. (2008)
Critical and differential roles of NKp46- and NKp30-activating receptors
expressed by uterine NK cells in early pregnancy. J Immunol 181: 3009–3017. p
p
29. Waggoner SN, Cornberg M, Selin LK, Welsh RM (2011) Natural killer cells act
as rheostats modulating antiviral T cells. Nature 481:394–8. 30. Lopez-Verges S, Milush JM, Schwartz BS, Pando MJ, Jarjoura J, et al. (2011)
Expansion of a unique CD57NKG2Chi natural killer cell subset during acute
human cytomegalovirus infection. Proc Natl Acad Sci U S A 108: 14725–14732. 10. Hanna J, Goldman-Wohl D, Hamani Y, Avraham I, Greenfield C, et al. (2006)
Decidual NK cells regulate key developmental processes at the human fetal-
maternal interface. Nat Med 12: 1065–1074. 31. Vivier E, Raulet DH, Moretta A, Caligiuri MA, Zitvogel L, et al. (2011) Innate
or adaptive immunity? The example of natural killer cells. Science 331: 44–49. 11. Keskin DB, Allan DS, Rybalov B, Andzelm MM, Stern JN, et al. (2007)
TGFbeta promotes conversion of CD16+ peripheral blood NK cells into CD16-
NK cells with similarities to decidual NK cells. Proc Natl Acad Sci U S A 104:
3378–3383. 32. Markel G, Wolf D, Hanna J, Gazit R, Goldman-Wohl D, et al. (2002) Pivotal
role of CEACAM1 protein in the inhibition of activated decidual lymphocyte
functions. J Clin Invest 110: 943–953. J
33. Mselle TF, Howell AL, Ghosh M, Wira CR, Sentman CL (2009) Human
uterine natural killer cells but not blood natural killer cells inhibit human
immunodeficiency virus type 1 infection by secretion of CXCL12. J Virol 83:
11188–11195. 12. Koopman LA, Kopcow HD, Rybalov B, Boyson JE, Orange JS, et al. Supporting Information dNK cells in
contact with uninfected fibroblasts are represented by black line,
dNK cells in contact with HCMV-infected fibroblasts are
represented by shaded gray. Dotted gray line represents isotype-
matched control Ig. (B) dNK cells were co-cultured with Figure S7
HCMV infection modulates Fibroblasts cyto-
kine/chemokine production. Decidual fibroblasts were kept Figure S7
HCMV infection modulates Fibroblasts cyto-
kine/chemokine production. Decidual fibroblasts were kept April 2013 | Volume 9 | Issue 4 | e1003257 15 PLOS Pathogens | www.plospathogens.org Uterine NK Cell Response to HCMV Infection uninfected (AD1692, gray) or AD169-infected (AD169+, black) for
48 h with HCMV-AD169 strain. Cytokines were quantified in the
supernatants using a 42-multi-plexed cytokine assay. Representa-
tive histograms from specific cytokines-chemokines that are
produced by uninfected and HCMV-infected decidual fibroblasts
are presented. Normalized data points are given as mean values 6
S.D. calculated from four independent experiments. (TIF) Video
S2
dNK
cells
infiltrate
and
form
‘‘immune
synapse-like structures’’ with AD-169 infected autolo-
gous trophoblasts. Three-dimensional reconstruction of dNK
cell infiltrating HCMV-infected chorionic villi organ explant
shown in Figure 6. Volume rendering reconstruction and
animation were obtained as in video S1. Images are at 5
frames/s; Scale bar: 100 mm. (AVI) Video
S2
dNK
cells
infiltrate
and
form
‘‘immune
synapse-like structures’’ with AD-169 infected autolo-
gous trophoblasts. Three-dimensional reconstruction of dNK
cell infiltrating HCMV-infected chorionic villi organ explant
shown in Figure 6. Volume rendering reconstruction and
animation were obtained as in video S1. Images are at 5
frames/s; Scale bar: 100 mm. (AVI) Acknowledgments We would like to thank C. Davrinche for providing HCMV strains and for
helpful discussions. We thank D. Gonzales-Dunia and R. Al Daccak for
critically reading the manuscript. We thank M. March for IHC
experiments. We are grateful for the help from UMR 5282’s cell imaging
and flow cytometry facilities. We also thank patients and MDs from Paule
de Viguier Hospital for providing clinical material to this study. Video S1
dNK cells are localized at the external boarder
of uninfected autologous trophoblasts. Three-dimensional
reconstruction of dNK cell infiltration of chorionic villi organ
explant shown in Figure 6. Volume rendering reconstruction and
animation were obtained from two-photon Z-stack taken at 10 mm
slice intervals using Imaris software of 200 mm section. dNK cells
(Cell tracker Red), dapi staining of explants’ nuclei (cyan). Images
are at 5 frames/s; Scale bar: 100 mm. References (2003)
Human decidual natural killer cells are a unique NK cell subset with
immunomodulatory potential. J Exp Med 198: 1201–1212. 13. Le Bouteiller P, Siewiera J, Casart Y, Aguerre-Girr M, El Costa H, et al. (2011)
The human decidual NK-cell response to virus infection: what can we learn
from circulating NK lymphocytes? J Reprod Immunol 88: 170–175. 34. Orange JS (2008) Formation and function of the lytic NK-cell immunological
synapse. Nat Rev Immunol 8: 713–725. g
y
p
y
J
p
14. Santoni A, Carlino C, Gismondi A (2008) Uterine NK cell development,
migration and function. Reprod Biomed Online 16: 202–210. 14. Santoni A, Carlino C, Gismondi A (2008) Uterine NK cell 35. Mentlik AN, Sanborn KB, Holzbaur EL, Orange JS (2010) Rapid lytic granule
convergence to the MTOC in natural killer cells is dependent on dynein but not
cytolytic commitment. Mol Biol Cell 21: 2241–2256. migration and function. Reprod Biomed Online 16: 202–210. 15. Vacca P, Cantoni C, Vitale M, Prato C, Canegallo F, et al. (2010) Crosstalk
between decidual NK and CD14+ myelomonocytic cells results in induction of
Tregs and immunosuppression. Proc Natl Acad Sci U S A 107: 11918–11923. 36. Rak GD, Mace EM, Banerjee PP, Svitkina T, Orange JS (2011) Natural killer
cell lytic granule secretion occurs through a pervasive actin network at the
immune synapse. PLoS Biol 9: e1001151. g
pp
16. Cooper MA, Fehniger TA, Caligiuri MA (2001) The biology of human natural
killer-cell subsets. Trends Immunol 22: 633–640. 37. Blaschitz A, Weiss U, Dohr G, Desoye G (2000) Antibody reaction patterns in
first trimester placenta: implications for trophoblast isolation and purity
screening. Placenta 21: 733–741. 17. Hanna J, Bechtel P, Zhai Y, Youssef F, McLachlan K, et al. (2004) Novel
insights on human NK cells’ immunological modalities revealed by gene
expression profiling. J Immunol 173: 6547–6563. 38. Toledo MS, Suzuki E, Handa K, Hakomori S (2005) Effect of ganglioside and
tetraspanins in microdomains on interaction of integrins with fibroblast growth
factor receptor. J Biol Chem 280: 16227–16234. p
p
g J
18. Male V, Hughes T, McClory S, Colucci F, Caligiuri MA, et al. (2010) Immature
NK cells, capable of producing IL-22, are present in human uterine mucosa. J Immunol 185: 3913–3918. 39. Davis DM, Chiu I, Fassett M, Cohen GB, Mandelboim O, et al. (1999) The
human natural killer cell immune synapse. Proc Natl Acad Sci U S A 96: 15062–
15067. Author Contributions Conceived and designed the experiments: NJF. Performed the experi-
ments: JS HEC JT. Analyzed the data: JS NJF. Contributed reagents/
materials/analysis tools: AB GC PB. Wrote the paper: JS NJF. Conceived and designed the experiments: NJF. Performed the experi-
ments: JS HEC JT. Analyzed the data: JS NJF. Contributed reagents/
materials/analysis tools: AB GC PB. Wrote the paper: JS NJF. References 20. King A, Kalra P, Loke YW (1990) Human trophoblast cell resistance to decidual
NK lysis is due to lack of NK target structure. Cell Immunol 127: 230–237. 1. Cannon MJ (2009) Congenital cytomegalovirus (CMV) epidemiology and
awareness. J Clin Virol 46 Suppl 4: S6–10. 21. Kopcow HD, Allan DS, Chen X, Rybalov B, Andzelm MM, et al. (2005)
Human decidual NK cells form immature activating synapses and are not
cytotoxic. Proc Natl Acad Sci U S A 102: 15563–15568. 2. Gohring K, Dietz K, Hartleif S, Jahn G, Hamprecht K (2010) Influence of
different extraction methods and PCR techniques on the sensitivity of HCMV-
DNA detection in dried blood spot (DBS) filter cards. J Clin Virol 48: 278–281. 3. Berger A, Reitter A, Harter PN, Buxmann H, Allwinn R, et al. (2011) Problems
and challenges in the diagnosis of vertical infection with human cytomegalovirus
(CMV): lessons from two accidental cases. J Clin Virol 51: 285–288. 22. Lopez-Botet M, Angulo A, Guma M (2004) Natural killer cell receptors for
major histocompatibility complex class I and related molecules in cytomegalo-
virus infection. Tissue Antigens 63: 195–203. 4. Enders G, Daiminger A, Bader U, Exler S, Enders M (2011) Intrauterine
transmission and clinical outcome of 248 pregnancies with primary cytomeg-
alovirus infection in relation to gestational age. J Clin Virol 52: 244–246. 23. Kuijpers TW, Baars PA, Dantin C, van den Burg M, van Lier RA, et al. (2008)
Human NK cells can control CMV infection in the absence of T cells. Blood
112: 914–915. alovirus infection in relation to gestational age. J Clin Virol 52: 244 24. Lanier LL (2008) Up on the tightrope: natural killer cell activation and
inhibition. Nat Immunol 9: 495–502. 5. Pereira L, Maidji E (2008) Cytomegalovirus infection in the human placenta:
maternal immunity and developmentally regulated receptors on trophoblasts
converge. Curr Top Microbiol Immunol 325: 383–395. 25. Saez-Borderias A, Romo N, Magri G, Guma M, Angulo A, et al. (2009) IL-12-
dependent inducible expression of the CD94/NKG2A inhibitory receptor
regulates CD94/NKG2C+ NK cell function. J Immunol 182: 829–836. 6. Manaster I, Mandelboim O (2010) The unique properties of uterine NK cells. Am J Reprod Immunol 63: 434–444. Am J Reprod Immunol 63: 434–444. 26. Alter G, Altfeld M (2009) NK cells in HIV-1 infection: evidence for their role in
the control of HIV-1 infection. J Intern Med 265: 29–42. 7. Uterine NK Cell Response to HCMV Infection (2001) Il-
6 and its soluble receptor orchestrate a temporal switch in the pattern of
leukocyte recruitment seen during acute inflammation. Immunity 14: 705–714. 46. Dumortier J, Streblow DN, Moses AV, Jacobs JM, Kreklywich CN, et al. (2008)
Human cytomegalovirus secretome contains factors that induce angiogenesis
and wound healing. J Virol 82: 6524–6535. y
g
y
62. Nordoy I, Muller F, Nordal KP, Rollag H, Aukrust P, et al. (2000) Chemokines
and soluble adhesion molecules in renal transplant recipients with cytomega-
lovirus infection. Clin Exp Immunol 120: 333–337. g J
47. Kuhn JR, Poenie M (2002) Dynamic polarization of the microtubule
cytoskeleton during CTL-mediated killing. Immunity 16: 111–121. p
63. Muntasell A, Magri G, Pende D, Angulo A, Lopez-Botet M (2010) Inhibition of
NKG2D expression in NK cells by cytokines secreted in response to human
cytomegalovirus infection. Blood 115: 5170–5179. y
g
g
y
48. Trambas CM, Griffiths GM (2003) Delivering the kiss of death. Nat Immunol 4:
399–403. 64. Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei XQ, et al. (2002) Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15
regulation of NK cell responses to viral infection. J Immunol 169: 4279–4287. 49. Stinchcombe JC, Majorovits E, Bossi G, Fuller S, Griffiths GM (2006)
Centrosome polarization delivers secretory granules to the immunological
synapse. Nature 443: 462–465. 65. Gur C, Doron S, Kfir-Erenfeld S, Horwitz E, Abu-Tair L, et al. (2012) NKp46-
mediated killing of human and mouse hepatic stellate cells attenuates liver
fibrosis. Gut 61: 885–893. 50. Stinchcombe JC, Salio M, Cerundolo V, Pende D, Arico M, et al. (2011)
Centriole polarisation to the immunological synapse directs secretion from
cytolytic cells of both the innate and adaptive immune systems. BMC Biol 9: 45. 66. Ohira M, Ishiyama K, Tanaka Y, Doskali M, Igarashi Y, et al. (2009) Adoptive
immunotherapy with liver allograft-derived lymphocytes induces anti-HCV
activity after liver transplantation in humans and humanized mice. J Clin Invest
119: 3226–3235. 51. Straszewski-Chavez SL, Abrahams VM, Funai EF, Mor G (2004) X-linked
inhibitor of apoptosis (XIAP) confers human trophoblast cell resistance to Fas-
mediated apoptosis. Mol Hum Reprod 10: 33–41. p p
p
52. Kalkunte SS, Mselle TF, Norris WE, Wira CR, Sentman CL, et al. (2009)
Vascular endothelial growth factor C facilitates immune tolerance and
endovascular activity of human uterine NK cells at the maternal-fetal interface. J Immunol 182: 4085–4092. 67. Uterine NK Cell Response to HCMV Infection 55. Eagle RA, Traherne JA, Hair JR, Jafferji I, Trowsdale J (2009) ULBP6/
RAET1L is an additional human NKG2D ligand. Eur J Immunol 39: 3207–
3216. 40. Inoue H, Miyaji M, Kosugi A, Nagafuku M, Okazaki T, et al. (2002) Lipid rafts
as the signaling scaffold for NK cell activation: tyrosine phosphorylation and
association of LAT with phosphatidylinositol 3-kinase and phospholipase C-
gamma following CD2 stimulation. Eur J Immunol 32: 2188–2198. 56. Bennett NJ, Ashiru O, Morgan FJ, Pang Y, Okecha G, et al. (2010) Intracellular
sequestration of the NKG2D ligand ULBP3 by human cytomegalovirus. J Immunol 185: 1093–1102. 41. Arnon TI, Achdout H, Levi O, Markel G, Saleh N, et al. (2005) Inhibition of the
NKp30 activating receptor by pp65 of human cytomegalovirus. Nat Immunol 6:
515–523. 57. Kaiser BK, Barahmand-Pour F, Paulsene W, Medley S, Geraghty DE, et al. (2005) Interactions between NKG2x immunoreceptors and HLA-E ligands
display overlapping affinities and thermodynamics. J Immunol 174: 2878–2884. 42. Prod’homme V, Tomasec P, Cunningham C, Lemberg MK, Stanton RJ, et al. (2012) Human cytomegalovirus UL40 signal peptide regulates cell surface
expression of the NK cell ligands HLA-E and gpUL18. J Immunol 188: 2794–
2804. p y
pp g
y
58. Hanna J, Gonen-Gross T, Fitchett J, Rowe T, Daniels M, et al. (2004) Novel
APC-like properties of human NK cells directly regulate T cell activation. J Clin
Invest 114: 1612–1623. 43. Tomasec P, Braud VM, Rickards C, Powell MB, McSharry BP, et al. (2000)
Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by
human cytomegalovirus gpUL40. Science 287: 1031. 59. Zingoni A, Sornasse T, Cocks BG, Tanaka Y, Santoni A, et al. (2004) Cross-talk
between activated human NK cells and CD4+ T cells via OX40-OX40 ligand
interactions. J Immunol 173: 3716–3724. 44. Guma M, Angulo A, Lopez-Botet M (2006) NK cell receptors involved in the
response to human cytomegalovirus infection. Curr Top Microbiol Immunol
298: 207–223. 60. Slinger E, Maussang D, Schreiber A, Siderius M, Rahbar A, et al. (2010)
HCMV-encoded chemokine receptor US28 mediates proliferative signaling
through the IL-6-STAT3 axis. Sci Signal 3: ra58. 45. Guma M, Budt M, Saez A, Brckalo T, Hengel H, et al. (2006) Expansion of
CD94/NKG2C+ NK cells in response to human cytomegalovirus-infected
fibroblasts. Blood 107: 3624–3631. g
g
61. Hurst SM, Wilkinson TS, McLoughlin RM, Jones S, Horiuchi S, et al. References 19. Male V, Trundley A, Gardner L, Northfield J, Chang C, et al. (2010) Natural
killer cells in human pregnancy. Methods Mol Biol 612: 447–463. April 2013 | Volume 9 | Issue 4 | e1003257 April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org PLOS Pathogens | www.plospathogens.org 16 Uterine NK Cell Response to HCMV Infection Uterine NK Cell Response to HCMV Infection Rauwel B, Mariame B, Martin H, Nielsen R, Allart S, et al. (2010) Activation of
peroxisome proliferator-activated receptor gamma by human cytomegalovirus
for de novo replication impairs migration and invasiveness of cytotrophoblasts
from early placentas. J Virol 84: 2946–2954. 53. Chen X, Trivedi PP, Ge B, Krzewski K, Strominger JL (2007) Many NK cell
receptors activate ERK2 and JNK1 to trigger microtubule organizing center and
granule polarization and cytotoxicity. Proc Natl Acad Sci U S A 104: 6329–
6334. 68. Didier C, Merdes A, Gairin JE, Jabrane-Ferrat N (2008) Inhibition of
proteasome activity impairs centrosome-dependent microtubule nucleation
and organization. Mol Biol Cell 19: 1220–1229. 69. Jabrane-Ferrat N, Campbell MJ, Esserman LJ, Peterlin BM (2006) Challenge
with mammary tumor cells expressing MHC class II and CD80 prevents the
development of spontaneously arising tumors in MMTV-neu transgenic mice. Cancer Gene Ther 13: 1002–1010. 54. Delahaye NF, Rusakiewicz S, Martins I, Menard C, Roux S, et al. (2011)
Alternatively spliced NKp30 isoforms affect the prognosis of gastrointestinal
stromal tumors. Nat Med 17: 700–707. April 2013 | Volume 9 | Issue 4 | e1003257 PLOS Pathogens | www.plospathogens.org 17
|
https://openalex.org/W3084414150
|
https://jurnal.ugm.ac.id/v3/DB/article/download/343/96
|
Indonesian
| null |
Analisa Teknik Penerjemahan pada kalimat Deklaratif oleh Auto Translation dalam fitur Closed Captions (CC) pada Video di Youtube.com
|
Deleted Journal
| 2,019
|
cc-by-sa
| 2,771
|
Deskripsi Bahasa
Vol. 2 (1) Maret 2019 P-ISSN: 2615-7349 https://jurnal.ugm.ac.id/db https://jurnal.ugm.ac.id/db https://jurnal.ugm.ac.id/db Pribadi Fitra Utama
Mahasiswa Pascasarjana Prodi Linguistik UGM
Surel: pfutama@gmail.com Keywords: Teknik penerjemahan; auto translation; closed captions; Youtube.com Keywords: Teknik penerjemahan; auto translation; closed captions; Youtube.com INTISARI INTISARI Penerjemahan otomatis yang terdapat di dalam fitur auto translation pada Youtube.com memiliki
alogaritma tersendiri dalam menerjemahkan subtitle yang akan ditampilkan. Pada luarannya, didapati
bahwa ada pengaplikasian teknik-teknik penerjemahan yang dilakukan. Penelitian ini menganalisa teknik
penerjemahan yang terdapat pada terjemahan otomatis oleh fitur Closed Captions yang terdapat pada
video di situs Youtube.com yang berjudul 'President Donald Trump URGENT Speech to the United Nations
General Assembly'. Penelitian ini berbasis pada metode kualitatif deskriptif dan perolehan datanya didapat
melalui proses transkripsi. Hasil dari penelitian ini menunjukkan bahwa terdapat 9 teknik terjemahan
ditemukan pada luaran dari fitur auto-translation pada Youtube.com dimana teknik penerjemahan harfiah
mendominasi dengan 56.9%, teknik peminjaman diposisi kedua dengan persentasi 26,9%, teknik
padanan lazim diposisi ketiga dengan 8,6%, teknik kompresi linguistik diposisi keempat dengan 2,15%
serta teknik kalke, modulasi, partikularisasi, transposisi dan teknik kompensasi yang mengisi posisi lima
sampai sembilan dengan persentase 1,07%. Analisa Teknik Penerjemahan
pada kalimat Deklaratif oleh
Auto Translation dalam fitur
Closed Captions (CC) pada
Video di Youtube.com
INTI Mahasiswa Pascasarjana Prodi Linguistik UGM
Surel: pfutama@gmail.com LATAR BELAKANG Secara harfiah, KBBI daring mengartikan video sebagai rekaman gambar hidup atau program
televisi untuk ditayangkan lewat pesawat televisi. Media penyebaran informasi melalui gambar
bergerak atau yang lazim disebut video tersebut telah lama digunakan oleh manusia modern. Perkembangan zaman telah merevolusi metode penyebaran berita bergerak dari era VHS sampai
pada era streaming. Pada era on-demand seperti sekarang, setiap orang yang ingin memperoleh
berita atau sekedar ingin mendapatkan hiburan hanya perlu memiliki perangkat seperti telpon
pintar dan akses internet. Di internet, terdapat satu portal video yang sangat popular yakni
Youtube.com Youtube.com adalah portal video yang dimiliki oleh raksasa internet dunia Google.inc. Dari laman
utama Youtube, semua orang dapat mengakses video secara bebas sesuai dengan kategori yang
mereka inginkan. Video-video yang ada pada Youtube.com berasal dari kontributor-kontributor
yang berasal dari seluruh dunia. Selain kebebasan akses yang sangat fleksibel, Youtube juga dibekali
oleh fitur yang sangat canggih, yakni translasi otomatis pada videonya. Fitur ini sangat memudahkan
penggunanya karena tidak semua pengguna menggunakan Bahasa yang sama. Bila berbicara tentang Bahasa dan translasi, tentu ada celah untuk meneliti bagaimana Bahasa luaran
dari hasil fitur tersebut. Pada penelitian ini, objek yang dijadikan bahan kajian adalah video pada
Youtube.com yang berjudul 'President Donald Trump URGENT Speech to the United Nations General
Assembly' yang diunggah oleh kanal resmi Golden State Times. Video ini berisi tentang pidato yang
dibawakan oleh presiden Amerika Serikat pada acara Perserikatan Bangsa-Bangsa tahun 2018
dengan total durasi 40:04 menit. Di dalam pidatonya, kalimat yang diujarkan oleh Donald Trump
bersifat deklaratif, yakni menyebarkan berita atau informasi tertentu. Bahasa sumber dari video
tersebut adalah Bahasa Inggris dan luaran yang dihasilkan oleh fitur translasi otomatis adalah Bahasa
Indonesia. Pada translasi otomatis yang dimuat pada video tersebut, terdapat Teknik-teknik
penerjemahan yang digunakan. [57-61] Analisa Teknik Penerjemahan pada kalimat Deklaratif oleh Auto Translation dalam fitur Closed Captions (CC)
pada Video di Youtube.com Analisa Teknik Penerjemahan pada kalimat Deklaratif oleh Auto Translation dalam fitur Closed Captions (CC)
pada Video di Youtube.com Berdasarkan celah tersebut, maka teori yang cocok untuk digunakan sebagai dasar penelitian
adalah teori dari penelitian ini menggunakan teori Molina dan Albir (2002). Molina dan Albir
(2002:509) mendefiniskan teknik penerjemahan sebagai prosedur untuk menganalisis dan
mengklasifikasikan bagaimana kesepadanan terjemahan terjadi dan dapat diterapkan pada berbagai
satuan lingual. LATAR BELAKANG Berikut adalah Teknik-teknik penerjemahan yang dikemukakan oleh Molina dan
Albir (2002:509-511): adaptasi, amplifikasi, peminjaman langsung/natural, kalke, kompensasi,
deskripsi, kreasi diskursif, kesepadanan lazim, generalisasi, amplifikasi, kompresi, penerjemahan
harfiah, modulasi, partikulasi, reduksi, substitusi, variasi dan transposisi. Analisis dari teknik-teknik
tesebut dilakukan untuk mengetahui bagaimana translasi otomatis menerjemahkan ujaran dari
Bahasa sumber ke dalam Bahasa sasaran demi mencapai kesepadanan dan kesamaan makna. Teori
yang digunakan pada penelitian ini juga didukung oleh teori lain seperti teori penerjemahan dan
kesepadanan oleh Larson (1984). Larson (1984:3) mendefinisikan penerjemahan sebagai pengalihan makna dari Bahasa sumber (BSu)
kedalam bahasa sasaran (BSa). Menurutnya, Makna merupakan inti yang harus dipertahankan dan
dialihkan sedangkan struktur atau bentuk Bahasa bisa diubah dan disesuaikan dengan Bahasa
targetnya. Larson memberi Batasan tentang definisinya tentang penerjemahan yakni penerjemahan
sebagai proses pengalihan makna yang tidak selalu berusaha mempertahankan bentuk BSu tetapi
maknalah yang harus disampaikan dalam bentuk yang berterima dalam BSa. Lalu, Larson
menjelaskan tentang teori mengenai kesepadanan yang bermanfaat untuk memberikan pemahaman
mengenai pentingnya kesepadanan dalam penerjemahan dengan memperhatikan konteks. Penelitian ini termasuk dalam jenis penelitian dengan pendekatan kualitatif deskriptif dan dalam
bentuk studi kasus terpancang. Penelitian ini dikategorikan penelitian dasar (basic research) karena
bertujuan untuk memahami suatu masalah yang mengarah pada manfaat teoretik tidak pada
manfaat praktis (Sutopo, 2006). Jenis ini sering disebut dengan penelitian akademik. Hal ini sejalan
dengan penelitian ini yang merupakan penelitian akademik dan bertujuan untuk mengidentifikasi,
mendeskripsikan dan mengkaji teknik penerjemahan subtitling pada satuan frasa, klausa dan kalimat
serta kaitannya dengan kualitas penerjemahan dilihat dari aspek keakuratannya. Data pada penelitian ini adalah subtitle dari ujaran berbentuk deklaratif yang diujarkan oleh
presiden Amerika Serikat Donald Trump pada pidatonya yang berjudul 'President Donald Trump
URGENT Speech to the United Nations General Assembly'. Sumber data tersebut diakses melalui
Youtube.com dengan tautan https://www.youtube.com/watch?v=8Fx1yqcaxjA&t=563s. Pengambilan
data pada video tersebut menggunakan metode transkripsi yakni menyimak dan mencatat kalimat,
frasa atau kata yang dikategorikan sebagai data melalui subtitle dari fitur Closed Captions (CC). PEMBAHASAN Pribadi Fitra Utama Pribadi Fitra Utama Dari hasil yang ditunjukkan pada tabel di atas, ditemukan beberapa teknik penerjemahan yang
digunakan oleh alogaritma auto translation dalam fitur Closed Captions pada translasi otomatis,
yakni: Dari hasil yang ditunjukkan pada tabel di atas, ditemukan beberapa teknik penerjemahan yang
digunakan oleh alogaritma auto translation dalam fitur Closed Captions pada translasi otomatis,
yakni: 1. Teknik yang paling sering digunakan adalah teknik penerjemahan harfiah dengan 56,9%
dari total keseluruhan data. Dari acuan data, contoh dari teknik tersebut adalah sebagai
berikut: 1) America's so true yang diterjemahkan menjadi negara amerika sangat benar
(DTS/6), 2) didn't expect that reaction but that's okay diterjemahkan menjadi tidak
mengharapkan reaksi itu tapi itu baik (DTS/7), 3) since my election we've added 10 trillion
dollars in wealth diterjemahkan menjadi sejak pemilihan saya, kami telah menambahkan 10
triliun dolar dalam kekayaan (DTS/9), 4) we have past the biggest tax cuts and reforms in
american history diterjemahkan menjadi kita memiliki masa lalu pemotongan pajak terbesar
dan reformasi di amerika sejarah (DTS/14), 5) our military will soon be more powerful than it
has ever been before diterjemahkan menjadi militer akan segera lebih kuat daripada itu pernah
ada sebelumnya (DTS/19), 6) we are standing up for america and for the american people
diterjemahkan menjadi kami berdiri untuk amerika dan untuk rakyat amerika (DTS/22). Menurut Molina dan Albir (2002:510), Penerjemahan harfiah atau literal translation merupakan
Teknik penerjemahan dimana penerjemah (dalam hal ini mesin) menerjemahkan ungkapan kata
demi kata. Pada terjemahan oleh mesin, hal ini lazim ditemukan pada awal-awal pengembangan dan
fitur translasi otomatis ini juga masih pada awal pengembangannya. Alogaritma dari fitur ini
terkesan jauh dari sempurna karena hasil dari terjemahannya terlihat kaku karena teknik
penerjemahan harfiah mendominasi. 2. Teknik kedua yang banyak terdapat setelah penerjemahan harfiah adalah teknik
peminjaman atau borrowing Berdasarkan data, persentase dari teknik peminjaman adalah
26,9% dengan 25 data. Dari 25 data, terdapat 9 data teknik peminjaman yang bersifat
murni dan 16 data teknik peminjaman yang sudah dinaturalisasi. Fungsi dari teknik ini
adalah untuk menjelaskan istilah, kata atau frasa yang tidak memiliki padanan yang sepadan
pada Bahasa sasaran. PEMBAHASAN Setelah analisis data pada video presiden Amerika Serikat Donald Trump dalam pidatonya yang
berjudul 'President Donald Trump URGENT Speech to the United Nations General Assembly' dilakukan,
terdapat temuan berupa 9 teknik penerjemahan yang digunakan oleh algoaritma translasi otomatis
pada Youtube.com. dibawah ini adalah tabel dari jumlah berikut dengan akumulasi penggunaannya: Teknik Penerjemahan
Jumlah
Persentase
Peminjaman
25
26,9%
Penerjemahan Harfiah
53
56.9%
Padanan Lazim
8
8,6%
Kalke
1
1,07%
Modulasi
1
1,07%
Partikularisasi
1
1,07%
Kompresi Linguistik
2
2,15%
Transposisi
1
1,07%
Kompensasi
1
1,07%
Jumlah
93
100% 58| https://doi.org/10.22146/db.v1i2.47xxx Pribadi Fitra Utama Berikut adalah contoh dari peminjaman yang bersifat murni: 1)
America’s economy is booming like never before diterjemahkan menjadi ekonomi amerika
sedang booming seperti tidak pernah sebelumnya (DTS/8), 2) That is why america will always
use independence and cooperation over global governance control and domination
diterjemahkan menjadi itulah mengapa amerika akan melakukannya selalu menggunakan
kemandirian dan kerja sama atas kontrak tata kelola global dan dominasi (DTS/29). Pada contoh 1, kata Booming dipinjam dari BSu ke BSa tanpa adanya penerjemahan. Ini
dilakukan untuk mempertahankan makna dan konteks agar kalimat tersebut dapat utuh
secara makna. Pada contoh 2, kata Global tidak diterjemahkan menjadi luas karena
maknanya akan menjadi berbeda. Berikut contoh dari teknik peminjaman yang bersifat naturalisasi: 1) The sanctions will stay
in place until denuclearization occurs diterjemahkan menjadi Sanksi akan tetap berlaku
sampai denuklirisasi terjadi (DTS/49), 2) Our shared goals must be the escalation of military
conflict along with a political solution that honors the will of the syrian people diterjemahkan
menjadi Tujuan kita bersama ekskalasi konflik militer bersama dengan solusi politik itu
menghormati kehendak rakyat suriah (DTS/66). Pada data 1 dan 2, penerjemahan Sanctions menjadi Sanksi dan escalation menjadi
ekskalasi termasuk pada peminjaman yang dinaturalisasi, karena pada hakikatnya kata
tersebut tidak ada pada Bahasa Indonesia. Menurut Molina dan Albir (2002:510), Peminjaman adalah Teknik penerjemahan dimana
penerjemah meminjam kata atau ungkapan dari BSu. Peminjaman bisa bersifat murni (pure
borrowing) dan peminjaman yang sudah dinaturalisasi (naturalized borrowing). 3. Teknik penerjemahan yang memiliki persentase terbanyak ketiga adalah padanan lazim
dengan 8,6%. Terdapat 8 data padanan lazim pada video pidato Donald Trump ini. Contoh
dari teknik padanan lazim adalah sebagai berikut: 1) i presented a vision to achieve a brighter
future for all of humanity diterjemahkan menjadi saya disajikan visi untuk mencapai masa Deskripsi Bahasa, Volume 2 (1) Maret 2019|59 Analisa Teknik Penerjemahan pada kalimat Deklaratif oleh Auto Translation dalam fitur Closed Captions (CC)
pada Video di Youtube.com depan yang cerah bagi semua manusia (DTS/3), 2) in less than two years, my administration
has accomplished more than almost administration in the history of our country diterjemahkan
menjadi di kurang dari dua tahun administrasi, saya telah mencapai lebih dari hampir semua
administrasi dalam sejarah kami (DTS/5), 3) jobless claims are at a 50-year low diterjemahkan
menjadi klaim pengangguran berada pada titik terendah dalam 50 tahun (DTS/11), 4) we
had highly productive conversations and meetings diterjemahkan menjadi kami sangat
produktif percakapan dan pertemuan (DTS/58). Pribadi Fitra Utama Pada contoh 1, kata vision diterjemahkan menjadi visi. Penerjemahan ini dilakukan karena
visi termasuk pada kata yang lazim digunakan pada Bahasa Indonesia dan banyak ditemui
pada penggunaan sehari-hari. Begitupun dengan contoh 2 yakni Administration yang
diterjemahkan menjadi administrasi. Contoh 3 dan 4 yakni claims menjadi klaim dan
productive menjadi produktif adalah contoh lain dari kata yang lazim digunakan pada BSa
dan memiliki padanan yang sama dengan makna yang ada pada BSu-nya. Menurut Molina & Albir (2002:510), Kesepadanan lazim adalah Teknik penerjemahan yang lazim
digunakan untuk menerjemahkan istilah atau ekspresi yang sudah dikenal dalam kamus atau
digunakan dalam kehidupan sehari-hari. 4. Pada urutan keempat, teknik yang digunakan sebanyak 2,15% adalah teknik kompresi
linguistik atau linguistic compression. Pada penelitian ini, ditemukan 2 data mengenai
kompresi linguistik, yakni: 1) in the middle east, our new approach is also yielding great and
very historic change diterjemahkan menjadi di timur tengah, pendekatan baru kami juga bagus
dan perubahan yang sangat bersejarah (DTS/54), 2) thanks to the united states military and
our partnership with many of your nations diterjemahkan menjadi terima kasih ke militer
amerika serikat dan kami kemitraan dengan banyak dari anda (DTS/62). Molina dan Albir (2002:510) mendefinisikan teknik kompresi linguistik sebagai Teknik
penerjemahan yang biasa diterapkan dalam pengalihbahasaan simultan atau dalam penerjemahan
teks film dengan cara mensistesa unsur-unsur linguistik dalam teks Bsa. Sesuai Namanya, kompresi
yang dimaksud adalah dengan memapatkan makna dari BSu kepada BSa sesuai konteks. Contoh 1
merupakan kompresi makna dari yielding great yang diterjemahkan menjadi Bagus. Arti dari frasa
dan kedua kata tersebut memiliki kesamaan makna. Pada contoh kedua, frasa your Nations pada
BSa hanya diterjemahkan menjadi Anda. Jika melihat dari konteks, tidak ada perbedaan yang
signifikan dari pemampatan kata tersebut. 5. Teknik selanjutnya adalah Kalke. Pada penelitian ini hanya terdapat 1 data saja dan
persentasenya hanya 1.07 %. Contoh pada penelitian ini adalah : 1) African-American,
Hispanic-American, and Asian-American unemployment have all achieve their lowest
ever recorded yang diterjemahkan menjadi Afrika-Amerika, Hispanik-Amerika Dan
Pengangguran Asia-Amerika memiliki segalanya mencapai level terendah mereka direkam
(DTS/12). Teknik kalke adalah Teknik penerjemahan yang merujuk pada penerjemahan secara literal, baik
leksikal maupun structural. Pada data (DTS/12), penerjemahan African-American menjadi Afrika-
Amerika terkesan secara literal tanpa ada perubahan secara struktur. Ini menandakan bahwa kata
tersebut termasuk dalam kategori penerjemahan kalke. Teknik berikutnya adalah teknik kompensasi. Pribadi Fitra Utama Molina dan Albir (2002:510) menyebutkan bahwa
Kompensasi adalah Teknik penerjemahan dimana penerjemah memperkenalkan unsur-unsur
informasi atau pengaruh statistik teks BSu di tempat lain dalam Teks BSa. 6. Penerapan teknik Kompensasi lazim dilakukan ketika ada makna yang tidak bisa
diterjemahkan kedalam Bahasa sasaran. Berikut adalah contoh pada data: 1) i addressed
the threats facing our world yang diterjemahkan menjadi saya berbicara tentang ancaman
menghadapi dunia kita (DTS/2). Dari data di atas, kata Adressed yang diterjemahkan
menjadi berbicara adalah contoh dari teknik kompensasi. Secara literal, kedua kata ini
memiliki arti yang bebeda. Tetapi, secara konteks, BSa tersebut bisa menyamai arti dari 60| Pribadi Fitra Utama https://doi.org/10.22146/db.v1i2.47xxx BSu karena konteksnya masih pada makna yang sama. Teknik ini hanya mendapat
persentase 1.07%. BSu karena konteksnya masih pada makna yang sama. Teknik ini hanya mendapat
persentase 1.07%. 7. Teknik selanjutnya adalah teknik partikularisasi. Partikularisasi merupakan teknik
penerjemahan dimana penerjemah menggunakan istilah yang lebih konkrit, dari
superordinat ke subordinat. Realisasi dari Teknik ini adalah dengan menggunakan istilah
yang presisi. Pada Analisa yang telah dilakukan, terdapat 1 data yang mewakili persentase
1,07%. Data tersebut adalah: 1) we believe that when nations respect the right of their
neighbors and defend the interests of their people yang diterjemahkan menjadi kita percaya
bahwa ketika negara menghormati hak-hak negara tetangga mereka dan membela kepentingan
orang-orang mereka (DTS/25). Pada data DTS/25, kata Neighbors diterjemahkan menjadi kata yang lebih presisi dan kongkrit,
yakni Negara tentangga, tidak hanya dengan ‘tetangga’ saja. 8. Teknik selanjutnya adalah teknik modulasi. Pada Analisa, teknik ini juga memperoleh
persentase 1,07% saja. Berikut adalah datanya: 1) we are also standing up for the world
diterjemahkan menjadi kita juga membela dunia (DTS/23). Teknik modulasi adalah teknik penerjemahan dimana penerjemah mengubah sudut pandang, fokus
atau kategori kognitif dalam kaitannya dengan BSu. Perubahan sudut pandang tersebut dapat
bersifat leksikal atau structural. Teknik modulasi adalah teknik penerjemahan dimana penerjemah mengubah sudut pandang, fokus
atau kategori kognitif dalam kaitannya dengan BSu. Perubahan sudut pandang tersebut dapat
bersifat leksikal atau structural. 9. Teknik terakhir yang ditemukan pada analisis adalah teknik transposisi. Hanya ditemukan
1 data pada teknik ini. Berikut adalah contoh dari teknik transposisi:1) the most
compassionate policy is to place refugees as close to their homes as possible yang diterjemahkan
mernjadi paling kebijakan welas asih adalah tempat pengungsi sedekat mungkin dengan rumah
mereka (DTS/71). Menurut Molina dan albir (2002:510), Transposisi merupakan Teknik penerjemahan dengan
mengubah kategori gramatikal. Pribadi Fitra Utama Teknik ini sama degan Teknik pergeseran kategori, struktur dan
unit. Kata kerja dalam BSu diubah menjadi kata benda dalam BSa ataupun sebaliknya. Pada BSa,
kata compassionate diterjemahkan menjadi Welas Asih. Compassionate merupakan kata adjektifa
yang diterjemahkan menjadi kata nomina. KESIMPULAN Hasil penelitian ini menunjukkan bahwa penerjemahan oleh mesin pada fitur translasi otomatis
dalam CC di Youtube.com menerapkan beberapa teknik penerjemahan yang dikemukakan oleh
Molina dan Albir (2002). Terdapat 9 teknik penerjemahan yang diterapkan oleh alogaritma dari
fitur translasi otomatis tersebut. Dari Sembilan teknik tersebut, teknik yang paling mendominasi
adalah teknik penerjemahan Harfiah. Hal ini dapat dimaklumi karena fitur ini tergolong baru
diimplementasikan pada teknologi subtitling dan juga pionir dari fitur translasi secara otomatis pada
video daring. Penerjemahan secara literal dan terkesan kata per kata sangatlah kuat. Teknik
peminjaman menempati posisi kedua, teknik padanan lazim mengisi posisi ketiga, teknik kompresi
linguistik pada posisi ke empat dan 5 teknik lainnya menempati urutan yang sama karena memiliki
jumlah frekuensi yang sama. DAFTAR PUSTAKA Albir, A.H and Molina, L. 2002. Translation Technique Revisited: A Dynamic and Functionalist
Approach. Meta, Vol. XLVII, No. 4. Kamus Besar Bahasa Indonesia Online. https://kbbi.kemdikbud.go.id/. Diakses pada tanggal 20
November 2018 pukul 15.19 WIB. Larson, Mildred L. 1984. Meaning-Based Translation: A Guide to Cross Language Equivalence. Larson, Mildred L. 1984. Meaning-Based Translation: A Guide to Cross Language Equivalence. Sutopo. 2006. Metodologi Penelitian Kualitatif. Surakarta: UNS Sutopo. 2006. Metodologi Penelitian Kualitatif. Surakarta: UNS Trump, Donald. President Donald Trump URGENT Speech to the United Nations General Assembly. Diunggah
oleh
Golden
State
Times
pada
tautan
https://www.youtube.com/watch?v=8Fx1yqcaxjA&t=563s. Deskripsi Bahasa, Volume 2 (1) Maret 2019|61
|
https://openalex.org/W2117891008
|
https://bioone.org/journals/journal-of-insect-science/volume-10/issue-6/031.010.0601/Use-of-Sleeve-Nets-to-Improve-Survival-of-the-Boisduval/10.1673/031.010.0601.pdf
|
English
| null |
Use of Sleeve Nets to Improve Survival of the Boisduval Silkworm,<i>Anaphe panda</i>, in the Kakamega Forest of Western Kenya
|
Journal of insect science
| 2,010
|
cc-by
| 5,268
|
Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Use of Sleeve Nets to Improve Survival of the Boisduval
Silkworm, Anaphe panda, in the Kakamega Forest of
Western Kenya Use of Sleeve Nets to Improve Survival of the Boisduval
Silkworm, Anaphe panda, in the Kakamega Forest of
Western Kenya Authors: Mbahin, N., Raina, S. K., Kioko, E. N., and Mueke, J. M. Source: Journal of Insect Science, 10(6) : 1-10
Published By: Entomological Society of America
URL: https://doi.org/10.1673/031.010.0601 Authors: Mbahin, N., Raina, S. K., Kioko, E. N., and Mueke, J. M. Source: Journal of Insect Science, 10(6) : 1-10
Published By: Entomological Society of America
URL: https://doi.org/10.1673/031.010.0601 BioOne Complete (complete.BioOne.org) is a full-text database of 200 subscribed and open-access titles
in the biological, ecological, and environmental sciences published by nonprofit societies, associations,
museums, institutions, and presses. Your use of this PDF, the BioOne Complete website, and all posted and associated content indicates your
acceptance of BioOne’s Terms of Use, available at www.bioone.org/terms-of-use. Usage of BioOne Complete content is strictly limited to personal, educational, and non - commercial use. Commercial inquiries or rights and permissions requests should be directed to the individual publisher as
copyright holder. BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit
publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to
critical research. Journal of Insect Science: Vol. 10 | Article 6
Mbahin
! Use of sleeve nets to improve survival of the Boisduval
silkworm, Anaphe panda, in the Kakamega Forest of
western Kenya
N. Mbahin1,2a*, S. K. Raina1b, E. N. Kioko1c and J. M. Mueke2d
1Commercial Insects Programme, ICIPE-African Insect Science for Food and Health, P. O. Box: 30772-0010
Nairobi, Kenya
2Department of Zoological Sciences, Kenyatta University, P. O. Box: 43844, Nairobi, Kenya Mbahin et al. Journal of Insect Science: Vol. 10 | Article 6 N. Mbahin1,2a*, S. K. Raina1b, E. N. Kioko1c and J. M. Mueke2d 1Commercial Insects Programme, ICIPE-African Insect Science for Food and Health, P. O. Box: 30772-00100
Nairobi, Kenya
2Department of Zoological Sciences, Kenyatta University, P. O. Box: 43844, Nairobi, Kenya 2Department of Zoological Sciences, Kenyatta University, P. O. Box: 43844, Nairobi, Kenya Abstract Prospects for development of a wild silk industry in Africa would be improved if silkworm
survival during mass production could be improved. A study on the survival of the Boisduval
silkworm, Anaphe panda (Boisduval) (Lepidoptera: Thaumetopoeidae) was conducted with
and without protection by net sleeves in two different forest habitats (natural and modified) in
the Kakamega forest of western Kenya. Overall, cohort survival was significantly higher (P <
0.001) in the natural than in the modified forest, but larval survival was improved over three-
fold by protection with net sleeves in both habitat types. In the modified forest, only 16.8% of
unprotected larvae survived to the pupal stage and formed cocoons, whereas 62.3% survived in
the same environment when they were protected with net sleeves. In the natural forest, 20.4%
of unprotected larvae survived, whereas 67.7% survived in net sleeves. There was also a
significant effect of season; cohorts of larvae that eclosed in the wet season had significantly
lower survival than those eclosing in the dry season (P = 0.02). Sources of mortality appeared
to be natural enemies (parasites, predators and diseases) and climatic factors. Key words: conservation, exclusion, mortality, silk farming, silkmoth
Corresponcence: a* mnorber@icipe.org, b sraina@icipe.org, c ekioko@icipe.org, d jmueke@yahoo.com, *Corresponding
author
Associate Editor: J.P. Michaud was editor of this paper
Received : 8 December 2007 Accepted : 16 November 2008
Copyright : This is an open access paper. We use the Creative Commons Attribution 3.0 license that permits
unrestricted use, provided that the paper is properly attributed. ISSN: 1536-2442 | Vol. 10, Number 6
Cite this paper as:
Mbahin N, Raina SK, Kioko EN, Mueke JM. 2010. Use of sleeve nets to improve survival of the Boisduval silkworm,
Anaphe panda, in the Kakamega Forest of western Kenya. Journal of Insect Science 10:6, available online:
insectsicence.org/10.6 Journal of Insect Science: Vol. 10 | Article 6
Introduction Mbahin et al. Anaphe ambrizia Butler. Anaphe are
polyphagous moths but Bridelia micrantha
(Hochst) (Malpighiales: Euphorbiaceae) is
the preferred host plant of A. panda in the
Kakamega Forest. In nature, B. micrantha
can be found scattered over a large area of
the Kakamega forest and it flourishes in
both natural tracts and those mixed with
exotic species. With very little care it can
be raised from seed or from cuttings that
propagate rapidly and are ready for use as
host plants in about a year (Gowdey 1953;
Jolly et al. 1979). In the Kakamega Forest,
A. panda silkmoths lay egg clusters under
the leaves of B. micrantha. In many parts of the developing world,
people seek diversified sources of income,
especially those that are sustainable and
environmental friendly. Due to current
population growth in Kenya, pressure on
the Kakamega forest is growing because
the forest plays an important role in
satisfying the daily needs and income of
local people. Wild silk farming is a unique,
ecologically friendly industry with a great
potential
for
employment
generation,
artisanal development and export earnings
(Kioko et al. 1999). African species of
silkmoths provide strong silk of high
commercial value (Raina 2004). The
Boisduval
silkworm,
Anaphe
panda
(Boisduval)
(Lepidoptera: The potential of the African indigenous
silkmoth species for wild silk production
has been well documented in Nigeria
(Ashiru 1991), Uganda (Kato 2000) and
Kenya (Kioko et al. 2000; Mbahin et al. 2008). However, the wild silk industry in
Kenya will not be commercially viable
unless technologies are developed to reduce
silkworm mortality due to natural enemies
and help farmers develop sustainable mass
production of A. panda cocoons. Some
studies have recommended the use of
sleeve nets to protect young larvae (Kioko
et al. 1999; Raina 2000; 2004; Ngoka
2003), but there are no reliable data
available on the benefit of using sleeve nets
to improve Anaphe silkworm survival in
the field. The present study was conducted
in two forest habitats to test whether the
survival rate of A. panda silkworms can be
improved by protection with sleeve nets. Thaumetopoeidae) shows the best potential
for wild silk production since it produces a
huge cocoon that is communally weaved by
20-200 silkworm larvae. Wild silk farming
is among the industries that might assist
resource-poor farmers of this region to
escape a vicious cycle of poverty, while
providing
an
incentive
for
forest
conservation (Kioko et al. 2000). Journal of Insect Science: Vol. 10 | Article 6
Introduction In
countries where rural communities depend
on subsistence farming, wild silkmoth
cultivation can be a supplementary activity
for income generation while simultaneously
conserving biodiversity. Silk has been used for textiles for
thousands of years (Raina 2004). In Africa,
most of the wild silkmoths belong to the
families Saturniidae, Lasiocampidae, and
Thaumetopoeidae. Anaphe species are
widely distributed in the intertropical
regions of continental Africa such as
Nigeria, Uganda, Kenya, Cameroon, Congo
and Togo. The important species used in
the production of Anaphe silk are Anaphe
infracta
Walsingham,
Anaphe
venata
Butler, Anaphe panda Boisduval, Anaphe
reticulata
Walker,
Anaphe
carteri
Walsingham, Anaphe moloneyi Druce and Journal of Insect Science | www.insectscience.org Journal of Insect Science | www.insectscience.org 1! Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Journal of Insect Science: Vol. 10 | Article 6
Introduction Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Study sites The Kakamega Forest is a tropical
rainforest that covers a total area of
approximately 265 km2 and is located
between latitudes 0˚ 10 and 0˚ 21 North
and longitudes 34˚ 47 and 34˚ 58 East,
respectively (Figure 1). It comprises several
separate blocks of forest of which Isecheno Journal of Insect Science | www.insectscience.org Journal of Insect Science | www.insectscience.org 2 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Journal of Insect Science: Vol. 10 | Article 6
(415 ha) belongs to the Lunyu sub-location
whereas Ikuywa (380 ha) belongs to the
Ikuywa sub-location. Two sites were used
for the study: Musembe village, located in
Ikuywa
(a
natural
forest
comprised
exclusively of indigenous species), and
Chirobani village, located in Isecheno (a
modified indigenous forest comprised of a
combination of native and exotic species). The exotic species are mainly pines, Acacia
spp. (Fabaceae) and Eucalyptus spp. (Myrtaceae)
(Mbahin
et
al. 2008). Indigenous plants comprise about 150
species of woody trees, 90 species of
dicotyledonous
herbs,
80
species
of
monocotyledonous herbs of which about 60
are orchids, and a further 62 species of
ferns, totalling to about 380 identified
species of vascular plants (KIFCON 1994). Mbahin et al. am; 12 am; 3 pm and 9 pm) at both sites
throughout the period of study. A rain
gauge was used for recording rainfall data. ta data
hygrothermometer
Apparatus
Figure 1: Study sites in the Kakamega Forest of western Kenya. High quality figures are available online.
Figure 1: Study sites in the Kakamega Forest of western Kenya. High quality figures are available online. Environmental data
A
di it l
h
th Environmental data
A
digital
hygrothermometer
(Zheda
Electric
Apparatus
Inc.,
http://www.zjlab.com)
was
used
for
recording daily temperature (maximum and
minimum) and measurements of relative
humidity were recorded four times daily (6 Silkworm survival Two naturally-laid egg clusters of A. panda
were selected on each of 150 B. micrantha
trees that had canopies of about 10 cubic
feet (Figure 2). Any additional egg clusters
were removed. Each tree was divided into
two experimental areas: one with a net
sleeve measuring 1.5 x 1.5 x 2 m covering
one cohort of larvae and a control cohort of
larvae that were not covered. The net
sleeves were tied closed on the branches of
the host plant and a 2 m long zippered
aperture \ permitted access for purpose of
observation and data collection (Figure 3). Branches were selected among those that
bore sufficient leaves to provide adequate
food. Of 300 clusters of eggs selected, eggs
hatched in a total of 221 that were included
in the experiment (105 protected, 116
exposed). Observations on the possible
causes of mortality were made twice
weekly and counts of the surviving larvae
in net sleeves and on control branches from
June 2005 to June 2007. Protected cohorts
were moved to new branches of the same
tree two or three times during the course of
development as required to ensure an
adequate food supply. Only cohorts that Journal of Insect Science | www.insectscience.org Journal of Insect Science | www.insectscience.org Journal of Insect Science | www.insectscience.org 3 3 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Journal of Insect Science: Vol. 10 | Article 6 Mbahin et al. completed all seven larval instars with
some survivors (Figure 4) were included in
the analysis. The mortality rate (by instar)
was calculated as follows:
Figure 2: Eclosing egg mass of Anaphe panda. High quality
figures are available online. Mortality rate = Sini S fin
Sini
100 With: S ini = Number of larvae alive at the
beginning of the instar and S fin = Number
of larvae alive at the end of the instar. The instantaneous death rate is the
boundary of the expression below when
. 0
t probability fora particular larva todiebetween t and t + t instar
(
)
t probability fora particular larva todiebetween t and t + t instar
(
)
t Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare
overall cohort survival between forest
types. Mbahin et al. Journal of Insect Science: Vol. 10 | Article 6
Data analysis
The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment'
(protected/unprotected), forest
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare
overall cohort survival between forest
types. Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use probability fora particular larva todiebetween t and t + t instar
(
)
t Figure 2: Eclosing egg mass of Anaphe panda. High quality
figures are available online. The 'force of mortality' (incidence rate) for
each instar can be calculated as follows:
Figure 3: Branch of Bridelia micrantha with net sleeve used
to enclose developing cohorts of Anaphe panda silkworms. High quality figures are available online. Incidencerate = Dper
Sexp
100 Incidencerate = Dper
Sexp
100 Where D per = Number of dead larvae in the
specific instar and S
exp = Number of
surviving larvae exposed to risk in that
instar.
Figure 3: Branch of Bridelia micrantha with net sleeve used
to enclose developing cohorts of Anaphe panda silkworms. High quality figures are available online.
Figure 4: 7th instar larvaof Anaphe panda on its host plant, Bridelia micrantha. High quality figures are available online.
nstar larvaof Anaphe panda on its host plant, Bridelia micrantha. High quality figures are available online. Figure 4: 7th instar larvaof Anaphe panda on its host plant, Bridelia micrantha. High quality figures a Journal of Insect Science | www.insectscience.org 4 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Journal of Insect Science: Vol. 10 | Article 6
Data analysis
The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment'
(protected/unprotected), forest
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare
overall cohort survival between forest
types. Figure 5: Rainfall, temperature and relative humidit
figures are available online. Journal of Insect Science: Vol. 10 | Article 6
Data analysis
The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment'
(protected/unprotected), forest
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare
overall cohort survival between forest
types. Journal of Insect Science: Vol. 10 | Article 6
Data analysis
The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment'
(protected/unprotected), forest
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Larval survival There was no effect of 'year' on the number
of larvae pupating (F = 0.09; df = 2,177; P
= 0.910) so data were pooled across years
for further analysis. In both forest types,
mortality rates tended to be higher for
young larvae (1st to 4th instar) than for older
instars (5th to 7th) (Table 1), but the main
effect of forest type was not significant (F
= 2.49; df = 1, 178; P = 0.117). However,
the effect of the sleeve net protection
treatment was highly significant (F =
229.26; df = 1,178; P < 0.001) and cohorts
eclosing in the wet season (2nd brood) had
significantly lower survival than those
eclosing in the dry season (1st brood) (F =
12.49; df = 1,178; P = 0.02). The forest
type*treatment
interaction
was
not
significant (F = 0.24; df = 3,176; P =
0.624), nor was the treatment*brood
interaction (F = 2.34; df = 3,176; P =
0.128), nor the forest*brood interaction (F
= 0.10; df = 3,176; P = 0.757), nor the
three-way interaction (F = 2.34; df = 7,172;
P = 0.440). Discussion The 'force of mortality' (incidence rate) for
each instar can be calculated as follows: 100
exp
= S
D
rate
Incidence
per Where D per = Number of dead larvae in the
specific instar and S
exp = Number of
surviving larvae exposed to risk in that
instar. Journal of Insect Science: Vol. 10 | Article 6 180.9 to 265.9 cm in the modified forest
(Isecheno), and from 188.6 to 224.7 cm in
the natural forest (Ikuywa). The annual
number of rainy days ranged from 196 –
219, and from 207 – 209 in the modified
and natural forests, respectively. Mean
monthly maximum temperature ranged
from 15.5° C to 36.8° C at Isecheno, and
from 16.5 – 35.6º C at Ikuywa. Mean
monthly maximum humidity ranged from
45.4 – 86.2 % in the modified forest and
from 35.6 – 80.9 % in the natural forest. Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Environmental data Data
on
mean
monthly
average
temperature, relative humidity, and rainfall
for the two study sites are reported in
Figures 5 and 6, respectively. Note that
rainfall is bimodal in the Kakamega Forest,
with a period of "long rains" from April
through June, and "short rains" in August
through November. During the study, the
annual rainfall ranged from (p
p
),
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare
overall cohort survival between forest
types. es.
Figure 5: Rainfall, temperature and relative humidity in the Isecheno modified forest (2005-2007). High quality
figures are available online. Figure 6: Rainfall, temperature and relative humidity in the Ikuywa natural forest (2005-2007). High quality figures
are available online.
Figure 5: Rainfall, temperature and relative humidity in the Isecheno modified forest (2005-2007). High quality
figures are available online.
Figure 5: Rainfall, temperature and relative humidity in the Isecheno modified forest (2005-2007). High quality
figures are available online. Figure 6: Rainfall, temperature and relative humidity in the Ikuywa natural forest (2005-2007). High quality figures
are available online. Figure 6: Rainfall, temperature and relative humidity in the Ikuywa natural forest (2005-2007). High quality figures
are available online. Figure 6: Rainfall, temperature and relative humidity in the Ikuywa natural forest (2005-2007). High quality figures
are available online. Journal of Insect Science | www.insectscience.org 5 5 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Mbahin et al. natural forest 76 of 78 (97.4 %) protected
cohorts survived, compared to 51 of 57
(89.5 %) control cohorts. Considering total
numbers of larvae, only 2,557 of 15,198
(16.8%) unprotected silkworms survived to
pupation in the modified forest, whereas
7,757 out of 12,447 (62.3%) survived when
protected with sleeve nets. In the natural
forest, 3,511 out of 17,213 (20.4%)
unprotected silkworms survived, compared
to 8,888 out of 13,124 (67.7%) in the
sleeve nets. Thus, protection with sleeve
nets increased survival 3.7 and 3.3 fold in
the
modified
and
natural
forests,
respectively. The Nelson-Aalen cumulative
hazard function (Figure 7) reveals that the
protective environment provided by net
sleeves significantly reduced (F = 202.04;
df = 1,125; P < 0.001) silkworm larval
mortality across all instars, although
mortality was greater in early instars than
in later ones. Journal of Insect Science: Vol. 10 | Article 6 Data analysis The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment'
(protected/unprotected), forest
(natural/modified), and 'brood' (1st/2nd) as
independent
variables. Analysis
of
survival-time data was carried out with the
software Stata7 (STATACORP, 2004) and
a Chi-Square test was used to compare The data on numbers of larvae surviving to
form cocoons were analyzed by factorial
ANOVA (SAS Institute, 2003) with 'year'
(2005/2006/2007), 'treatment' The overall survival of silkworm cohorts
was significantly higher in the natural
forest compared to the modified forest (c2 =
36.6, P < 0.001). In the Ischeno modified
forest, 38 out of 48 cohorts (79.2%)
survived to spin cocoons in the sleeve net
treatment, compared to 15 out of 38 (39.5 Journal of Insect Science | www.insectscience.org
p
(
%) for unprotected controls. In the Ikuywa Journal of Insect Science | www.insectscience.org
%) for unprotected controls. In the Ikuywa Journal of Insect Science | www.insectscience.org
%) for unprotected controls. In the Ikuywa 6 Mbahin et al. interaction (F = 2.34; df = 3,176; P =
0.128), nor the forest*brood interaction (F
= 0.10; df = 3,176; P = 0.757), nor the
three-way interaction (F = 2.34; df = 7,172;
P = 0.440). Journal of Insect Science: Vol. 10 | Article 6
overall cohort survival between forest
types. Larval survival There was no effect of 'year' on the number
of larvae pupating (F = 0.09; df = 2,177; P
= 0.910) so data were pooled across years
for further analysis. In both forest types,
mortality rates tended to be higher for
young larvae (1st to 4th instar) than for older
instars (5th to 7th) (Table 1), but the main
effect of forest type was not significant (F
= 2.49; df = 1, 178; P = 0.117). However,
the effect of the sleeve net protection
treatment was highly significant (F =
229.26; df = 1,178; P < 0.001) and cohorts
eclosing in the wet season (2nd brood) had
significantly lower survival than those
eclosing in the dry season (1st brood) (F =
12.49; df = 1,178; P = 0.02). The forest
type*treatment
interaction
was
not
significant (F = 0.24; df = 3,176; P =
0.624), nor was the treatment*brood Environmental data The overall survival of silkworm cohorts
was significantly higher in the natural
forest compared to the modified forest (c2 =
36.6, P < 0.001). In the Ischeno modified
forest, 38 out of 48 cohorts (79.2%)
survived to spin cocoons in the sleeve net
treatment, compared to 15 out of 38 (39.5
%) for unprotected controls. In the Ikuywa
natural forest 76 of 78 (97.4 %) protected
cohorts survived, compared to 51 of 57
(89.5 %) control cohorts. Considering total
numbers of larvae, only 2,557 of 15,198
(16.8%) unprotected silkworms survived to
pupation in the modified forest, whereas
7,757 out of 12,447 (62.3%) survived when
protected with sleeve nets. In the natural
forest, 3,511 out of 17,213 (20.4%)
unprotected silkworms survived, compared
to 8,888 out of 13,124 (67.7%) in the
sleeve nets. Thus, protection with sleeve
nets increased survival 3.7 and 3.3 fold in
the
modified
and
natural
forests,
respectively. The Nelson-Aalen cumulative
hazard function (Figure 7) reveals that the
protective environment provided by net
sleeves significantly reduced (F = 202.04;
df = 1,125; P < 0.001) silkworm larval
mortality across all instars, although
mortality was greater in early instars than
in later ones. Data
on
mean
monthly
average
temperature, relative humidity, and rainfall
for the two study sites are reported in
Figures 5 and 6, respectively. Note that
rainfall is bimodal in the Kakamega Forest,
with a period of "long rains" from April
through June, and "short rains" in August
through November. During the study, the
annual rainfall ranged from 180.9 to 265.9
cm in the modified forest (Isecheno), and
from 188.6 to 224.7 cm in the natural forest
(Ikuywa). The annual number of rainy days
ranged from 196 – 219, and from 207 – 209
in the modified and natural forests,
respectively. Mean monthly maximum
temperature ranged from 15.5° C to 36.8° C
at Isecheno, and from 16.5 – 35.6º C at
Ikuywa. Mean monthly maximum humidity
ranged from 45.4 – 86.2 % in the modified
forest and from 35.6 – 80.9 % in the natural
forest. Discussion The climatic conditions observed in this
study (Figures 5 & 6) were consistent with
reports by Muriuki and Tsingalia (1990)
and Kokwaro (1988). As poikilothermic
organisms,
the
life
cycle,
activity,
distribution and abundance of Lepidoptera
are influenced by temperature (Hill et al. 1999). Pollard and Yates (1985) found that
temperature and rainfall were likely to Journal of Insect Science | www.insectscience.org 7 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Mbahin et al. Journal of Insect Science: Vol. 10 | Article 6 |
Figure 7: Survival function of Anaphe panda silkworms in natural and modified Kakamega Forests when protected
with a sleeve net (dashed line) or unprotected (solid line). High quality figures are available online.
Figure 7: Survival function of Anaphe panda silkworms in natural and modified Kakamega Forests when protected
with a sleeve net (dashed line) or unprotected (solid line). High quality figures are available online. Figure 7: Survival function of Anaphe panda silkworms in natural and modified Kakamega Forests when
with a sleeve net (dashed line) or unprotected (solid line). High quality figures are available online.
Figure 7: Survival function of Anaphe panda silkworms in natural and modified Kakamega Forests when protected
with a sleeve net (dashed line) or unprotected (solid line). High quality figures are available online. Predation likely contributed to mortality of
young larvae, and disease to mortality of
older
instars. The
larval
parasitoids
Exorista
cardinalis
F. (Diptera:
Tachinidae)
and
Cryptus
leucopygus
Granenhorst
(Hymneoptera:
Ichneumonidae)
were
recovered
in
mummified larvae during the course of the
study. These findings are consistent with influence the survival of butterflies directly
and indirectly through the effects on plant
growth, disease, predation or other factors. In light of the present study, further work is
warranted to understand why a forest insect
like A. panda periodically develops high
populations in certain well-defined types of
forest habitat, but not in all habitats where
it occurs. Table 1: Mean instar-specific mortality (perecent ±SEM) of Anaphe panda silkworms either enclosed in net sleeves
(protected) or exposed (control) in modified versus natural tracts in Kakamega Forest, Kenya.
Brood: 1st = eclosion during dry season, 2nd = eclosion during rainy season, n = no. of cohorts followed. Journal of Insect Science | www.insectscience.org Discussion n
Control
n
Protected
n
Control
n
Protected
1st
26
46.0 ± 13.3
25
16.7 ± 4.0
27
37.2 ± 6.8
24
10.5 ± 8.1
2nd
21
65.6 ± 19.7
18
17.4 ± 6.8
20
61.9 ± 10.5
19
14.8 ± 2.7
1st
26
40.4 ± 11.0
25
13.3 ± 5.7
27
33.3 ± 10.4
24
8.3 ± 4.5
2nd
21
53.8 ± 12.8
18
11.1 ± 5.5
20
53.6 ± 13..6
19
12.4 ± 5.4
1st
26
34.9 ± 13.9
25
12.6 ± 5.0
27
30.3 ± 11.8
24
8.4 ± 6.4
2nd
21
49.2 ± 16.3
18
9.4 ± 3.8
20
49.4 ± 16.7
19
9.2 ± 3.7
1st
26
31.8 ± 7.5
25
8.1 ± 4.6
27
29.1 ± 6.3
24
6.1 ± 1.5
2nd
21
44.8 ± 7.3
18
5.2 ± 4.0
20
45.6 ± 18.1
19
8.9 ± 5.6
1st
26
28.2 ± 13.2
25
4.3 ± 0.7
27
25.9 ± 11.4
24
4.8 ± 1.8
2nd
21
34.7 ± 13.2
18
3.8 ± 1.2
20
32.6 ± 15.4
19
5.4 ± 1.3
1st
26
24.2 ± 7.6
25
3.7 ± 1.2
27
24.9 ± 9.4
24
3.9 ± 1.7
2nd
21
30.3 ± 11.5
18
3.6 ± 1.6
20
31.9 ± 15.6
19
5.2 ± 1.2
1st
26
27.3 ± 9.2
25
4.0 ± 0.9
27
22.3 ± 8.8
24
3.5 ± 1.2
2nd
21
30.5 ± 17.5
18
4.3 ± 1.0
20
30.7 ± 11.3
19
4.9 ± 1.6
7th
Larval instar Brood
Isecheno (modified forest)
3rd
4th
5th
6th
1st
2nd
Ikuywa (natural forest) 8 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Mbahin et al. Journal of Insect Science: Vol. 10 | Article 6
parasitoids recovered from silkmoths by
Kioko et al. (1999) and Raina (2004) and
indicate that sleeves nets can be used to
reduce silkworm mortality due to natural
enemies, especially during the rainy season
when larval mortality tends to be higher. This simple technology has the potential to
improve the commercial viability and
sustainability of the wild silk industry in
Africa. The introduction of wild silk
production to the Kakamega forest may
offer important economic incentives to
farmers surrounding the forest. More than
12,400 ha are suitable for silkworm food
plants in the Kakamega Forest and could be
utilized for the cultivation of B. micrantha
(Mbahin et al. 2008). Acknowledgments The authors express their sincere thanks to
Deutscher Akademischer Austauschdienst
(DAAD) for providing the scholarship and
to International Fund for Agricultural
Development (IFAD) and GEF (Global
Environmental
Facility)
for
logistical
assistance. Thanks are also due to ICIPE
for providing facilities. Kioko EN, Raina SK, Mueke JM. 2000. Survey on diversity of wild silk moths
species in East Africa. East African Journal
of Science 2 (1): 1-6. Kokwaro JO. 1988. Conservation status of
the Kakamega forest in Kenya. The Eastern
most relic of the equatorial rain forest of
Africa. Monographs of Systematics and
Botanical Gardens 25: 471-489. Kokwaro JO. 1988. Conservation status of
the Kakamega forest in Kenya. The Eastern
most relic of the equatorial rain forest of
Africa. Monographs of Systematics and
Botanical Gardens 25: 471-489. Discussion Although the use of
sleeve nets greatly improved the survival
rate of silkworms in both forest habitats,
overall cohort survival was somewhat
higher in the natural forest than in the
modified forest that contained introduced
tree species. Thus, reforestation with
indigenous species such as B. micrantha
will not only favor the conservation of
indigenous biodiversity, but also enhance
the productivity of the wild silkmoth
industry. Gowdey CC. 1953. On the utilisation of an
indigenous silkworm (Anaphe infracta
Walsingham)
in
Uganda. Bulletin of
Entomological Research 3: 269-274. Gowdey CC. 1953. On the utilisation of an
indigenous silkworm (Anaphe infracta
Walsingham)
in
Uganda. Bulletin of
Entomological Research 3: 269-274. Hill JK, Thomas CD, Huntley B. 1999. Climate and habitat availability determine
20th century changes in a butterfly’s range
margins. Proceeding of the Royal Society
226: 1197-1206. Jolly MS, Sen SK, Sonwalker TN, Prasad
GK. 1979. Non-mulberry silks. F.A.O. Agricultural services bulletin 29. Kato H. 2000. Structure and thermal
properties of Anaphe, Cricula and Attacus
cocoon filaments. International Journal of
wild silkmoths and silk 5: 11-20. KIFCON 1994. Kenya Indigenous Forest
Conservation Programme. Phase 1 Report. Karura
Forest
Station,
Center
for
Biodiversity, Nairobi. Kioko EN, Raina SK, Mueke JM. 1999. Conservation of the African wild silkmoths
for
economic
incentives
to
rural
communities of the kakamega forest in
Kenya. International Journal of wild
silkmoths and silk 4: 1-5. Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use References Ashiru MO. 1991. Adult morphology of the
silkworm
Anaphe
veneta
Butler
(Lepidoptera: Notonidae). In: H. Akai and
M. Kiuchi, editors. Wild silk moth, pp. 89-
90. International Society for wild silk
moths, Ibaraki, Japan. Mbahin N, Raina SK, Kioko EN, Mueke
JM. 2008. Spatial distribution of cocoon
nests and egg clusters of the silkmoth
Anaphe panda (Boisduval) (Lepidoptera: Journal of Insect Science | www.insectscience.org Journal of Insect Science | www.insectscience.org 9 Mbahin et al. Mbahin et al. Journal of Insect Science: Vol. 10 | Article 6
Thaumetopoeidae) and its host plant
Bridelia micrantha (Euphorbiaceae) in the
Kakamega Forest of western Kenya. International Journal of Tropical Insect
Science 27 (3, 4): 138-144 Muriuki JH, Tsingalia MH. 1990. A new
population of De Brazza’s monkey in
Kenya. Oryx 24: 157–162. Ngoka MB. 2003. A study on the biology
and the impact of natural enemies on the
African wild silkmoth, Gonometa sp. at
Kamaguti, Uasin Gishu District. M. Sc. thesis, Kenyatta University, Nairobi Kenya. Pollard E, Yates TJ. 1985. Monitoring
butterflies for Ecology and conservation. 274 pp. Chapman and Hall. Raina SK. 2000. The economics of
apiculture and sericulture modules for
income generation in Africa. International
Bee Research Association, London. Raina SK. 2004. On developing incentives
for community participation in forest
conservation
through
the
use
of
commercial insects in Kenya. ICIPE, pp. 213. First training course (19th November
10th December 2004). SAS Institute. 2003. SAS/STAT users’
guide, version 8, 6th ed., vol. 2. SAS
Institute. Cary, NC. 10 10 10 Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use Statacorp 2004. Stata Statistical Software.
Release 8.0. Stata Corporation. Journal of Insect Science | www.insectscience.org Downloaded From: https://bioone.org/journals/Journal-of-Insect-Science on 23 Oct 2024
Terms of Use: https://bioone.org/terms-of-use
|
W1977350613.txt
|
https://escholarship.org/content/qt8rg1c3hx/qt8rg1c3hx.pdf?t=of8qib
|
en
|
Modulation of an optical needle’s reflectivity alters the average photon path through scattering media
|
Journal of biomedical optics
| 2,006
|
cc-by
| 5,738
|
UC Irvine
UC Irvine Previously Published Works
Title
Modulation of an optical needle's reflectivity alters the average photon path through
scattering media
Permalink
https://escholarship.org/uc/item/8rg1c3hx
Journal
JOURNAL OF BIOMEDICAL OPTICS, 11(1)
ISSN
1083-3668
Authors
Simonson, P
D'Amico, E
Gratton, E
Publication Date
2006
DOI
10.1117/1.2168167
Copyright Information
This work is made available under the terms of a Creative Commons Attribution License,
availalbe at https://creativecommons.org/licenses/by/4.0/
Peer reviewed
eScholarship.org
Powered by the California Digital Library
University of California
Journal of Biomedical Optics 11共1兲, 014023 共January/February 2006兲
Modulation of an optical needle’s reflectivity alters the
average photon path through scattering media
Paul Simonson
Enrico D’Amico
Enrico Gratton
University of Illinois at Urbana–Champaign
Department of Physics
Laboratory for Fluorescence Dynamics, MC-704
1110 West Green Street
Urbana, Illinois 61801-3080
E-mail: psimonso@uiuc.edu
Abstract. We introduce the concept of deliberate placement of absorbers to alter the average path of photons through tissue for a biomedical optical device. By changing the reflectivity of a needle that
separates a source and detector, the average photon path through a
turbid medium can be changed. Totally reflective needles have photon scattering density functions similar to a point source and detector
in an infinite medium. An absorbing needle moves the average photon path of photons that reach the detector away from the needle.
Thus, by modulating the reflectivity of the needle, it is possible to
modify the sensitive volume, and simple tomography data should be
possible. These results are confirmed by Monte Carlo simulations and
experiment. Experiments include moving a black target relative to an
optical “needle” and measuring the resulting intensity and phase lag
of light reaching a detector at the distal end of the needle. © 2006 Society
of Photo-Optical Instrumentation Engineers. 关DOI: 10.1117/1.2168167兴
Keywords: needle; reflectivity; Monte Carlo simulation; diffusion approximation;
turbid media.
Paper 04212RR received Nov. 5, 2004; revised manuscript received Sep. 27, 2005;
accepted for publication Oct. 19, 2005; published online Jan. 31, 2006.
1
Introduction
Optical biopsy needles are currently being investigated by
Lubawy and Ramanujam for use in breast cancer diagnosis.1
These needles offer the advantages of being real-time and
minimally invasive probes that offer detailed descriptions of
breast tissue. Diagnosis is virtually immediate, and accuracy
is reported to be greater than 90%.
These needles consist of hollow biopsy needles threaded
with optical fibers. At one end of the needle, light is emitted
from a source optical fiber.1 Light is multiply scattered
through breast tissue until it enters the detector optical fiber at
the distal end of the biopsy needle.1 These optical needles
have simple, fixed geometries that have one defining average
photon path and, as shown here, a relatively small measurement volume.
In this paper, we investigate how changing the reflectivity
of a needle between the source and detector alters the average
photon path through a turbid medium. We demonstrate that by
decreasing the reflectivity of the needle, the average photon
path through the turbid medium is effectively shifted away
from the needle. This is due to the fact that photons that travel
near to and collide with the needle are absorbed. These absorbed photons do not contribute to the average path of photons that reach the detector, which in turn shifts the average
path that photons travel in reaching the detector away from
the needle. The ability to modify the average photon path is
significant because, if it is possible to control the volume of
tissue that photons visit, simple tomography using optical
needles is possible. Because of the cylindrical symmetry of
Address all correspondence to Paul Simonson, Physics, University of Illinois at
Urbana–Champaign, Lab. for Fluor. Dyn., Dept. Physics, MC-704, 1110 W.
Green St., Urbana, IL 61801-3080.
Journal of Biomedical Optics
the needle, it is possible to differentiate volumes at different
distances away from the black needle but not volumes rotated
around the principal axis of the needle; hence the qualification
“simple tomography.”
We use diffusion theory, Monte Carlo simulations, and experiment to investigate the change in average photon paths.
We begin by using diffusion approximation theory after the
manner of Patterson et al.2 to construct photon density functions. These photon density functions describe the probability
that a photon visits a point in space r⬘ and eventually reaches
a detector, all the while not being absorbed by the needle. We
define probability functions by modeling the end of the source
optical fiber as an isotropic point source in a turbid, infinite
medium. We model the optical needle as a negative line segment source; its intensity along the length of the needle is
dependent on its distance away from the point source. The
Green’s functions of these two sources are used to calculate
the relative probability that photons pass through a point r⬘
relative to the absorbing needle and constitute the first probability function. A similar Green’s function is used to calculate the relative probability that photons leaving r⬘ reach the
detector, giving our second probability function. Then, the
photon density function is the product of these two probabilities. Numerical methods are used to produce 2-D images demonstrating these photon density functions.
Next, we use Monte Carlo simulations to construct photon
scattering density functions 共PSDFs兲 using methods outlined
by Bevilacqua et al.3 and Wang et al.4 Monte Carlo PSDFs
should be considered more correct than the functions obtained
from our diffusion approximations because 共1兲 they are accurate in both the diffusive and nondiffusive regimes,3 and 共2兲
1083-3668/2006/11共1兲/014023/9/$22.00 © 2006 SPIE
014023-1
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
they model the needle as a hard shell cylinder with a defined
radius, rather than as an infinitely thin line segment source.
We use the simulations to explore the effect that changing the
needle reflectivity produces on the PSDFs, as well as demonstrating how moving an inhomogeneity through the turbid medium near the needle changes the intensity of light arriving at
the detector.
Finally, we use the methods outlined by Patterson et al.2 to
experimentally produce images that relate to the photon density functions and PSDFs obtained using diffusion theory and
Monte Carlo simulations. We use a blackened optical fiber
immersed in milk to simulate an absorbing needle in scattering tissue. A small, black target is used to probe the photon
density near the “needle.” We also collect frequency domain
information that demonstrates how the phase lag of light
reaching the detector changes as a function of the position of
the black target. The images produced correlate with our predictions from diffusion approximation and Monte Carlo simulations.
We end with a discussion of the significance of this experiment for biomedical optics, especially for optical breast cancer diagnosis.
2
Diffusion Approximations
In this paper, we derive only dc diffusion approximations for
the cylindrical geometry of the needle. We consider the diffusion approximations for our work as giving an idea of what
the geometry of the photon paths should look like. For calculations of the photon paths for this geometry that are accurate
in both the diffusive and nondiffusive regimes and are calculated in the frequency domain, Monte Carlo simulations are
the method of choice. For diffusion approximation derivations
in the frequency domain in the presence of a cylindrical inhomogeneity, we refer to Walker et al.5
The diffusion equation has been used to predict with good
results the migration of photons through turbid media.2,3,6–11
The diffusion equation is a good approximation for media in
which s Ⰷ a and for distances away from sources, boundaries, and detectors that are much greater than the mean free
path for light scattering.5 Although this paper does not suggest
a solution to the diffusion equation for the black needle geometry 共we use a line segment to model the needle兲, it does
make qualitative predictions about the migration of photons in
the absorbing needle geometry.
Light propagation in turbid media is modeled by linear
transport theory using the diffusion approximation
equations6,7
U共r,t兲
+ aU共r,t兲 + ⵜ · J共r,t兲 = q0共r,t兲 ,
t
ⵜU共r,t兲 +
3J共r,t兲 J共r,t兲
+
= 0,
2 t
D
共1a兲
共1b兲
where U共r , t兲 is the density of photons, J共r , t兲 is the photon
current density, is the speed of light in the medium, D is the
diffusion coefficient, a is the absorption coefficient, and s
is the scattering coefficient; but the diffusion equation can
only be solved for a limited number of geometries.1,2 Solutions to the diffusion equation have been found for infinite
Journal of Biomedical Optics
media, semiinfinite media bounded by a plane, a semiinfinite
plane immersed in an infinite medium, and so forth, where the
geometries of the boundary conditions are relatively
simple.5–7,12,13 In the case of semiinfinite slabs, the diffusion
equation is solved by introducing images that counteract the
sources so as to produce a planar boundary where the photon
fluence is zero.2,6,7 However, the geometry of the black needle
problem does not lend itself well to the method of images. An
analogous image method that creates a boundary corresponding to the radius of the black needle is apparently not possible.
We do, however, model the absorbing needle as a negative
source of photons.
For the absorbing needle problem, the probability that a
photon visits a volume dV at r⬘ and goes on to the detector is
the product of two probability functions.2,3 The first is the
probability that a photon is emitted from the source at r0 and
reaches r⬘, and it is given by P共r0 → r⬘兲. The second is the
probability that a photon leaves r⬘ and reaches the detector at
rd and is given by P共r⬘ → rd兲. Calculating the probability that
a photon visits a given region of space before detection is
equivalent to calculating the corresponding Green’s functions
using linear transport theory.2 Note that P共r0 → r⬘兲 is directly
proportional to the photon fluence rate at r⬘ and can be denoted as 共r⬘兲. If we model the absorbing need as a negative
line segment source, the fluence rate at r⬘ can actually be
considered the superposition of fluence due to two sources;
the first source is a point source corresponding to the source
optical fiber, and the second source is a line segment source
corresponding to the absorbing needle. The fluence rate of an
isotropic point source in a highly scattering at r⬘ can be given
by
S共 r ⬘兲 =
exp关− eff兩r⬘ − r0兩兴
,
兩 r ⬘ − r 0兩
共2兲
where eff is the effective attenuation constant,2,7 eff
= 关3a共a + s⬘兲兴1/2. The absorbing needle can be considered a
negative source used in a similar manner to image sources
used for plane geometries. Because of the linear nature of the
diffusion equation, the line segment source’s Green’s function
can be constructed by integrating the Green’s function of a
point source over a line segment,12 multiplied by a term that
accounts for the linear intensity density 关in this case, 共l⬘兲兴,
which results in
N共 r ⬘兲 =
冕
rd
r0
共 l ⬘兲
exp关− eff兩r⬘ − l⬘兩兴
dl⬘ ,
兩 r ⬘ − l ⬘兩
共3兲
where 共l⬘兲 is a linear density function of the source intensity.
Since the absorbing needle cannot emit more negative photons than real photons that collide with it, we hypothesize that
共l⬘兲 is dependent on the fluence through the needle at l⬘ due
to the positive point source. If the reflectivity is constant
along the length of the needle between the source and detector, then 共l⬘兲 has the form
014023-2
共 l ⬘兲 = S共 r ⬘兲
qN
.
兩 r d − r 0兩
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
共4兲
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Fig. 1 Plot of s共x , 0 , z兲Es共x , 0 , z兲, left, demonstrates what the PSDF would look like without the influence of the light-absorbing needle. On the
right is the plot of 共x , 0 , z兲E共x , 0 , z兲 corresponding to the diffusion approximation for the absorbing needle. In these two plots L = 4 cm, a
= 0.05 cm−1, and s = 8 cm−1. Pixels near the source and detector were set to zero 共to make the scale such that viewing the changes in the rest of
the PSDF easier兲.
If we choose to place our point source at the origin and the
detector at 共0 , 0 , L兲, and if we connect those two points with
a line segment that represents the absorbing needle, Eqs. 共2兲
and 共3兲 can be rewritten as
S共x,y,z兲 =
exp关− eff共x2 + y 2 + z2兲1/2兴
,
共x2 + y 2 + z2兲1/2
冕
L
0
qN exp兵− eff关x2 + y 2 + 共z − l兲2兴1/2其
dl.
L
关x2 + y 2 + 共z − l兲2兴1/2
共5b兲
The total fluence at r⬘ is then
共x,y,z兲 = N共x,y,z兲 + S共x,y,z兲
共5c兲
The second probability function that we must calculate that
corresponds to P共r⬘ → rd兲 is the escape function. The escape
function E共r⬘ → rd兲 is proportional to the fluence rate at the
detector. If we now consider there to be a point source at r⬘,
the derivation of E共r⬘ → rd兲 is equivalent to the derivation of
共r⬘兲 and is given by
ES共x,y,z兲 =
exp兵− eff关x2 + y 2 + 共L − z兲2兴1/2其
,
关x2 + y 2 + 共L − z兲2兴1/2
Journal of Biomedical Optics
冕
L
0
qN exp共− eff兩L − l兩兲
dl, 共6b兲
L
兩L − l兩
E共x,y,z兲 = ES共x,y,z兲 + EN共x,y,z兲 .
共5a兲
N共x,y,z兲 = S共x,y,z兲
⫻
EN共x,y,z兲 = ES共x,y,z兲
共6a兲
共6c兲
The visitation density function is then proportional to
共x , y , z兲 E共x , y , z兲. The careful observer will notice that the
integral in Eq. 共6b兲 blows up at the end points and is not
descriptive of reality; however, the integral is constant for a
given problem setup. In our numerical calculations, the integral is simply replaced by a constant that does not include
integration near the endpoints.
Using numerical methods, it is possible to produce density
plots that demonstrate how changing the reflectivity changes
the sensitive volume of the optical needle 共see Fig. 1兲. Doing
so demonstrates that the introduction of an absorbing needle
shifts the average photon path away from the needle, while
absence of the needle allows the average photon path to follow a straight path from source to detector.
In our technique, the needle is used as support for the
optical fibers to perform a 1-D tomography of the needle surroundings. For our method to work, we require relatively
large source-detector separations, since our purpose is to explore the volume relatively far from the needle. 共For the semiinfinite geometry, increasing the source, detector separation
distance increases the average depth of tissue visited by photons in diffuse reflectance measurements.14兲 However, the
014023-3
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
needle cylinder, and the inner surface of each concentric ring
was 1 mm away from the inner surface of the ring just inside
of it. The resulting number of counts in each ring voxel was
divided by the volume of the voxel to give a PSDF that is
related to the density of photons in a plane that cuts through
the z axis of the needle.
Fig. 2 Monte Carlo needle setup. The cylinder representing the
needle had a radius of 3 cm. The absorbing cylinder part had a length
of 4 cm, and the detector had a length of 0.2 cm. The photons were
launched from the side of the needle cylinder at the base of the absorbing cylinder, initially propagating in the x direction.
concept of having an absorbing needle will influence the light
density also if the source-detector distance is a few millimeters. The size of this effect depends on the regime of light
transport. If there is multiple scattering, the absorbing material of the needle will have an effect.
3
Monte Carlo Simulations
3.1 Description of the Monte Carlo Model Geometry
For our Monte Carlo simulations, we modeled the optical
needle as being broken up into four sections. The absorbing
needle section was modeled as a hard cylinder, 4 cm long,
pointing along the z axis. The detector was also modeled as a
hard cylinder, 0.2 cm long, and it was placed at the end of the
needle’s cylinder. Two totally reflecting, semiinfinitely long
hard cylinders were placed before the absorbing needle and
after the detector cylinder so that all the cylinders were
aligned and were of equal radius 共0.3 cm兲, making one long
“needle” 共see Fig. 2兲.
Photons were injected at a point at the bottom edge of the
absorbing needle cylinder with an initial propagation direction
along the x axis. We used absorption and scattering coefficients of 0.05 and 8 cm−1, respectively; these values are similar to those of brain and breast tissue.3,15 Photons were propagated through the “tissue” isotropically.4,14,16 Frequency
domain information was collected using the modified shortcut
method described by Testorf et al.17 A slightly different setup
was used to measure radius dependence 共the radius varied,
and the reflection off of the cylinder was diffuse instead of
specular兲.
3.2 Construction of the PSDFs
The simulation programs were written in C⫹⫹ and modeled
after the popular MCML program used for photon-tissue
simulations.4,18 PSDFs were3 constructed by counting collisions in voxels that were essentially concentric, hollow cylinders. The voxels were 1 mm in length along the z axis of the
Journal of Biomedical Optics
3.3 Photon Path Dependence on Needle Reflectivity
PSDFs were constructed to compare the average photon paths
for several needle reflectivities 共Fig. 3兲. The needle reflectivity was modeled by calculating a specular reflection off of the
needle and assigning a probability of reflection by the needle.
A successive collision with the needle would produce a new
value for the probability of reflection, and if the new value
was less than the previous, then the value was updated. If the
photon eventually reached the detector, an object containing a
linked list of the photon’s history was passed to an object that
updated data arrays for the photon density and phase. PSDFs
were constructed for several reflectivity values, and the relative drop in total intensity was also recorded. The results demonstrated that a totally reflective needle 共reflectivity= 1兲 has a
PSDF similar to the PSDF of a point source and detector in an
infinite medium, as found by the diffusion equation, and a
totally absorbing needle 共reflectivity= 0兲 has a PSDF similar
to our model diffusion approximation photon density function. Intermediate values of reflectivity had PSDFs that varied
between the two extremes. Also note that the overall intensity
drops as the reflectivity is decreased 共Fig. 4兲.
3.4 Photon Path Dependence on Needle Radius
Monte Carlo data was also collected for several different
needle radii. It became apparent that as the radius of the
needle increases, the distance away from the edge of the
needle at which the maximum photon density is found also
increases. The rate of change seems to be largest for small
radii. Also, the intensity of photons is seen to decrease at the
maximum 共Fig. 5兲.
4
Experimental
4.1 Materials and Methods
4.1.1 Equipment
To verify the results of the diffusion approximations and
Monte Carlo simulations that describe the average photon
path from source to detector, we used a three-axis table micropositioner, a scattering medium 共2% milk兲, and a laser diode and two photomultiplier tube 共PMT兲 channels of an ISS
frequency domain tissue oximeter 共Oximeter, ISS, Champaign, Illinois兲. We used 2% milk fat milk for the scattering
medium because it does not have the settling problems of
TiO2 mixtures,2 has scattering and absorption coefficients
similar to human tissues,7 and perhaps most importantly, is
readily available. The laser diode is modulated at a frequency
of 110 MHz, and the second dynode of the PMTs is modulated at 110.005 MHz to demodulate the high frequency.5 The
laser diode light 共670 nm兲 was conducted to and from the
milk through optical fibers. Part of the detector fiber was colored black or white as a means to change the reflectivity of
the needle. We measured the positional influence of a small,
black target object on the intensity and phase lag of light
014023-4
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Fig. 3 Monte Carlo PSDFs with needle reflectivity= 0, 0.5, 0.75, and 1.0. Pixels associated with the source and detector were set to zero 共to make
the scale such that viewing the changes in the rest of the PSDF easier兲.
Journal of Biomedical Optics
014023-5
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Fig. 4 Intensity as a function of reflectivity based on Monte Carlo
absorbing needle simulations. Here the source-detector separation
was 4 cm, and the needle radius was 0.3 cm.
reaching the distal detector by moving the target through the
milk using the micropositioner. See Fig. 6 for a representation
of the setup.
4.1.2 Spatial intensity measurement
Measurements were taken in a raster fashion for positions of
the black target relative to the black needle. The small black
target is the absorption object used to probe the photon density. It is assumed that if the detected light intensity goes
down more when the black target moves into one particular
region of space than it does when the target moves into a
different region of space, then the photon density in the first
region of space is greater. Hence, we use the positioning of
Fig. 5 Comparison of Monte-Carlo-generated photon scattering as a
function of distance away from perfectly absorbing needles that vary
in the size of their radii. The number of photons reaching the detector
for each radius is scaled to n = 1000. Distance away from edge of
needle is measured as the radial distance from the lengthwise center
of the absorbing needle. The number of counts has also been scaled
for increasing voxel-size dependence on radius.
Journal of Biomedical Optics
Fig. 6 Experimental setup. The raster scanning device moves a black
target through the x-z plane where the black needle 共in this case, the
blackened end of a fiber optic兲 is pointing along the z axis. The light
source and detectors are part of an ISS oximeter 共ISS, Champaign,
Illinois兲 with a laser diode source 共630 nm兲 and PMT detectors. For
the intensity measurements, light intensity was measured at the distal
end of the needle. The phase measurements used both detectors, the
detector near the light source being the reference detector.
the black target to map out the photon density. Optical fibers
connected to the oximeter were inserted into the milk. The
end of the detector’s fiber optic cable was colored black with
a black marker to simulate an absorbing needle 共reflectivity
= 0兲. The end of the fiber optic leading from the laser diode
was positioned four centimeters above the end of the detector
fiber optic, and white tape held the two optical fibers together.
Black cloth was placed around the setup to minimize noise
from the room lights. The black target was attached to the
micropositioner through a transparent capillary tube. This
black target was moved in a raster fashion along the length of
and away from the “needle” to produce a two dimensional
map of the influence of the target position on the intensity of
light reaching the detector 共Fig. 7, right兲.
To verify that the average photon path shifts closer to more
reflective needles, white tape was placed around the “black
needle.” Another image was collected 共Fig. 7, left兲. It was
seen that the image thus created was similar to the image
produced by the simple analytical model for source and detector in the absence of a black needle 共Fig. 1兲 and the totally
reflective Monte Carlo simulation 共Fig. 3兲.
4.1.3 Spatial phase lag measurement
To measure the spatial influence of the black target on the
phase lag, a reference detector fiber optic was placed next to
the source, in addition to the detector found at the distal end
of the black needle. The “black needle” length was reduced to
3.5 cm in this experiment so that more light would reach the
detector and thus reduce the shot noise 共with the expectation
that the phase lag is a much more sensitive measurement兲.
The light source was again modulated at 110 MHz. Frequency domain information was collected for both detector
channels. The difference between the phases of the signals in
both channels was considered the phase lag. It is assumed that
as the target moves into volume elements with higher photon
014023-6
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Fig. 7 Left: Image produced by placing white tape around the “black needle.” This was done to verify that, for more reflective needles, the average
photon path is shifted nearer to the needle. Right: Observed changes in photon count for a raster scan in the x-z plane. The blackened “needle” is
directed along the z axis and is found near the left edge of the image. This image shows the expected shift of the photon path away from the needle.
The source-detector separation distance is 4 cm for both images. Compare with the Monte Carlo results in Fig. 3.
visitation probabilities, the average total path length for a photon to reach the distant detector must change because the average path of the photons is significantly changed. The results
showed that the phase lag increases as the black target crosses
the average photon path 共Fig. 8兲.
4.2 Results
Figure 7 maps light intensity reaching the detector for positions of the target relative to the needle. It is assumed that the
amount of light reaching the detector is less when the black
object is found impeding the average path of photons through
the scattering medium. If this is indeed the case, then Fig. 7,
right, clearly demonstrates that the most-visited volume elements are found at distances well away from the needle. Figure 7, left, is the result of the control experiment: when white
tape is placed around the exterior of the black needle, the
intensity map appears to be more like what is to be expected
for a source and a detector in an infinite medium without a
black needle. Thus, Fig. 7 demonstrates that the presence of
the black needle does indeed change the average path of photons that leave the source, pass through the milk, and arrive at
the detector.
5
Detection of Inhomogeneities
To test the usefulness of the needle in detecting tissue inhomogeneities, we wrote a Monte Carlo simulation that introduced inhomogeneities. This Monte Carlo simulation was the
same as our simulation described in Sec. 3.3, except we introduced a second layer of tissue around the tissue that surJournal of Biomedical Optics
Fig. 8 Left: Map of the phase dependence on the position of the black
target for the black needle. Right: corresponding intensity dependence
on the position of the black target for the same experiment. Sourcedetector separation is 3.5 cm. By inspection we can see that the phase
lag has a spatial dependence similar to that of the intensity.
014023-7
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Fig. 9 Graph demonstrating how changing the reflectivity of the
needle changes the intensity of light arriving at the detector when a
second, cylindrical layer of tissue is introduced around the needle.
The radii at which the boundary between the two layers is found is
indicated in the legend at the bottom of the figure.
rounded the absorbing needle. This new layer of tissue was
given scattering and absorption coefficients double the values
of the coefficients of the original tissue. The two layers of
tissue were separated by a cylindrical boundary. We varied
both the reflectivity of the needle and the radius of the cylindrical boundary between the two tissue layers. The intensities
of light detected after the second tissue layer was introduced
were compared with the detected light intensities before the
second layer was introduced. When the tissue boundary was
far from the needle, there was little or no change in the ratio
of the intensity with or without the tissue inhomogeneity.
When the inhomogeneity boundary was closer to the needle,
the ratio of the intensity with and without the inhomogeneity
changed significantly with respect to needle reflectivity. This
demonstrates that modulating the needle reflectivity makes
detection of inhomogeneities at different radial distances possible. See Fig. 9.
All our experiments were done with needles of different
reflectivity. However, we plan to change the reflectivity by
designing a needle with an external jacket that can be rotated
to change the surface reflectivity. Thus, in practice we will be
able to modulate the reflectivity of the needle in situ, even
though we do not show it here.
6
Summary, Discussion, and Conclusions
We showed through diffusion approximations, Monte Carlo
simulations, and direct experiment that the average photon
path changes as the reflectivity of an optical needle changes.
It is clear that as reflectivity decreases, the average photon
path for photons reaching the detector shifts away from the
absorbing needle. Experiments also indicate that modeling the
optical needle as a negative photon line segment source gives
qualitative results that compare well with Monte Carlo simulations and experiment.
We effectively presented a method to alter photon paths by
modulating the reflectivity of an absorbing needle; however,
Journal of Biomedical Optics
Fig. 10 Proposed insertion of needle into breast tissue for 1-D tomography. The source and detector are both located along the z axis of the
needle. When the reflectivity of the needle is changed, the average
photon path is shifted away from the needle. This is depicted on the
left as shells of light densities that correspond to different reflectivities.
By changing the reflectivity of the needle, tissue can be measured at
varying radii away from the needle.
the method could also be applied to other geometries. The
optical needle is particularly useful for medical applications
because it is minimally invasive but can still reach many parts
of the body. Selectivity in the reflectivities of optical needles
should make it possible to manufacture a variety of needles
doctors can choose from to select the most appropriate sensitive volume for a given application. Perhaps more exciting,
though, is that the modulation of reflectivity of optical needles
opens the door for internal, site-specific optical tomography,
which could be useful in breast cancer diagnosis 共Fig. 10兲. By
gradually changing the reflectivity of an optical needle, it is
possible to change the average photon path, and hence, the
volume of tissue explored. This opens the door for internal
optical tomography, which may be useful in cases when optical tomography measurements taken by placing source and
detector on the skin are not sufficient.
Acknowledgments
This research was jointly supported by National Institutes of
Health 共NIH兲 PHS 9ROI, Grant No. EB00559, and NIH,
NTROI-1U54CA105480-01.
References
1. C. Lubawy and N. Ramanujam, “Endoscopically compatible nearinfrared photon migration probe,” Opt. Lett. 29, 2022–2024 共2004兲.
2. M. S. Patterson, S. Anderson-Engels, B. C. Wilson, and E. K. Osei,
“Absorption spectroscopy in tissue-simulating materials: a theoretical
and experimental study of photon paths,” Appl. Opt. 34, 22–30
共1995兲.
3. F. Bevilacqua, J. S. You, C. K. Hayakawa, and V. Venugopalan,
“Sampling tissue volumes using frequency-domain photon migration,” Phys. Rev. E 69, 051908 共2004兲.
4. L. Wang, S. L. Jacques, and L. Zheng, “MCML—Monte Carlo modeling of light transport in multi-layered tissues,” Comput. Methods
Programs Biomed. 47, 131–146 共1995兲.
5. S. A. Walker, D. A. Boas, and E. Gratton, “Photon density waves
scattered from cylindrical inhomogeneities: theory and experiments,”
Appl. Opt. 37, 1935–1944 共1998兲.
6. S. Fantini, M. A. Franceschini, and E. Gratton, “Semi-infinitegeometry boundary problem for light migration in highly scattering
media: a frequency-domain study in the diffusion approximation,” J.
014023-8
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
Simonson, D’Amico, and Gratton: Modulation of an optical needle’s reflectivity…
Opt. Soc. Am. B 11, 2128–2138 共1994兲.
7. J. B. Fishkin and E. Gratton, “Propagation of photon-density waves
in strongly scattering media containing an absorbing semi-infinite
plane bounded by a straight edge,” J. Opt. Soc. Am. A 10, 127–140
共1993兲.
8. J. S. Maier, S. Walker, and E. Gratton, in Biomedical Optical Instrumentation and Laser-Assisted Biotechnology, pp. 121–142 共1996兲.
9. M. S. Patterson, B. Chance, and B. C. Wilson, “Time resolved reflectance and transmittance for the non-invasive measurement of tissue
optical properties,” Appl. Opt. 28, 2331–2323 共1989兲.
10. S. Fantini, M. A. Franceschini-Fantini, J. S. Maier, S. Walker, B.
Barbieri, and E. Gratton, “Frequency-domain multichannel optical
detector for noninvasive tissue spectroscopy and oximetry,” Opt.
Eng. 34, 32–42 共1995兲.
11. M. Wolf, U. Wolf, J. H. Choi, V. Toronov, L. A. Paunescu, A. Michalos, and E. Gratton, “Fast cerebral functional signal in the 100-ms
range detected in the visual cortex by frequency-domain near-infrared
spectrophotometry,” Psychophysiology 40, 521–528 共2003兲.
12. K. M. Case and P. F. Zweifel, Linear Transport Theory, Addison-
Journal of Biomedical Optics
Wesley, Reading, MA 共1967兲.
13. M. L. Shendeleva, “Green functions for diffuse photon-density waves
generated by a line source in two nonabsorbing turbid media in contact,” Appl. Opt. 43, 1638–1642 共2004兲.
14. G. H. Weiss, R. Nossal, and R. F. Bonner, “Statistics of penetration
depth of photons re-emitted from irradiated tissue,” J. Mod. Opt. 36,
349–359 共1989兲.
15. W. F. Cheong, S. A. Prahl, and A. J. Welch, “A review of the optical
properties of biological tissues,” IEEE J. Quantum Electron. 26,
2166–2185 共1990兲.
16. S. A. Dupree and S. K. Fraley, A Monte Carlo Primer: A Practical
Approach to Radiation Transport, Kluwer Academic/Plenum Publishers, New York 共2002兲.
17. M. Testorf, U. Osterberg, B. Pogue, and K. Paulsen, “Sampling of
time- and frequency-domain signals in Monte Carlo simulations of
photon migration,” Appl. Opt. 38, 236–245 共1999兲.
18. S. L. Jacques and L. Wang, in Optical-Thermal Response of LaserIrradiated Tissue, A. J. Welch and M. J. C. van Gemert, Eds., pp.
73–100, Plenum Press, New York 共1995兲.
014023-9
January/February 2006
Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/29/2016 Terms of Use: http://spiedigitallibrary.org/ss/termsofuse.aspx
쎲
Vol. 11共1兲
|
|
https://openalex.org/W4390953908
|
https://link.springer.com/content/pdf/10.1007/s10649-023-10290-5.pdf
|
English
| null |
Structure sense in students’ quantity comparison and repeating pattern extension tasks: an eye-tracking study with first graders
|
Educational studies in mathematics
| 2,024
|
cc-by
| 10,491
|
Educational Studies in Mathematics
https://doi.org/10.1007/s10649-023-10290-5 Educational Studies in Mathematics
https://doi.org/10.1007/s10649-023-10290-5 Abstract There is growing evidence that the ability to perceive structure is essential for students’
mathematical development. Looking at students’ structure sense in basic numerical and
patterning tasks seems promising for understanding how these tasks set the foundation
for the development of later mathematical skills. Previous studies have shown how stu-
dents use structure sense in enumeration tasks. However, little is known about students’
use of structure sense in other early mathematical tasks. The main aim of this study is to
investigate the ways in which structure sense is manifested in first-grade students’ work
across tasks, in quantity comparison and repeating pattern extension tasks. We investigated
students’ strategies in quantity comparison and pattern extension tasks and how students
employ structure sense. We conducted an eye-tracking study with 21 first-grade students,
which provided novel insights into commonalities among strategies for these types of tasks. We found that for both tasks, quantity comparison and repeating pattern extension tasks,
strategies can be distinguished into those employing structure sense and serial strategies. Keywords Eye tracking · Quantity comparison · Repeating pattern extension · Structure
sense · Serial strategies Structure sense in students’ quantity comparison
and repeating pattern extension tasks: an eye‑tracking study
with first graders Demetra Pitta‑Pantazi1 · Eleni Demosthenous1 · Maike Schindler2 ·
Achim J. Lilienthal3,4 · Constantinos Christou1 Accepted: 11 December 2023
© The Author(s) 2024 Accepted: 11 December 2023
© The Author(s) 2024 * Demetra Pitta‑Pantazi
dpitta@ucy.ac.cy 1 Introduction The term “structure sense” describes the ability to recognize how a mathematical whole
consists of parts as well as the relationships between these parts (Hoch & Dreyfus, 2004;
Lüken, 2012). It was first used by Linchevski and Livneh (1999), and subsequently, the idea
was developed and refined by Hoch and Dreyfus (2004). Structure sense seems important * Demetra Pitta‑Pantazi
dpitta@ucy.ac.cy
1
University of Cyprus, Nicosia, Cyprus
2
University of Cologne, Cologne, Germany
3
Örebro University, Örebro, Sweden
4
TU München, Munich, Germany 56789)
1 3 D. Pitta‑Pantazi et al. across various mathematical content domains, especially in algebra and arithmetic (Mul-
ligan et al., 2006). Lüken (2012) found that structure sense at the beginning of first grade is
an early predictor of arithmetical competence at the end of second grade.fi across various mathematical content domains, especially in algebra and arithmetic (Mul-
ligan et al., 2006). Lüken (2012) found that structure sense at the beginning of first grade is
an early predictor of arithmetical competence at the end of second grade.fi So far, there is insufficient knowledge of how structure sense is manifested in students’
work in early mathematics. Thus, there is a need to deepen the understanding of young
students’ structure sense and the processes related to the awareness of this structure. In this
context, eye-tracking studies can provide important insights into young students’ strategies
for solving mathematical tasks (Obersteiner & Tumpek, 2016). Eye tracking is a technique
that captures participants’ eye movements using the anatomic feature of human vision that
the eyes need to move so that an observer sees objects or regions of interest in high res-
olution. According to Radford (2010), attending to something in a certain way requires
an intentional act that he calls “domestication of the eye” (p. 4), and this attention allows
students to recognize things from a mathematical perspective. Hence, examining students’
strategies by tracking the eye gaze as an indicator of visual attention is promising in reveal-
ing insights into how students engage with mathematical tasks in their early years.f There is a growing consensus that “eye tracking offers unique ways to understand cogni-
tive processes in mathematics education” (Strohmaier et al., 2020, p.167). For the investi-
gation of students’ strategies in early years, this is particularly beneficial for two reasons. First, it is possible to observe the strategies without interrupting students (e.g., Weijden
et al., 2018). 1 Introduction Second, it is possible to explore strategies that may not be consciously acces-
sible or that young students may not be able to communicate (e.g., Ott et al., 2018; Schin-
dler & Lilienthal, 2018).i Sprenger and Benz (2020) used eye tracking and found that when five-year-old students
enumerate quantities, they are aware of structures, and some of them can even use these
structures to determine the cardinality of sets. Ten-year-old students also use strategies
such as enumerating all the dots simultaneously or enumerating groups of dots (Schindler,
Schovenberg, & Schabmann, 2020), which suggests that students can identify structures in
the visual representation of quantities. In these studies (Schindler, Schovenberg, & Schab-
mann, 2020; Sprenger & Benz, 2020), it was the use of an eye-tracking methodology that
allowed inferences about students’ structure sense to enumerate quantities. However, researchers need to understand better how structure is used in other mathemat-
ical activities. Since structure sense was found to be a significant predictor of mathematical
learning (Lüken, 2012), in this paper, we study how structure sense can be identified across
different tasks and how it can be assessed with eye tracking. We study students’ structure
sense when students compare quantities, an ability often developed after enumeration, and
when students extend repeating patterns, a type of task primarily linked with structure. The main aim of our study is to investigate the ways that structure sense is manifested in
first-grade students’ work across tasks, in particular, in quantity comparison and repeating
pattern extension tasks. 2.1 Structure sense When we refer to structure, we adopt Battista’s definition. In his view, “spatial structur-
ing is the mental operation of constructing an organization or form for an object or set of
objects. It determines the object’s nature, shape, or composition by identifying its spatial
components, relating, and combining these components, and establishing interrelationships
between components and the new object” (Battista, 1999, p.418). Lüken (2012) describes
early structure sense as an individual’s ability to (a) identify a configuration as a familiar
structure or pattern (e.g., dots on a dice), (b) break a pattern into sub-structures, (c) recog-
nize and find connections and relationships between sub-structures (i.e., similarities and
differences, detect regularity), and (d) integrate substructures to see a pattern as an entity
(e.g., extend a pattern). Many researchers relate patterning ability or even equate it with the ability to perceive
and use structures (Hutchinson, 2011; Lüken, 2012). According to Lüken (2012), students
may consciously and/or subconsciously use structure sense to determine a quantity or
extend a pattern. It seems likely that students’ structure sense might be manifested when
working with other types of tasks, such as quantity comparison. Although several studies
identify students’ structure sense in enumeration tasks through students’ actions and verbal
responses (Schindler, Schovenberg, & Schabmann, 2020; Sprenger & Benz, 2020), it is not
clear whether it is possible to identify structure sense through eye tracking in quantity com-
parison and pattern extension tasks. So far, there is evidence that students use structure sense when enumerating quantities. The ability to enumerate quantities (i.e., to grasp sets of items and say how many there are)
is crucial for children in preschool and the beginning of primary school. Research studies
that investigated students’ actions and verbal responses in enumeration tasks led research-
ers to the identification of a set of students’ strategies such as counting, subitizing, and
groupitizing (Schleifer & Landerl, 2011; Starkey & McCandliss, 2014). Counting is the
“one-to-one mapping between a set of objects and number words” (Schleifer & Landerl,
2011, p. 280). Subitizing is the ability to enumerate small quantities fast and precisely
without counting and is considered an essential requirement for arithmetic learning (Fis-
cher et al., 2008). Finally, groupitizing involves understanding the concepts of numbers and
part-whole schema (Starkey & McCandliss, 2014) and the idea of composing and decom-
posing (Clements, 1999). In groupitizing, children perceive sets in subsets even at a young
age (Clements, 1999). 2 Literature review In our literature review, we first focus on structure sense. Then, we present findings from
students’ use of structure sense in enumeration tasks, with and without eye tracking,
because these findings guided the investigation of eye-tracking strategies in quantity com-
parison and pattern tasks. Following this, we explore findings on quantity comparison and 1 3 Structure sense in students’ quantity comparison and repeating… pattern tasks. Finally, we present how the current study draws on the existing findings and
state the research questions. pattern tasks. Finally, we present how the current study draws on the existing findings and
state the research questions. 2.2 Quantity comparison Comparing quantities and identifying equal and unequal sets is another common topic
among early education curricula (e.g., Department of Education, 2013) and commonly a
part of mathematics instruments for students’ number development (e.g., Beltrán-Navarro
et al., 2018). Human beings intuitively make perceptual judgments about the relative mag-
nitude of quantities (Sarama & Clements, 2008). Such judgments about the magnitude of quantities (i.e., which set of objects has more/
less) are based on two non-symbolic cognitive numerical systems. According to the first
system, the comparison is executed through object tracking, in which small sets (less than
4) are enumerated based on subitizing without counting serially (Trick & Pylyshyn, 1994). The second system is referred to as the approximate number system (ANS) and is used for
larger numbers. The ANS supports the estimation of the magnitude of a set without relying
on language or symbols; instead, it is ratio-dependent (Nieder & Dehaene, 2009). Huntley-
Fenner and Cannon (2000) suggest that 3- to 5-year-old children’s decisions about numeri-
cal magnitude are mediated by a similar mechanism, which does not depend on their abil-
ity to count verbally. Huntley-Fenner and Cannon (2000) also show that comparison tasks with a 2:3 ratio in
the number of dots of the two sets were more difficult than the ones with a 1:2 ratio. It was
also observed that error rates and response times for number comparison increased when
the ratio of the smaller over the larger number increases (Moyer & Landauer, 1967). In
non-symbolic comparison tasks with numerosity of 5–22 dots, 7- to 9-year-old students
showed that those with high ANS acuity tended to have high achievement scores (Inglis
et al., 2011). It seems that preschoolers do not use counting often to compare sets of items (Clements
& Sarama, 2007). Children who are approximately 3.5 years old can match homogenous
visual sets; when they are approximately 4.5 years old, they can match equivalent collec-
tions of heterogeneous objects (Sarama & Clements, 2008). Initially, Piaget and Szeminska
(1952) and later Fuson (1988) corroborated that children at the age of 4 to 5 years, when
comparing sets, focus on misleading length cues and do not use counting. Even though
children may count two sets to compare them, they tend to still decide which set is bigger
based on appearance and extension (Piaget & Szeminska, 1952). 2.1 Structure sense Eye-tracking studies with 10-year-old students on enumeration tasks found that students
use simultaneous enumeration and enumeration through the use of structures for the canon-
ical arrangement of 2–9 dots (Schindler, Schovenberg, & Schabmann, 2020). For random
arrangements of 2–4 dots, students again used simultaneous enumeration and enumeration
of groups of dots. For random arrangements of 5–9 dots, students used quasi-simultaneous
enumeration and partial enumeration of groups of dots. In this paper, we classified these
strategies, which draw on simultaneous or quasi-simultaneous enumeration or the use of
structures, under strategies employing structure sense. On the contrary, we classified the
enumeration of single dots (i.e., counting all dots one by one) as a serial strategy. Sprenger
and Benz (2020) also found that 5-year-old students used structures to determine the car-
dinality of sets when they were asked to find how many eggs were presented in an egg 3 3 D. Pitta‑Pantazi et al. carton. For example, strategies employing structure sense included that of enumerating in
groups, (de)composing, and subitizing (Sprenger & Benz, 2020). carton. For example, strategies employing structure sense included that of enumerating in
groups, (de)composing, and subitizing (Sprenger & Benz, 2020). While studies on students’ enumeration appear to identify students that use structure
sense or serial strategies, to the best of our knowledge, there are no eye-tracking studies
that investigate students’ use of structure sense across tasks, in this case, across quantity
comparison and patterning tasks. This is the focus of our study. 1 3 2.3 Patterning Patterning is the ability to discover regularities among ordered sets of units (Clements &
Sarama, 2007). To foster their patterning skills, students are often taught (a) repeating pat-
terns, which contain a discernible unit (Threlfall, 1999) generated by the alteration of a
smaller part based on objects (e.g., numbers, letters, shapes) and/or their characteristics
(e.g., color, size) (Liljedahl, 2004; Papic, 2015); and (b) growing patterns which increase
or decrease systematically. In this study we focus on repeating patterns. Papic et al. (2011) investigated students’ strategies when dealing with repeating patterns
in an interview setting. They found that 4- to 5-year-old students use one of the following
strategies: random arrangement of the pattern elements without attention to the pattern,
direct comparison by matching one element at a time, alternation by focusing on succes-
sive elements, and identification of the repeating unit. In extending patterns, 5- to 6-year-
old students may only notice the changes between object characteristics, for example, “the
colour yellow comes after green and green comes after yellow,” thus identifying only the
element that follows. A shift in their understanding occurs when they are able to identify
the repeating unit, for example, “yellow, green” (Economopoulos, 1998).i p
g
,
p , y
, g
(
p
,
)
According to Lüken (2012), first-grade students could perceive the succession of col-
ours in repeating patterns. However, not all of them were able to relate the figure to the
mathematical aspects in ways that connect the spatial structure of the pattern with its
numerical one (e.g., two red and two yellow). Lüken (2012) based her findings on students’
use of specific vocabulary or actions that indicated an awareness or lack of awareness of
the repeating units during interviews. Hutchinson (2011) found that pre-primary school
students may apply structure sense consciously and/or subconsciously. Hence, students
may be in a position to successfully complete pattern tasks but may not be able to commu-
nicate how they have reached their answer (Lüken, 2012; Van Nes, 2009). Such difficulties
in communicating structure sense or not being consciously aware of it create methodologi-
cal obstacles in the interpretation of findings. This is one of the reasons why eye tracking
can provide complementary or otherwise unobtainable insights into students’ thought pro-
cesses. 2.2 Quantity comparison For example, they may
count the sets, recognize that the number is the same, but still mention that one set has
more items based on the spatial extension of the item arrangement. All in all, several stud-
ies indicate that only over time do children come to trust the results of the counting process
to compare the magnitude of sets. Only a few studies studied quantity comparison strategies using eye-tracking method-
ology (e.g., Fuson, 1988). These studies examined how adults respond to non-symbolic
comparison when varying the ratio effect (Huntley-Fenner & Cannon, 2000) and the cumu-
lative area (Odic & Halberda, 2015). 1 3 Structure sense in students’ quantity comparison and repeating… 2.3 Patterning Yilmaz’s (2019) eye-tracking study on repeating patterns showed that 4- to 5-year-
old students had extended unfocused gazes on the overall given patterns (AB, ABB, ABC)
while they primarily focused on the last repeating unit of the pattern. The findings indicate
that students may be implicitly aware of the repeating unit (Yilmaz, 2019), while other
studies have suggested that students compare one-to-one the middle elements of the pat-
terns and the elements at the beginning of the pattern (Collins & Laski, 2015; Threlfall,
1999). It is worth mentioning that orientation of eye movements could be overt (can be
observed through eye tracking) or covert (cannot be observed) (Posner, 1980). In the case
of covert orientation, information can be perceived using peripheral vision based on extra-
foveal processes (Posner, 1980; Shvarts et al., 2019), which may play a role when students
identify the repeating unit without focusing on it visually. 3.1 Participants All thirty first-grade students at a primary school in Cyprus were invited to participate in
the study, and 21 students (mean age: 6.5 years) agreed to participate. All students were
proficient in Greek and, according to their teachers, performing well in mathematics. The
research took place during two consecutive days in the first trimester of the first grade. This
specific period was chosen to investigate the strategies students use at the beginning of pri-
mary school. All students were taught the same mathematics curriculum, and none of them
received any supplementary mathematics instruction. Before the study, the students’ parents, teachers, and the school principal were informed
about the study and the interview procedure. Parent’s written consent was necessary for
their child to participate and the eye-tracking videos to be published anonymously. All
parents were also informed that they and their children could withdraw from the research
study at any point without any consequences. Additionally, specific steps were taken to
ensure the anonymity and confidentiality of all participants. 2.4 Research questions The present study set the stage for the framework of the Digital identification and support
of under-achieving students project (DIDUNAS). The DIDUNAS project, which was con-
ducted from 2020–2023, addressed the identification of under-achieving students in math-
ematics in Grade 1 (see www.didunas.eu). In previous publications based on DIDUNAS 1 3 D. Pitta‑Pantazi et al. studies, we investigated the types of strategies students use in pattern tasks (Baumanns
et al., 2022; Baumanns et al., 2023; Demosthenous et al., 2022). In this paper, we inves-
tigate for the first time—with the use of eye tracking—students’ strategies and the use of
structure sense across tasks. Specifically, we explore students’ strategies and use of struc-
ture across quantity comparison and patterning tasks. Therefore, the two research questions
are: (1) What strategies do first-grade students employ to respond to quantity comparison
and repeating pattern extension tasks, and in what ways is structure sense manifested in
students’ strategies? (1) What strategies do first-grade students employ to respond to quantity comparison
and repeating pattern extension tasks, and in what ways is structure sense manifested in
students’ strategies? (2) Is there a relationship between students’ correct answers and the strategies they use? (2) Is there a relationship between students’ correct answers and the strategies they use? 3.3 Tasks Students worked on quantity comparison tasks and repeating pattern extension tasks, as
shown in Fig. 1. All tasks were given to all students in the same order. Before each task,
all students were asked to first look at a star on the screen to ensure that all students’
eyes were fixated on the same point before attempting the task. 3.2 Setting To record students’ eye movements, we used a remote eye tracker, Tobii × 3–120, with
a sampling rate of 120 Hz (infrared, binocular, 9-point calibration). The eye tracker was
connected to a 22’’ Full HD computer monitor. The eye-tracking arrangement was free
from distractions and permitted head movements, which allowed the young students to
express natural behaviors. The eye-tracking accuracy in our study was 0.51° on average
(SD 0.17°), with a minimum of 0.28° and a maximum of 0.88°. The computer screen was
placed approximately 70 cm from the eyes of the students, which means the imprecision on
the screen amounted to around 0.62 cm on average (max. 1.08 cm). We accounted for this
imprecision by designing the tasks accordingly (e.g., the dots in the quantity comparison
tasks had a distance of more than 1 cm from one another). In the data collection, the individual students sat in a comfortable chair, and its height
could be adapted to accommodate the different heights of the students. A researcher gave
the instructions and asked the questions. The students responded to the tasks while look-
ing at the screen monitor. Additionally, the utterances were recorded by an audio recording
device. 1 3 1 3 Structure sense in students’ quantity comparison and repeating… 4 Results In the following section, we respond to the two research questions of the study. First, we
respond to the research question by presenting the strategies that first-grade students used
in quantity comparison and repeating pattern extension tasks. Then we refer to the relation-
ship between students’ correct answers and the strategies they use. 3.3.2 Repeating pattern extension tasks Students were presented with a picture of a tower of 6 unifix blocks (Fig. 1). The colors of
the first tower were red, yellow, red, yellow, red, yellow; the second: blue, green, yellow,
blue, green, yellow; and the third: orange, red, red, orange, red, red. Students were asked to
say which block comes next as the tower is built. 3.3.1 Quantity comparison tasks Students worked on six quantity comparison tasks, and in each task, there were two
sets, one with green dots and one with yellow dots. In three tasks, the two sets were in
columns, while the other three were in rows. In four tasks, the groups were equal, and in
the other two, there was a difference of one dot. There were (a) two equal groups of the
same length, (b) two unequal groups of the same length and (c) two equal groups of dif-
ferent length (see Fig. 1). The students were asked to say which set had more dots (the
yellow or the green) or whether the sets were equal and to answer as fast as possible. In the comparison tasks, dots were arranged in this manner based on previous research
(Piaget & Szeminska, 1952; von Aster et al., 2006) and on the appearance of these types
of tasks in the mathematics textbooks of this age group. 1 3
Fig. 1 Types of tasks Fig. 1 Types of tasks Fig. 1 Types of tasks 1 3 3 D. Pitta‑Pantazi et al. 3.4 Analysis We collected eye-tracking data and transcribed the recordings of students’ verbal responses. For the eye-tracking video analysis, we produced gaze-overlaid videos. Although more
time-consuming and tedious for researchers, gaze-overlaid videos allowed observations
that would not have been possible in the investigation of students’ strategies if we recorded
only the number of eye fixations.i i
To answer the first research question, our analysis followed the four stages described by
Schindler et al. (2019). In the first stage, we watched the gaze-overlaid videos and assigned
initial categories according to the strategy used for each task. These initial categories were
labeled according to the respective common strategy. In the second stage, through a con-
stant comparative method, we reached saturation and finalized the description of the cat-
egories by preparing a codebook with a description and gaze plot of each strategy. In the
third stage, all gaze-overlaid videos were coded as correct or wrong based on the audio
recordings of students’ verbal responses. In the fourth stage, 25% of all the data were coded
by two raters independently (Mayring, 2014) to establish inter-rater reliability. For the cod-
ing of students’ strategies, the inter-rater reliability was calculated using Cohen’s kappa
(Cohen, 1988) and found to be 0.96 for the comparison tasks and 0.83 for the pattern exten-
sion tasks, which is considered an almost perfect and substantial agreement, respectively. To answer the second research question, a chi-square (X2) statistic test was applied to
investigate whether there was a relationship between students’ strategy use and the correct-
ness of their solutions to the tasks. 4.1 Students’ strategies in quantity comparison and repeating patterns For each type of task, we describe the strategies and illustrate them with an exemplary
scanpath. While the term scanpath can be defined “as the route of oculomotor events
through space within a certain timespan” (Holmqvist et al., 2011, p. 254), we analyzed
dynamic scanpath visualizations (gaze-overlaid videos), but for visualization purposes in
this paper use static visualizations (gaze plots). We respond to the first research question
by grouping students’ strategies into those that employ structure sense and those that are
serial. To the best of our knowledge, no other research study has identified categories of
student strategies for quantity comparison tasks based on scanpath analysis in particular. 1 3 Structure sense in students’ quantity comparison and repeating… 1 3
e
s
n
e
s
er
utc
u
rts
g
niy
olp
m
e
s
eig
etart
S
(1a) Simultaneous
comparison of the
two sets
The gaze moves from one
dot or two dots of one set
(e.g., green) to another
dot in the other set (e.g.,
yellow). 9
(1b) Partial
comparison of
groups of dots of the
two sets
The gaze goes (i) to parts
of dots (indicating use of
groups of dots) (Example
i) or (ii) to individual dots
of subset(s), of one set
(green dots) (Example ii). Then, the gaze goes to
parts of dots or in-
between spaces
(indicating the use of
groups) of the other set
(e.g., yellow dots). The
gaze makes one transition
between the two sets. Example i
Example ii
37
(1c) Extended partial
comparison of dots
of the two sets
The gaze goes to subset(s)
of dots of one set (e.g.,
green dots). Then, the
gaze goes to subset(s) of
dots of the other set (e.g.,
yellow dots). The gaze
goes back and forth
several times between
dots of the same set or
between the two sets of
dots. 52
s
eig
etarts
laire
S
(2a) Attending to/
comparing all
The gaze goes to every
dot or to all dots but one
in each set and in some
cases moves back-and-
forth between the two
sets. The gaze may follow
a sequential order
(Example ii) or not
(Example i). Example i
33
Example ii
(2b) One-to-one
correspondence
The gaze moves between
pairs of dots (e.g., one
from the green group and
one from the yellow
group of dots), in a back-
and-forth movement. The
gaze goes to every pair of
dots. 4.1 Students’ strategies in quantity comparison and repeating patterns 1 Example i
Example ii
37 Example ii ded partial
n of dots
sets
The gaze goes to subset(s)
of dots of one set (e.g.,
green dots). Then, the
gaze goes to subset(s) of
dots of the other set (e.g.,
yellow dots). The gaze
goes back and forth
several times between
dots of the same set or
between the two sets of
dots. 52 s
eig
etarts
laire
S
(2a) Attending to/
comparing all
The gaze goes to every
dot or to all dots but one
in each set and in some
cases moves back-and-
forth between the two
sets. The gaze may follow
a sequential order
(Example ii) or not
(Example i). Example i
33
Example ii Example ii 3 D. Pitta‑Pantazi et al. The scanpaths that we identified, with indicative examples and the frequency of appear-
ance among students’ responses, are presented in Table 1. Examples of all the strategies
presented in Table 1 are illustrated with videos that can be reached through the URL in the
respective references. For example, the URL for Strategy 1(a) can be found in the reference
Pitta-Pantazi et al. (2023a). 4.1.1 Students’ strategies in quantity comparison tasks In Strategy 1(a), students compare the two sets at once, either identify a difference or match
dots between the two sets, and then decide which set has more dots (Pitta-Pantazi et al.,
2023a). In Strategy 1(b), students make partial comparisons of groups of dots of the two
sets. They identify groups of dots or individual dots in one set and then move to the other
set and look again, either at groups of dots or at individual dots (Pitta-Pantazi et al., 2023b,
c). In Strategy 1(c) (Pitta-Pantazi et al., 2023d), students make extended partial compari-
sons of the two sets. The gaze goes to subsets of dots of one of the sets and then back and
forth to a subset of dots of the other set. For Strategies 1(a), 1(b), and 1(c), students relied
on selected dots or parts of dots. Therefore, we grouped these strategies and labeled them
strategies employing structure sense. In Strategy 2(a), students’ gazes go to each dot of
each set, either in a sequential or non-sequential order, implying that the student was enu-
merating the dots one by one (Pitta-Pantazi et al., 2023e, f). In Strategy 2(b) (Pitta-Pantazi
et al., 2023g), the gaze focuses on one-to-one correspondences between the dots of the two
sets. We grouped Strategies 2(a) and 2(b) and labeled them serial strategies since students
focused on all the dots when comparing the two sets. The gaze moves from one dot in one
of the sets to a corresponding dot in the other set. Both strategies, employing structure sense or serial strategies, could lead to correct or
erroneous responses. For example, a student’s gaze may indicate structure sense in a com-
parison task (e.g., 1b) since it indicates a partial comparison between subsets of the green
and yellow dots. If the respective student is focusing on the length of the two sets of dots,
this may lead to an erroneous response (if there are two unequal sets but the dots are spread
to the same length). An error with enumeration may occur if, for example, a student double
counts or misses a dot. 4.1.2 Students’ strategies in pattern extension tasks In Strat-
egy 2 (Pitta-Pantazi et al., 2023j), students gazed at each block of the pattern before finding
how to extend the pattern; we labeled it serial strategy. them strategies employing structure sense since gazes focused on repeating units. In Strat-
egy 2 (Pitta-Pantazi et al., 2023j), students gazed at each block of the pattern before finding
how to extend the pattern; we labeled it serial strategy. 4.1.2 Students’ strategies in pattern extension tasks Students’ strategies, indicative examples, and the frequency of the strategies identified in
the pattern tasks are presented in Table 2. Among students’ responses, we found evidence
suggesting that some students focused on the last repeating unit of the pattern (Yilmaz,
2019) while other students appeared to look at each element of the pattern one by one (Col-
lins & Laski, 2015; Threlfall, 1999). Specifically, Strategy 1(a) (Pitta-Pantazi et al., 2023h)
involved directly identifying the repeating unit, while in Strategy 1(b) (Pitta-Pantazi et al.,
2023i), students identified the repeating unit, and then their gazes jumped to (an)other
repeating unit(s), implying a comparison between the repeating unit and another block or
group of blocks. When applying Strategies 1(a) or 1(b), the students seemed to rely on
selected elements of the pattern to decide how the pattern continued by identifying the
repeating unit. It appears that they identified the repeating unit at once and stopped their
gaze as soon as the next repeating unit started (1a), or they identified the repeating unit,
and then their gaze jumped to another repeating unit (not necessarily the next one) to make
a comparison in order to reach their answer (1b). We grouped these strategies and labeled Structure sense in students’ quantity comparison and repeating… Table 2 Students’ strategies in pattern extension tasks
Name
Description
Gazeplot
(Example)
Frequency
S
s
g
niy
olp
m
e
s
eig
etart
e
s
n
e
s
er
utc
u
rt
(1a) Simultaneous
identification of
the repeating unit
The gaze starts from the top
part of the pattern. The gaze
(a) goes only to one
repeating unit (e.g., red,
red, yellow) or (b) goes to
one repeating unit plus one
adjacent block from the
next repeating unit (e.g.,
red, red, yellow, red). 6
(1b) Identification
of the repeating
unit and
comparison with
other elements or
groups of
elements
(repeating unit)
The gaze goes to each block
of one repeating unit. Then,
the gaze jumps to the
bottom or middle part of
another group of blocks
(repeating unit), without
gazing at individual blocks. 26
Serial strategies
(2) Sequential,
continuous
“attending to all”
The gaze goes to all the
blocks of the pattern or
goes to all blocks but one. This gaze might repeat,
more than once, in different
directions (e.g., top to
bottom, bottom to top,
middle to bottom). 31 them strategies employing structure sense since gazes focused on repeating units. 4.2 Relationship between students’ correct answers and the strategies they used To further investigate what strategies the students employed, we explored the frequency of
the strategies employing structure sense and serial strategies in each type of task. Through
the chi-square test of independence, we examined whether the strategies employed by stu-
dents were likely to be related to correct or erroneous answers to the tasks. According to
the values of the chi-square test, there was no significant correlation between the strategies
the students used and their correct or erroneous solutions for the quantity comparison tasks
X2 (25, N = 21) = 16.42, p = 0.902 and the pattern tasks X2 (6, N = 21) = 10.55, p = 0.10. 4.1.3 The ways in which structure sense and serial strategies are manifested
in students’ strategies when working on quantity comparison and repeating
pattern extension tasks In the comparison tasks, strategies employing structure sense appeared more frequently
than serial strategies (Table 3). On one hand, it may be inferred that counting the dots,
one by one, sometimes led to erroneous solutions due to miscounting. On the other hand,
students who looked at the structure of the dot arrangements may sometimes have been
misled by the fact that unequal sets of dots were arranged at the same distance, or equal
sets of dots were not evenly distributed. The highest frequency of errors (f = 9 and f = 6)
was observed in the comparison of equal groups of dots, which were distributed in different
lengths. 3 D. Pitta‑Pantazi et al. Table 3 Frequencies of strategies employing structure sense and serial strategies in the comparison tasks
All strategies employing structure sense and serial strategies (74% + 26%) sum up to 100%. Correct and
wrong answers sum up to 100% (54% + 20% + 23% + 3%)
Strategies employing structure sense
Serial strategies
All
Correct
Wrong
All
Correct
Wrong
Total (Percent-
age)
93 (74%)
68 (54%)
25 (20%)
33 (26%)
29 (23%)
4 (3%) Table 4 Frequencies of strategies employing structure sense and serial strategies in the pattern tasks
All strategies employing structure sense and serial strategies (51% + 49%) sum up to 100%. Correct and
wrong answers sum up to 100% (48% + 3% + 44% + 5%)
Strategies employing structure sense
Serial strategies
All
Correct
Wrong
All
Correct
Wrong
Total (Percentage)
32 (51%)
30 (48%)
2 (3%)
31 (49%)
28 (44%)
3 (5%) Table 4 Frequencies of strategies employing structure sense and serial strategies in the pattern tasks In the pattern tasks, strategies employing structure sense were used with a frequency
of 51% compared to the serial strategies with 49% (Table 4). In one of the pattern tasks
(ΑΒ), strategies employing structure sense were more often used (f = 18 vs. f = 3), whereas,
in the other two pattern tasks (ABC and ABB), the frequency of the strategies employ-
ing structure sense was lower than the frequency of serial strategies (f = 7 vs. f = 14). In
the pattern tasks, the number of erroneous answers was generally low and almost equal
between those who employed structure sense and those who used serial strategies (3% and
5%, respectively). 4.1.3 The ways in which structure sense and serial strategies are manifested
in students’ strategies when working on quantity comparison and repeating
pattern extension tasks We observe that in the quantity comparison and pattern tasks, students applied either
strategies employing structure sense or serial strategies. 5 Discussion In our study, we used eye tracking to inquire into young learners’ use of structure sense
in different mathematical tasks. More concisely, we investigated first-graders’ use of
structure sense across tasks, in particular, across repeating pattern tasks and quantity 1 3 Structure sense in students’ quantity comparison and repeating… comparison tasks. Our study contributes to the research landscape in mathematics edu-
cation (a) through its insights into how young learners’ structure sense is employed
across two domains and (b) through—methodologically—demonstrating the potential of
eye tracking to gain insights into the distinction between strategies employing structure
sense and serial strategies (Hunting, 2003; Lüken & Sauzet, 2021; Schöner & Benz,
2017). )
How did the students employ structure sense? In the quantity comparison tasks, struc-
ture sense was identified when students compared quantities simultaneously or compared
sub-groups of dots from each set and made connections. On the other hand, we identified
serial strategies when students enumerated dots sequentially or performed one-to-one cor-
respondence comparisons between the two sets. Erroneous answers were more likely given
when students miscounted the number of dots. When employing structure sense strategies,
it is possible that erroneous answers were given when students were misled by the dots’
spatial arrangements. It seems that some students erroneously decided that an equal length/
width corresponded to the same number of dots or that a shorter length/width implied a
smaller number of dots. Our findings provide further insight into how the cumulative area
effect (i.e., the effect of varying the area occupied by a set of objects) (Odic & Halberda,
2015) appears when using the analogue number system (ANS) to estimate the magnitude
of two sets. For structure sense in students’ work with pattern tasks, we also grouped students’
strategies into those employing structure sense and those using serial strategies. Students
who appeared to identify the repeating unit at once (or identified the repeating unit and
made comparisons between units) demonstrated structure sense. In contrast, students who
attended to all the pattern elements sequentially, one by one, demonstrated a serial strategy. When investigating students’ strategies in repeating pattern tasks, Yilmaz (2019) found that
children focused on the last repeating unit of the pattern but also had extended unfocused
gazes. In our study, we did not notice such unfocused gazes, but we found students attend-
ing to the last repeating unit of the pattern. 5 Discussion Instead, some students can capture the repeating unit directly,
while other students compare a repeating unit with elements of another repeating unit. In
addition, in our study, we did not find evidence to indicate that students matched one ele-
ment of one repeating unit to the respective element of the following repeating unit, as sug-
gested by Yilmaz (2019). Students who used strategies employing structure sense in the pattern tasks could
directly spot the repeating unit, while in the comparison tasks, they could identify the
equality and inequality of the two sets without going through and attending to each element
(dots in the comparison tasks or blocks in the pattern tasks). This relates to Chumachemko
et al.’s (2014) findings that experts tend to make fewer saccades when dealing with coor-
dinate systems and rely on peripheral vision, for example, to perceive prototypical geo-
metric figures using the entire perceptual field (Shvarts et al., 2019). Referring to periph-
eral vision, the data of this study also indicate that students who used strategies employing
structure sense did not fixate serially on individual dots and seemed to rely on the extrafo-
veal perception of the display. For example, it can be assumed that some students who only
scanned the top of the pattern in the pattern task used peripheral vision to perceive the rest
of the pattern. This relates to the findings of Shvarts et al. (2019) that extrafoveal processes
were involved in the identification of squares and rectangles.i i
The findings of this study suggest that it is possible through the investigation of
dynamic scanpath visualizations, namely gaze-overlaid videos, to identify students who
employ structure sense and students who employ serial strategies in quantity comparison
and pattern tasks. We noticed that there were students who looked at the configuration and
searched for a structure with more efficient and flexible eye movements. For example, in
the comparison tasks, students simultaneously compared the two sets and identified the
equality or inequality of the two sets, while in the repeating pattern task, they found the
repeating unit all at once. In contrast, students with serial strategies used the more sequen-
tial and elaborate process of focusing on each dot in the comparison tasks or on each
colored block in the pattern tasks. 5 Discussion Another study using a revised set of pattern
tasks with a different group of students led to four categories (Baumanns et al., 2022). The first three respective categories were: identifying one repeating unit of the pattern,
identifying one repeating unit and validating/applying it, and looking at each element. An
additional category was the unsystematic jumping over the pattern.i In this study, we present findings from an exploration of students’ structure sense,
highlighting strategies employed across different domains (quantity comparison and pat-
tern tasks). What we observed across the two domains investigated in our study — both
of which involved visual tasks requiring perception and processing of given information
— was that the first-graders exhibited two predominant approaches: They either visually
attended to all given elements (serial strategies) or attended to parts of the given infor-
mation, utilizing structures to infer the information sought to determine the color of the
next element or to identify the larger quantity (using structure sense). It is noteworthy that
although the two domains under investigation, namely patterns (early algebra) and quantity
comparison (arithmetic), were inherently different, we could observe apparent commonali-
ties in students’ strategies. We also found that structure sense was utilized quite frequently,
occurring in more than half of the cases in both domains. It is interesting that although the
participants of the study were first-graders in the first months of primary school, they could
already rely on structure sense to a great extent. However, the use of structures did not
necessarily coincide with correct answers in either of the two domains. This was particu-
larly evident in the quantity comparison tasks, where an emphasis on spatial distribution
and associated inferences sometimes may have led to incorrect answers. Making use of 3 3 D. Pitta‑Pantazi et al. structure sense, thus, was not related to success in terms of correctness in these mathemati-
cal tasks. ucture sense, thus, was not related to success in terms of correctness in these mathemati-
tasks. Furthermore, our eye-tracking investigation provided further insights into the strategies
identified by previous researchers, which were conducted without the use of eye tracking,
such as matching one element at a time, focusing on successive elements, and identifying
the repeating unit (Collins & Laski, 2015; Lüken, 2012; Papic et al., 2011). We found that
the identification of the repeating unit does not necessarily result from attending to each
element of the whole pattern. 5 Discussion Students who showed structure sense were also able to
divide the dots or patterns into substructures when looking at groups of dots in the com-
parison tasks, while in the pattern tasks, their eye movements jumped between the repeat-
ing units. Furthermore, in the comparison tasks, they recognized and established connec-
tions between the two sets of dots when they compared them, either through comparison
at once or partial comparison. In the repeating pattern tasks, they identified the repeating
unit at once or first identified the repeating unit and then compared it with other elements
of the group. They seemed to have been able to do the comparison and extend the repeating
pattern by viewing the groups of dots and the pattern as an entity either simultaneously or
partially. All these actions appeared to be more efficient and flexible. In contrast, the serial
strategies, where students attended to each element one-by-one, were more sequential and
elaborate (Lüken, 2012). The findings of this study suggest that it is possible through eye tracking to identify
what Radford (2010) calls “the domestication of the eye” (p. 4). By “domestication of the
eye,” Radford (2010) means the lengthy procedure by which the eye recognizes things, in 1 3 Structure sense in students’ quantity comparison and repeating… our cases, dots or colored blocks, from a mathematical perspective. However, both Radford
(2010) and Lüken (2012) identified this “domestication” through students’ actions and ver-
bal responses, whereas in our study, we found that eye tracking allows this distinction as
well. With the explosion of the use of eye tracking in mathematics educational research, it
appears that in a few years, it may be possible not only to identify students’ strategies but to
be able to interpret them and analyze students’ thinking processes in more detail. In this study, we analyzed the eye-tracking data manually, which is time-consuming and
requires experts who have domain knowledge and are experienced in eye tracking. For fur-
ther research (especially with larger sample sizes) and practical applications, eye-tracking
data can also be examined using Artificial Intelligence (AI). Qualitative analyses, as pre-
sented in this article, can be supported by AI approaches, for example, by using unsuper-
vised machine learning (Schindler, Schaffernicht, & Lilienthal, 2020, 2022; Simon et al.,
2023). Supervised machine learning approaches can be used to automate the analysis of
eye-tracking data, as demonstrated, for example, by Schindler et al. 5 Discussion (2019).iif In this study, we did not find significant differences in students’ achievement in the
quantity comparison and pattern tasks based on the strategies used, whether structure sense
or serial. It may be possible that future studies could examine if this changes with a differ-
ent age group or different tasks. f
In the future, studies with larger sample sizes (including students at the lower end of
the performance level) may allow the examination of clusters of students (different ages or
abilities) to investigate whether students tend to use the same strategy across different types
of tasks. Furthermore, future studies could also investigate the relationship between stu-
dents’ use of strategies and mathematical performance. Researchers could examine which
kinds of tasks may best allow the early identification of students at risk or students with
exceptional abilities based on students’ strategies. A limitation of our study is that, due to relying only on eye tracking and no other source
of information, identifying structure sense may not automatically reveal students’ reason-
ing, as in the case of the comparison tasks. For instance, instead of seeing the structure
of the numerosity of objects, students could have concentrated on the space taken by the
objects. Another limitation of our study is that we did not use example tasks, and this may
have resulted in differences in students’ responses, especially to the first task. Lastly, it
would have been helpful to include more pattern tasks and patterns in a horizontal arrange-
ment to resemble the number of tasks, as well as the two orientations of dots appearing in
the comparison tasks. 6 Conclusion Even though the exploration of young students’ strategies with eye tracking is still at an
early stage, our study illustrated its potential. Through eye tracking, we inquired into stu-
dents’ use of structure sense and we observed that young students exhibited a variety of
strategies. Students seemed to either utilize structure sense or follow a serial process across
two types of tasks. The ability to shift attention from single elements to groups of elements
has been identified as fundamental for the development of students’ understanding of num-
bers, arithmetic operations, part-whole relationship, multiplication, and patterns (Hunting,
2003; Lüken, 2012; Schöner & Benz, 2017). The results of this study suggest that it is
possible through eye tracking, and specifically through the analysis of students’ scan paths
(here through video analysis), to identify students’ abilities to move their attention away 1 3 3 3 D. Pitta‑Pantazi et al. from single elements and towards a sense of structure. The eye-tracking method can be
seen as a tool to identify and assess students’ structure sense across tasks. Thus, it is pos-
sible to get a better idea of students’ work in these mathematical activities. This possibility
opens the door to numerous applications for eye tracking in various activities. Further stud-
ies and evidence that draw from various methodological approaches would contribute to
solidifying the theoretical basis for a comprehensive understanding of how students work
across tasks during the early years of education, what the involved processes are, how strat-
egy use relates to mathematical performance, and in what ways teaching tailored to the
individual strategy use profiles could enhance students’ learning. from single elements and towards a sense of structure. The eye-tracking method can be
seen as a tool to identify and assess students’ structure sense across tasks. Thus, it is pos-
sible to get a better idea of students’ work in these mathematical activities. This possibility
opens the door to numerous applications for eye tracking in various activities. Further stud-
ies and evidence that draw from various methodological approaches would contribute to
solidifying the theoretical basis for a comprehensive understanding of how students work
across tasks during the early years of education, what the involved processes are, how strat-
egy use relates to mathematical performance, and in what ways teaching tailored to the
individual strategy use profiles could enhance students’ learning. Funding Open access funding provided by the Cyprus Libraries Consortium (CLC). 6 Conclusion Funded by the
European Union under grant agreement No 2020-1-DE03-KA201-077597. However, views and opinions
expressed are those of the author(s) only and do not necessarily reflect those of the European Union or the
European Education and Culture Executive Agency (EACEA). Neither the European Union nor EACEA can
be held responsible for them. Availability of data and material The datasets generated and/or analysed during the current study are avail-
able from the corresponding authors upon reasonable request. Competing interests None. Competing interests None. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Com-
mons licence, and indicate if changes were made. The images or other third party material in this article
are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Battista, M. T. (1999). Fifth graders’ enumeration of cubes in 3D arrays: Conceptual progress in an inquiry-
based classroom. Journal for Research in Mathematics Education, 30(4), 417–449. https://doi.org/10.
2307/749708 Baumanns, L., Pitta-Pantazi, D., Demosthenous, E., Christou, C., Lilienthal, A. J., & Schindler, M. (2022). How do first-grade students recognize patterns? An eye-tracking study. In C. Fernández, S. Llinares,
Á. Gutiérrez, & N. Planas (Eds.), Proceedings of the 45th Conference of the International Group for
the Psychology of Mathematics Education (Vol. 2, pp. 59–66). PME. Baumanns, L., Pitta-Pantazi, D., Christou, C., Lilienthal, A. J., Simon, A. L., & Schindler, M. (2023). Adap-
tive strategy use in pattern-recognition of first graders with and without risk of developing mathemati-
cal difficulties: an eye-tracking study. In M. Ayalon, B. Koichu, R. Leikin, L. Rubel, & M. Tabach
(Eds.), Proceedings of the 46th Conference of the International Group for the Psychology of Math-
ematics Education (PME 46) (Vol. 2, pp. 75–82). PME. Beltrán-Navarro, B., Abreu-Mendoza, R. A., Matute, E., & Rosselli, M. (2018). Development of early
numerical abilities of Spanish-speaking Mexican preschoolers: A new assessment tool. Applied Neu-
ropsychology: Child, 7(2), 117–128. https://doi.org/10.1080/21622965.2016.1266940 p y
gy
( ),
p
g
Chumachemko, D., Shvarts, A., & Budanov, A. (2014). The development of the visual perception of the
cartesian coordinate system: An eye tracking study. In C. Nicol, P. Liljedahl, S. Oesterle, & D. Allan
(Eds.), Proceedings of the Joint Meeting of PME 38 and PME-NA 36 (Vol.2, pp. 313–320). PME. g
f
g f
pp
Clements, D. H. (1999). Subitizing: What is it? Why teach it? Teaching Children Mathematics, 5(7), 400–
405. https://doi.org/10.5951/TCM.5.7.0400 1 3 Structure sense in students’ quantity comparison and repeating… Clements, D. H., & Sarama, J. (2007). Early childhood mathematics learning. In F. K. Lester Jr. (Ed.), Sec-
ond handbook of research on mathematics teaching and learning (Vol. 1, pp. 461–555). Information
Age. g
Cohen, J. (1988). Statistical power analysis for the behavioral sciences (2nd ed.). Lawrence Erlbaum. https://doi.org/10.1016/c2013-0-10517-x Collins, M. A., & Laski, E. V. (2015). Preschoolers’ strategies for solving visual pattern tasks. Early Ch
hood Research Quarterly, 32, 204–214. https://doi.org/10.1016/j.ecresq.2015.04.004 Demosthenous, E., Pitta-Pantazi, D., Christou, C., Lilienthal, A. J., & Schindler, M. (2022). Repeating
patterns: Grade 1 primary school students’ strategies using eye-tracking. In V. Chrysikou, C. Statho-
poulou, T. Triantafillidis, C. Chatzikyriakou, A. Chronaki, & C. References Sdrolias (Eds.), Proceedings of the
9th Conference of the Greek Council of Researchers in Mathematics Education (EN.E.DI.M.). Univer-
sity of Thessaly. (In Greek). Department of Education. (2013). https://assets.publishing.service.gov.uk/government/uploads/system/
uploads/attachment_data/file/335158/PRIMARY_national_curriculum_-_Mathematics_220714.pdf. Accessed 12 Jan 2022. Economopoulos, K. (1998). Early childhood corner: What comes next? The mathematics of pattern in kin-
dergarten. Teaching Children Mathematics, 5(4), 230–233. https://doi.org/10.5951/TCM.5.4.0230 Fischer, B., Gebhardt, C., & Hartnegg, K. (2008). Subitizing and visual counting in children with proble
in acquiring basic arithmetic skills. Optometry and Vision Development, 39(1), 24–29. on, K. C. (1988). Children’s counting and concepts of number. Springer. https://doi.org/10.1007/
978-1-4612-3754-9f Hoch, M., & Dreyfus, T. (2004). Structure sense in high school algebra: The effect of brackets. In M. J. Høines & A. B. Fuglestad (Eds.), Proceedings of the 28th Conference of the International Group for
the Psychology of Mathematics Education (Vol. 3, pp. 49–56). PME. Holmqvist, K., Andrà, C., Lindström, P., Arzarello, F., Ferrara, F., Robutti, O., & Sabena, C. (2011). A
method for quantifying focused versus overview behavior in AOI sequences. Behavior Research Meth-
ods, 43, 987–998. https://doi.org/10.3758/s13428-011-0104-x p
g
Hunting, R. P. (2003). Part-whole number knowledge in preschool children. The Journal of Mathematical
Behavior, 22(3), 217–235. https://doi.org/10.1016/S0732-3123(03)00021-X Huntley-Fenner, G., & Cannon, E. (2000). Preschoolers’ magnitude comparisons are mediated by a pre-
verbal analog mechanism. Psychological Science, 11(2), 147–152. https://doi.org/10.1111/1467-9280.
00230 Hutchinson, E. (2011). Pre-school children’s understanding of mathematical patterns. South African Journal
of Childhood Education, 1(2), 92–111. https://doi.org/10.4102/sajce.v1i2.87 Inglis, M., Attridge, N., Batchelor, S., & Gilmore, C. (2011). Non-verbal number acuity correlates with
symbolic mathematics achievement: But only in children. Psychonomic Bulletin & Review, 18(6),
1222–1229. https://doi.org/10.3758/s13423-011-0154-1 p
g
Liljedahl, P. (2004). Repeating pattern or number pattern: The distinction is blurred. Focus on Learning
Problems in Mathematics, 26(3), 24–42. Linchevski, L., & Livneh, D. (1999). Structure sense: The relationship between algebraic and numerical
contexts. Educational Studies in Mathematics, 40(2), 173–196. https://doi.org/10.1023/A:1003606308
064 Lüken, M. M. (2012). Young children’s structure sense. Journal Für Mathematik-Didaktik, 33(2), 263–285. https://doi.org/10.1007/s13138-012-0036-8 Lüken, M. M., & Sauzet, O. (2021). Patterning strategies in early childhood: A mixed methods study exam-
ining 3- to 5-year-old children’s patterning competencies. Mathematical Thinking and Learning, 23(1),
28–48. https://doi.org/10.1080/10986065.2020.1719452 p
g
Mayring, P. (2014). Qualitative content analysis: Theoretical foundation, basic procedures, and software
solution. Beltz. Moyer, R. S., & Landauer, T. K. (1967). Time required for judgements of numerical inequality. Nature, 215,
1519–1520. https://doi.org/10.1038/2151519a0 p
g
Mulligan, J., Mitchelmore, M., & Prescott, A. (2006). References Integrating concepts and processes in early mathemat-
ics: The Australian Pattern and Structure Mathematics Awareness Project (PASMAP). In J. Novotná,
H. Moraová, M. Krátká, & N. Stehlíková (Eds.), Proceedings of the 30th Annual Conference of the
International Group for the Psychology of Mathematics Education (Vol. 4, pp. 209–216). PME. p f
y
gy f
pp
Nieder, A., & Dehaene, S. (2009). Representation of number in the brain. Annual Review of Neuroscience,
32(1), 185–208. https://doi.org/10.1146/annurev.neuro.051508.135550 p
g
Obersteiner, A., & Tumpek, C. (2016). Measuring fraction comparison strategies with eye-tracking. ZDM–
Mathematics Education, 48(3), 255–266. https://doi.org/10.1007/s11858-015-0742-z 1 3 D. Pitta‑Pantazi et al. Odic, D., & Halberda, J. (2015). Eye movements reveal distinct encoding patterns for number and cumula-
tive surface area in random dot arrays. Journal of Vision, 15(5), 1–16. https://doi.org/10.1167/15.15.5 Ott, N., Brünken, R., Vogel, M., & Malone, S. (2018). Multiple symbolic representations: The combination
of formula and text supports problem solving in the mathematical field of propositional logic. Learn-
ing and Instruction, 58, 88–105. https://doi.org/10.1016/j.learninstruc.2018.04.010 Papic, M. (2015). An early mathematical patterning assessment: Identifying young Australian Indigenous
children’s patterning skills. Mathematics Education Research Journal, 27(4), 519–534. https://doi.org/
10.1007/s13394-015-0149-8 Papic, M., Mulligan, J. T., & Mitchelmore, M. C. (2011). Assessing the development of preschoolers’ math-
ematical patterning. Journal for Research in Mathematics Education, 42(3), 237–268. https://doi.org/
10.5951/jresematheduc.42.3.0237 j
Piaget, J., & Szeminska, A. (1952). The child’s conception of number. Routledge & Kegan Paul. Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023a). Eye-move-
ments comparison task: Simultaneous comparison of the two sets. Retrieved from https://youtu.be/
nOiUzaPwZHQ Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023b). Eye-move-
ments comparison task: Partial comparison of groups of dots of the two sets (Example i). Retrieved
from https://youtu.be/E2E_jXBaZyc p
y
j
y
Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023c). Eye-
movements comparison task: Partial comparison of groups of dots of the two sets (Example ii). Retrieved from https://youtu.be/q94FmL-2uNE p
y
q
Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023d). Eye-move-
ments comparison task: Extended partial comparison of dots of the two sets. Retrieved from https://
youtu.be/19OdB3gyeO4 Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023e). Eye-move-
ments comparison task: Attending/comparing all (Example i). Retrieved from https://youtu.be/
rsMeBKqy-Ts qy
Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023f). References (2020). Enumeration processes of children with math-
ematical difficulties: An explorative eye-tracking study on subitizing, groupitizing, counting, and
pattern recognition. Learning Disabilities: A Contemporary Journal, 18(2), 193–211. Schleifer, P., & Landerl, K. (2011). Subitizing and counting in typical and atypical development: Subi-
tizing and counting. Developmental Science, 14(2), 280–291. https://doi.org/10.1111/j.1467-7687.
2010.00976.x Schöner, P., & Benz, C. (2017). “Two, three and two more equals seven” – Preschoolers’ perception and
use of structures in sets. In T. Dooley & G. Gueudet (Eds.), Proceedings of the 10th Congress of the
European Society for Research in Mathematics Education (pp. 1893 – 1900). European Society for
Research in Mathematics Education. Shvarts, A., Chumachenko, D., Drenyova, A., & Krichevets, A. (2019). From prototypical phenomenon
to dynamic functional system: Eye-tracking data on the identification of special quadrilaterals. In A. Shvarts (Ed.), Proceedings of the PME and Yandex Russian conference: Technology and Psychology
for Mathematics Education (pp. 122–129). HSE. Simon, A. L., Asghari, P., Lilienthal, A. J., & Schinder, M. (2023). Strategy use in number line tasks of stu-
dents with and without mathematical difficulties: A study using eye tracking and AI. In M. Ayalon, B. Koichu, R. Leikin, L. Rubel & M. Tabach (Eds.), Proceedings of the 46th Conference of the Interna-
tional Group for the Psychology of Mathematics Education (Vol. 4, pp. 211–218). PME 46. Sprenger, P., & Benz, C. (2020). Children’s perception of structures when determining cardinality of sets—
Results of an eye-tracking study with 5-year-old children. ZDM–Mathematics Education, 52(4), 753–
765. https://doi.org/10.1007/s11858-020-01137-x Starkey, G. S., & McCandliss, B. D. (2014). The emergence of “groupitizing” in children’s numerical cogni-
tion. Journal of Experimental Child Psychology, 126, 120–137. https://doi.org/10.1016/j.jecp.2014.03.
006 Strohmaier, A. R., MacKay, K. J., Obersteiner, A., & Reiss, K. M. (2020). Eye-tracking methodology in
mathematics education research: A systematic literature review. Educational Studies in Mathematics,
104(2), 147–200. https://doi.org/10.1007/s10649-020-09948-1 Threlfall, J. (1999). Repeating patterns in the primary years. In A. Orton (Ed.), Pattern in the teaching and
learning of mathematics (pp. 18–30). Cassell.f Trick, L. M., & Pylyshyn, Z. W. (1994). Why are small and large numbers enumerated differently? A lim-
ited-capacity preattentive stage in vision. Psychological Review, 101(1), 80–102. https://doi.org/10.
1037/0033-295X.101.1.80 van der Weijden, F. A., Kamphorst, E., Willemsen, R. H., Kroesbergen, E. H., & van Hoogmoed, A. H. (2018). Strategy use on bounded and unbounded number lines in typically developing adults and adults
with dyscalculia: An eye-tracking study. References Eye-move-
ments comparison task: Attending/comparing all (Example ii). Retrieved from https://youtu.be/
9m1drAGgW5w g
Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023g). Eye-
movements comparison task: One-to-one correspondence. Retrieved from https://youtu.be/hzGS5
6vLsDo Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023h). Eye-move-
ments pattern task: Simultaneous identification of the repeating unit. Retrieved from https://www.
youtube.com/watch?v=am2ynmMacnw Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023i). Eye-move-
ments pattern task: Identification of the repeating unit and comparison with other elements or groups
of elements (repeating unit). Retrieved from https://www.youtube.com/watch?v=pzanlwvQKpE f
p
g
p
y
p
p
Pitta-Pantazi, D., Demosthenous, E., Schindler, M., Lilienthal, A. J., & Christou, C. (2023j). Eye-move-
ments pattern task: Sequential, continuous “attending all”. Retrieved from https://www.youtube.
com/watch?v=yxGKpaEdPhA y
p
Posner, M. I. (1980). Orienting of attention. Quarterly Journal of Experimental Psychology, 32(1), 3–25. https://doi.org/10.1080/00335558008248231 Radford, L. (2010). The eye as a theoretician: Seeing structures in generalizing activities. For the Learn-
ing of Mathematics, 30(2), 2–7. g f
Sarama, J., & Clements, D. H. (2008). Mathematics in early childhood. In O. N. Saracho & B. Spodek
(Eds.), Contemporary perspectives on mathematics in early childhood education (pp. 67–94). Information Age.fi Schindler, M., & Lilienthal, A. J. (2018). Eye-tracking for studying mathematical difficulties—also in
inclusive settings. In E. Bergqvist, M. Österholm, C. Granberg, & L. Sumpter (Eds.), Proceedings
of the 42nd Conference of the International Group for the Psychology of Mathematics Education
(Vol. 4, pp. 115–122). PME. Schindler, M., Bader, E., Lilienthal, A. J., Schindler, F., & Schabmann, A. (2019). Quantity recogni-
tion in structured whole number representations of students with mathematical difficulties: An eye-
tracking study. Learning Disabilities: A Contemporary Journal, 17(1), 5–28.f Schindler, M., Doderer, J. H., Simon, A. L., Schaffernicht, E., Lilienthal, A. J., & Schäfer, K. (2022). Small
number enumeration processes of deaf or hard-of-hearing students: A study using eye tracking and
artificial intelligence. Frontiers in Psychology, 13, 1–17. https://doi.org/10.3389/fpsyg.2022.909775 1 3 Structure sense in students’ quantity comparison and repeating… Schindler, M., Schaffernicht, E., & Lilienthal, A. J. (2020). Identifying student strategies through eye
tracking and unsupervised learning: The case of quantity recognition. In M. Inprasitha, N. Chang-
sri, & N. Boonsena (Eds.), Proceedings of the 44th Conference of the International Group for the
Psychology of Mathematics Education (pp. 518–527). PME. ogy of Mathematics Education (pp. 518–527). PME Schindler, M., Schovenberg, V., & Schabmann, A. References Journal of Numerical Cognition, 4(2), 337–359. https://doi.
org/10.5964/jnc.v4i2.115 g
j
Van Nes, F. T. (2009). Young children’s spatial structuring ability and emerging number sense. Utrecht
University. von Aster, M., Weinhold Z. M., & Horn, R. (2006) Neuropsychologische Testbatterie für Zahlenverarbei-
tung und Rechnen bei Kindern (ZAREKI-R) [Neuropsychological Test Battery for Number Processing
and Calculation in Children]. Harcourt Test Services.f Yilmaz, N. (2019). The effect of the hypothetical learning trajectories and the contribution of eye-tracking
technology in understanding young children’s mathematical patterning recognition and generalization
[Doctoral dissertation, Middle East Technical University]. Middle East Technical University Research
Repository. https://open.metu.edu.tr/bitstream/handle/11511/46125/index.pdf. Accessed 12 Jan 2022. 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. 3 1
|
https://openalex.org/W3172462826
|
https://www.ajol.info/index.php/tjpr/article/download/207840/195916
|
English
| null |
Investigation of in vivo protective effect of orally administered vitamin E and selenium against gentamicininduced renal and hepatic toxicity
|
Tropical journal of pharmaceutical research
| 2,021
|
cc-by
| 4,849
|
Investigation of in vivo protective effect of orally
administered vitamin E and selenium against gentamicin-
induced renal and hepatic toxicity Amin A Al-Doaiss1,2, Yazun B Jarrar3*
1Department of Biology, College of Science, King Khalid University, Abha, Saudi Arabia,
2Anatomy and Histology Department,
Faculty of Medicine, Sana’a University, Sana’a, Republic of Yemen,
3Department of Pharmacy, College of Pharmacy,
Alzaytoonah University of Jordan, Amman, Jordan Amin A Al-Doaiss1,2, Yazun B Jarrar3*
1Department of Biology, College of Science, King Khalid University, Abha, Saudi Arabia,
2Anatomy and Histology Department,
Faculty of Medicine, Sana’a University, Sana’a, Republic of Yemen,
3Department of Pharmacy, College of Pharmacy,
Alzaytoonah University of Jordan, Amman, Jordan *For correspondence: Email: yazun.jarrar@zuj.edu.jo; Tel: 00962-795930283 *For correspondence: Email: yazun.jarrar@zuj.edu.jo; Tel: 00962-795930283 Revised accepted: 21 June 2019 Sent for review: 21 February 2019 Sent for review: 21 February 2019 Abstract Purpose: To investigate the protective effect of vitamin E (Vit E) and selenium (Se) combination against
gentamycin (GM)-induced renal and hepatic toxicity in rats. g
y
(
)
p
y
Methods: Forty-eight male Wistar albino rats were administrated GM at a dose of 80 mg/kg/day, with or
without Se (1.5 mg/kg/day), and/or Vit E (250 mg/kg/day) for a period of 4 weeks. Serum samples from
each rat were subjected to biochemical analysis for kidney and liver functions, while kidney and liver
biopsies were also investigated by histological examination. p
g
y
g
Results: GM significantly increased serum creatinine, urea, alanine aminotransferase (ALT), aspartate
aminotransferase (AST) and free radicals (p < 0.05). Moreover, GM induced significant histological and
ultrastructural alterations in the renal and hepatic tissues of the rats. Exposure to a combination of Vit E
and Se did not attenuate the GM-induced toxicity in renal and hepatic tissues. y
p
Conclusion: These results suggest that Vit E and Se combination have no significant protective role
against GM-induced hepatic and renal toxicity. Keywords: Antioxidants, Vitamin E, Selenium, Gentamicin, Toxicity Keywords: Antioxidants, Vitamin E, Selenium, Gentamicin, Toxicity This is an Open Access article that uses a fund-ing model which does not charge readers or their
institutions for access and distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0)
and
the
Budapest
Open
Access
Initiative
(http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus,
International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African
Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals
(DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts is related to its accumulation in the renal
proximal convoluted tubules leading to acute
renal failure and tubular necrosis [4]. Tropical Journal of Pharmaceutical Research July 2019; 18 (7): 1435-1442
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research July 2019; 18 (7): 1435-1442
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Tropical Journal of Pharmaceutical Research July 2019; 18 (7): 1435-1442
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria. Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v18i7.10 Original Research Article Trop J Pharm Res, July 2019; 18(7): 143
© 2019 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License Biochemical analyses At the end of the first and fourth weeks after
treatment with GM and combination of Vit E and
Se, blood samples were taken from the orbital
sinus of each rat, and serum was obtained for
measurement of biochemical parameters of
kidney function (urea and creatinine), and liver
function (ALT, AST, and alkaline phosphatase,
ALP). A blood Chemical Analyser (Reflotron,
Roche Co., Germany) was used for the
biochemical analysis, using the appropriate
assay kits. The levels of free radicals in rat blood
were determined with FRAS-4 (Iram-Param Co.,
Italy), as described previously [14]. Experimental design Vitamin E (Vit E) and selenium (Se) are essential
nutrients which function as antioxidants and
hence minimize cellular damage caused by free
radicals such as reactive oxygen species and
endogenous peroxides [9]. Vitamin E and Se
prevent
oxidative
damage
to
the
cellular
membrane
by
decreasing
hydroperoxide
formation [10]. Previous studies have reported
the nephro-protective effects of Vit E and Se in
rats fed high-cholesterol diets [11], and in
organo-phosphate-induced
toxicity
[12]
and
cadmium-induced renal toxicity [13]. In addition,
Vit E and Se are protective against malathion-
induced hepatotoxicity [14]. However, Vit E, in
combination with diphenyl-phenylenediamine, did
not exert protective effects against GM-induced
nephrotoxicity [15]. Indeed, a study has reported
that Vit E increased the risk of cancer
development [16]. Forty-eight male Wistar albino rats (Rattus
norvegicus) were randomly assigned to four
groups of 12 rats each as follows: Control: Each rat received daily 1 mL of 0.9 %
saline and 0.25 mL corn oil via gavage for a
period of 4 weeks. GM-treated: The rats were given GM at a dose of
80 mg/kg/day via intramuscular (im) injection for
a period of 4 weeks. GM-treated group with co-administration of Vit E
and Se: Single daily doses of Se (1.5 mg/kg) and
Vit E (250 mg/kg, dissolved in corn oil) were
administrated to the rats via gavage. One hour
later, GM was given at a dose of 80 mg/kg via im
injection for 4 weeks. Several
studies
have
reported
the
nephroprotective and hepatoprotective effects of
Vit E and Se, but the effect of Vit E and Se
combination on GM-induced nephrotoxicity and
hepatotoxicity
has
not
been
investigated. Therefore, the present study was conducted to
determine the in vivo influence of orally-
administered Vit E and Se on GM-induced
toxicity in renal and hepatic tissues. Vitamin E- and Se-treated: The rats in this group
received daily single dose of Se (1.5 mg/kg) and
single dose of Vit E (250 mg/kg) via gavage for 4
weeks. The doses of GM, Vit E and Se were
based on previous investigations [7,12,14]. Chemicals and drugs Gentamicin
was
obtained
from
Al-Qassim
Pharmaceutical Company of Saudi Arabia. Vitamin E (α-tocopherol, Evion 100 mg capsule)
and Se were purchased from Merck Limited
Company,
Germany,
and
Oxoid
Limited
Company (Basingstoke, UK), respectively. INTRODUCTION Gentamicin (GM) is still considered an important
aminoglycoside antibiotic which is widely used in
treatment of bacterial infections caused by gram
negative bacteria [1]. It is used clinically in
treatment
of
urinary
tract
infections
and
endocarditis [2]. However, treatment with this
antibiotic causes renal and hepatic toxicity
through induction of oxidative stress, apoptosis
and necrosis [3]. The GM-induced nephrotoxicity Extracts of medicinal plants, trace elements,
vitamins and antioxidants have been successfully
used to ameliorate GM-induced toxicity [5-7]. Several approaches have been attempted for
reducing GM toxicity. These efforts were mainly
focused on the use of plant antioxidant extracts
[6] or other agents with antioxidant properties [8]. Trop J Pharm Res, July 2019; 18(7): 14
© 2019 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License Al-Doaiss & Jarrar Effect of Vit E and Se on GM-induced
biochemical alterations Effect of Vit E and Se on GM-induced
biochemical alterations Table 1 shows that GM significantly elevated
serum ALT, ALP, AST, urea and creatinine levels
after 1 and 4 weeks of exposure (p value < 0.05). Co-administration of vitamin E and Se did not
ameliorate the GM-induced changes in kidney
and liver functions tests. Indeed, after 4 weeks of
treatment of the GM rats with Vit E and Se, ALT
and AST were more significantly elevated,
relative to rats treated with GM alone (p < 0.05). On the other hand, Vit E + Se- treated rats had
significantly lower levels of free radicals (p <
0.05) after 1 and 4 weeks of administration, when
compared with the GM-treated rats and GM rats
that received co-administration of Vit E and Se. Renal tubules in the group treated with GM and
Vit E+Se combination showed accumulation of
luminal debris by the end of the first week (Figure
2 d). In addition, lymphocyte infiltration after 4
weeks of co-administration of GM, Vit E and Se
was almost similar to that seen in the kidney of
rats exposed to GM only (Figure 2 e). At the end
of the first week, calcification of the necrotic renal
tubules was observed (Figure 2 f). Histopathological examination Adult male Wistar albino rats weighing 220 – 250
g were obtained from the Animal Care Centre in
College of Pharmacy, King Saud University. All
rats were housed under controlled conditions
which included controlled suitable temperature
(22 ± 1°C) and a 12h light/12h dark cycle. The
rats were fed laboratory animal diet and water ad
libitum. The protocol for the current study was
approved by the Research Ethics Committee of
College of Pharmacy, King Saud University, with
reference number 97-40. All the experimental
procedures were conducted in the Histology and
Cell Biology Laboratory at King Saud University,
Saudi Arabia. The rats were handled and treated
according to the guidelines of Canadian Council
on Animal Care [17]. Six rats from each group were sacrificed. The
kidney and liver specimens were excised for
histological examination at the end of first and
fourth weeks of treatment with GM and the
combination of Vit E and Se. Fresh biopsies of
both kidney and the liver from each rat were
taken and fixed in neutral buffered formalin (10
%), followed by dehydration in ascending
concentrations of ethanol (70 to 100 %). Dehydration was followed by clearing in xylene. Then, the tissue samples were impregnated in
molten paraffin wax, embedded and blocked. Thereafter, 4-µm tissue sections were sliced with
a microtome and stained with hematoxylin and Trop J Pharm Res, July 2019; 18(7): 1436 Al-Doaiss & Jarrar Figure 1: (a) Photomicrograph of kidney section of
control rat demonstrating normal structures (H & E,
×400); (b) Electron micrograph of kidney of control rat
with normal ultrastructure. Note the spherical nucleus
with euchromasia, together with normal mitochondria
and profuse apical microvilli (×5000). eosin (H & E). Histological changes were
observed and photographed under an Olympus
Microscope BX51 with Digital Camera (Japan). Statistical analysis There were no abnormalities in the histology and
ultrastructure of the kidneys from rats treated
with Vit. E and Se. In contrast, GM induced
marked tubular and interstitial alterations in the
kidneys of rats in the GM-treated group. The
tubular
alterations
due
to
GM
treatment
appeared early in the form of necrosis,
degeneration and vacuolization by the end of the
first week. The degenerative tubules had swelling
and loss of the proximal tubular brush border. These changes were seen in most of the
proximal convoluted tubules, and to a lesser
extent in the distal tubules (Figure 2a). Severe
tubular
necrosis
occurred
and
intertubular
lymphocytic infiltration appeared after one week
of GM treatment at dose 80 mg/kg (Figures 2 b
and 2 c). Moreover, lymphocytic infiltration was
prominent after 4 weeks of GM treatment. However, the damage was seen more in the
outer layer of the kidney cortex than in the
medulla. Data are presented as mean ± standard
deviations (SD). The data were statistically
analysed with SAS, 2002, using one-way
ANOVA test. Statistically significant changes in
comparison with the control group were indicated
as p < 0.05. Ultrastructural analysis For ultrastructural analysis, small, fresh tissue
blocks (1 - 3 mm3) were cut off from the kidney
and liver tissues. Tissue blocks were fixed in 3 %
glutaraldehyde solution which was embedded in
epoxy resin and processed for ultrastructural
analysis using electron microscopy. Figure 1: (a) Photomicrograph of kidney section of
control rat demonstrating normal structures (H & E,
×400); (b) Electron micrograph of kidney of control rat
with normal ultrastructure. Note the spherical nucleus
with euchromasia, together with normal mitochondria
and profuse apical microvilli (×5000). Trop J Pharm Res, July 2019; 18(7): 1438 Effect of Vit E and Se on GM-induced renal
histological alterations The kidneys of control rats showed normal
histological features of glomerular, tubular and
interstitial components of the cortex and the
medulla (Figure 1 a). In addition, ultra-thin
sections of the control kidneys showed normal
ultrastructure (Figure 1 b). The epithelial lining of
the PCTs had normal profuse apical microvilli
and spherical nuclei with euchromasia. Several
mitochondria were observed in the cytoplasm of
the renal cells. Relative to the ultrastructure of the control
kidneys, renal cells of the proximal convoluted
tubules (PCT) of rats that received GM only had
ultrastructural alterations such as necrosis,
cytoplasmic vacuolation and cellular debris
(Figure 3 a). The changes extended to the distal
convoluted tubules (DCTs) and collecting ducts
which showed a lot of lysosomal structures and
large phagocytic vacuoles (Figure 3 b). The renal
ultrastructure of rats in this group showed no
significant
recovery
from
the
GM-induced
alterations. Trop J Pharm Res, July 2019; 18(7): 1437 Al-Doaiss & Jarrar Table 1: Blood biochemical analysis (mean ± SD) at weeks 1 and 4 Parameters
Control (I)
Vit E (II)
GM 80 mg/kg (III)
GM 80+ GM and Se (IV)
Week 1
Week 4
Week 1
Week 4
Week 1
Week 4
Week 1
Week 4
ALP (μl/L)
419±00.10B
389±00.00D
361±0.00C*
590±00.00A*
320±00.00D*
557±25.98B*
323.67±19.66CD*
414±0.00C*
ALT (μl/L)
34.6±1.56B
52.7±4.85BC
34±5.54B
55.8±4.33B
47.03±4.21A*
47.43±0.93C
29.73±00.00B
151±0.00A*
AST (μl/L)
180.33±21.36CB
224±12.12B
137.67±4C
136.67±35.27C*
296±34.64A*
246±17.58B
145.33±22.81C
353±0.00A*
Creatinine
(mg/dl)
0.5±0.1D
0.50±0.02A
0.5±0.21D
0.50±0.15A
0.83±0.03D
0.60±0.06A
6.39±1.46A*
0.70±0.00A
Urea (mg/dl)
37.63±0.75D
49.53±6.12A
37.3±3.63D
40.63±0.63B*
89.73±10.62C*
55.17±5.61A
274±1.00B*
53.90±0.00A
Free radicals
(U Carr)
365±0.00A
322±0.00B
296±17.32A
253±0.00C*
360±0.00A
379.67±26.69A
*
290.33±83.72A
371±0.00A*
*Significant difference (p < 0.05, compared with control group. Different letters within the same row indicate significant differences (p < 0.05) between
experimental groups for the same biochemical parameter Al-Doaiss & Jarrar hepatic lobules, hepatocytes and hepatic portal
triads (Figure 4a). In addition, the ultrastructure
of the control liver appeared normal (Figure 4b). The hepatocytes showed normal nucleoplasm
with nuclei surrounded by even distinct nuclear
envelop showing heterochromatin adjacent to the
border. The cytoplasm of these hepatocytes
appeared
crowded
with
mitochondria,
endoplasmic reticulum and bounded ribosomes. Effect of Vit E and Se on GM-induced renal
histological alterations Figure 5: (a) Photomicrograph of liver section of GM-
treated rats showing- scattered hemorrhagic-necrosis
after one week of drug administration (H & E; ×1000);
(b)
focal
necrosis
with
lymphocytes
infiltration
microgranuloma)
(*)
after
one
week
of
GM
administration (×400); (c) haemorrhagic foci with
necrosis after 4 weeks of treatment (H & E; ×400) Figure 3: Electron micrograph showing epithelial cells
lining the PCT of (a) GM-treated rat after one week,
demonstrating necrosis and cytoplasmic vacuolations
containing cellular debris; (b) Electron micrograph
GM+Vit.E +Se-treated rats after one week, showing
lysosomal structures and large phagocytic vacuoles
(×4000) Figure 5: (a) Photomicrograph of liver section of GM-
treated rats showing- scattered hemorrhagic-necrosis
after one week of drug administration (H & E; ×1000);
(b)
focal
necrosis
with
lymphocytes
infiltration
microgranuloma)
(*)
after
one
week
of
GM
administration (×400); (c) haemorrhagic foci with
necrosis after 4 weeks of treatment (H & E; ×400) Figure 5: (a) Photomicrograph of liver section of GM-
treated rats showing- scattered hemorrhagic-necrosis
after one week of drug administration (H & E; ×1000);
(b)
focal
necrosis
with
lymphocytes
infiltration
microgranuloma)
(*)
after
one
week
of
GM
administration (×400); (c) haemorrhagic foci with
necrosis after 4 weeks of treatment (H & E; ×400) Figure 3: Electron micrograph showing epithelial cells
lining the PCT of (a) GM-treated rat after one week,
demonstrating necrosis and cytoplasmic vacuolations
containing cellular debris; (b) Electron micrograph
GM+Vit.E +Se-treated rats after one week, showing
lysosomal structures and large phagocytic vacuoles
(×4000) Similar to rats exposed to GM only, rats in the
group treated with GM in combination with Vit
and
Se
had
hepatocytes
necrosis
and
lymphocytic infiltration (Figures 6a-d). Effect of Vit E and Se on GM-induced renal
histological alterations Figure 4: a- Photomicrographs showing liver of control
rat with normal histological structure (H & E; ×400); b-
Electron micrograph showing liver of control rat with
normal ultrastructure (×6000) Figure 4: a- Photomicrographs showing liver of control
rat with normal histological structure (H & E; ×400); b-
Electron micrograph showing liver of control rat with
normal ultrastructure (×6000) Rats treated with a combination of Vit E and Se
had no significant changes in renal or hepatic
tissues. In contrast, after 4 weeks of treatment,
liver sections from GM-treated rats showed
congestion and haemorrhagic foci accompanied
by hepatic necrosis and lymphocytic infiltration
(Figures 5 a - 5c). Figure 2:
Photomicrographs
demonstrating:
(a)
accumulation of eosinophilic materials in the collecting
tubules of the medulla after one week of treatment
with GM + Vit E + Se; (b) severe necrosis and
accumulation of eosinophilic debris in the kidney
convoluted
tubules
after
one
week
of
GM
administration; (c) calcified necrotic tubules with
intraluminal accumulation of necrotic debris after one
week of treatment with GM + Vit E + Se); (d)
accumulation of eosinophilic materials in the collecting
tubules of the medulla after one week of GM+ Vit E +
Se
administration;
(e)
severe
necrosis
and
accumulation of eosinophilic debris in the kidney
convoluted
tubules
after
one
week
of
GM
administration; (f) necrotic tubules with intraluminal
accumulation of necrotic debris after one week of
treatment with GM + Vit E + Se. (H & E, × 400). Ultrastructural changes Co-administration of GM and Vit E + Se led to
mitochondrial swelling and crystolysis, necrosis,
endoplasmic reticulum disruption and occasional
cytoplasmic vacuolization, as shown on Figures
7 a and 7b. Creatinine and urea are considered biomarkers
of the efficiency of renal function [20]. Increased
blood creatinine and urea are strongly related to
renal
damage
[7]. Consistent
with
this
relationship, it was found exposure to GM
caused
significant
renal
alterations
and
significant elevations in serum creatinine and
urea. These results are in agreement with those
obtained in an earlier study in which creatinine
and urea levels were elevated after 5-7 days of
GM administration [7]. Figure 7: Electron micrographs of hepatocytes of rats
treated with Gm + Vit E + Se for 4 weeks showing (a)
necrotic foci (×5000); (b) swollen mitochondria with
damaged cristae (×10000) Figure 7: Electron micrographs of hepatocytes of rats
treated with Gm + Vit E + Se for 4 weeks showing (a)
necrotic foci (×5000); (b) swollen mitochondria with
damaged cristae (×10000) The GM-induced hepatotoxicity and nephron-
toxicity might be a result of the generation of
reactive oxygen species which increase lipid
peroxidation
and
lower
the
activities
of
antioxidant enzymes [21]. In addition, GM acts as
an iron chelator by forming an iron-GM complex
which is considered a potent catalyst of free
radical generation [22]. Some studies have
shown that antioxidant agents such as curcumin,
Arabic gum, probucol, vitamin C, lycopene and
grape seed extract can be used to ameliorate
GM-induced nephrotoxicity in rats [18,21,22]. Hepatic histological alterations The
hepatic
tissues
of
the
control
rats
demonstrated normal architecture with normal Trop J Pharm Res, July 2019; 18(7): 1439 Al-Doaiss & Jarrar Figure 6: Photomicrograph of liver section from rats
treated with GM+Vit E +Se showing (a) necrosis at the
end after one week (H & E; x1000); (b) focal necrosis
and pyknosis after one week (H & E; x1000); (c)
hemorrhage with edema after 4 weeks (H & E; x400);
(d) hemorrhagic foci with necrosis after 4 weeks (H &
E; x400). Several studies have reported that GM caused
hepatotoxic
and
nephrotoxic
effects
in
experimental animals [6,18]. The results of the
present study are in line with these previous
reports, since GM induced significant toxicity
after 1 week treatment, as shown in significant
increases in serum levels of ALT, AST, creatinine
and urea, and histological alterations in the
hepatic
and
renal
tissues
of
the
rats. Interestingly,
there
were
no
significant
differences in results of kidney and liver function
tests between 1-week GM administration and 4-
week GM administration. This finding may
indicate that 1-week GM administration is
enough to induce significant hepatotoxic and
nephrotoxic effects in rats. The observed renal
histological changes, and the accompanying
increased levels of ALP indicate functional
disorders in the liver and kidney, as postulated
by Kim and Moon study [19]. It has been
reported that GM induced renal toxicity and
significant elevation in ALP [3]. The hepatic
alterations observed in histological examination
in the present study confirm that significant
structural changes in the liver lead to significant
increase in the blood level of ALT. Figure 6: Photomicrograph of liver section from rats
treated with GM+Vit E +Se showing (a) necrosis at the
end after one week (H & E; x1000); (b) focal necrosis
and pyknosis after one week (H & E; x1000); (c)
hemorrhage with edema after 4 weeks (H & E; x400);
(d) hemorrhagic foci with necrosis after 4 weeks (H &
E; x400). DISCUSSION Gentamicin (GM)-induced toxicity limits chronic
administration of GM. The present
study
investigated the in vivo chemoprotective role of
oral
Vit
E
and
Se
against
GM-induced
hepatotoxicity and nephrotoxicity. The findings
revealed that daily administration of GM at a
dose of 80 mg/kg for 4 weeks led to significant
hepatotoxicity and nephrotoxicity in rats. In
addition, the study showed that co-administration
of oral Vit E and Se combination did not
ameliorate the GM-induced toxicity. These
findings may indicate that oral administration of
Vit E and Se does not protect against GM-
induced hepatotoxicity and nephrotoxicity. Vitamin E and Se are popularly used as
antioxidants, and some studies have reported
that these agents ameliorated drug-induced
toxicity [14]. The present study found that
combined treatment with Se and Vit. E lowered
free
radical
levels,
when
compared
to
corresponding values for the control and GM-
treated groups. However, co-administration of Trop J Pharm Res, July 2019; 18(7): 1440 Al-Doaiss & Jarrar GM with Vit E and Se did not ameliorate the
levels of free radicals, consistent with elevated
levels of kidney and liver function biomarkers,
and with abnormal histological alterations. These
findings may indicate that the antioxidant effects
of orally administrated Vit E and Se are not
sufficient to protect the liver and kidney from
oxidative stress induced by GM exposure. This
may be due to the incomplete oral absorption of
Vit E and Se, or due to other reasons which need
further investigation. The results of the current
study agree with a previous report which showed
that Vit E and diphenyl-phenylenediamine failed
to
offer
protection
against
GM-induced
nephrotoxicity [15]. However, the findings of the
present study are in agreement with those of a
previous study which indicated that Vit E
attenuated GM-induced nephrotoxicity in rats
[23]. It has been shown that Vit E and Se
combination might not be beneficial to health and
recovery from diseases. Moreover, it has been
demonstrated that the use of Vit E and Se
supplements did not offer any appreciable
benefits in prevention of prostate cancer [24]. Indeed, Vit E and Se supplementation has been
shown to significantly increase the risk of
prostate cancer among healthy men [16]. The
elevation of serum ALT and ALP levels as seen
in the present study may indicate that Vit E and
Se may have harmful impact on the liver. Conflict of interest No conflict of interest is associated with this
work. 7. Karahan I, Atessahin A, Yilmaz S, Ceribasi AO, Sakin F. Protective effect of lycopene on gentamicin-induced
oxidative stress and nephrotoxicity in rats. Toxicology
2005; 215(3): 198-204. CONCLUSION 3. Alarifi S, Al-Doaiss A, Alkahtani S, Al-Farraj SA, Al-Eissa
MS, Al-Dahmash B, Al-Yahya H, Mubarak M. Blood
chemical changes and renal histological alterations
induced by gentamicin in rats. Saudi J Biol Sci 2012;
19(1): 103-110. The results obtained in the present investigation
indicate that exposure to GM is capable of
inducing
significant
biochemical
alterations,
together with marked deleterious ultrastructural
alterations in renal and hepatic tissues. However,
oral administration of Vit E and Se combination
does not ameliorate GM-induced toxicity. 4. Pedraza-Chaverri J, Maldonado PD, Medina-Campos
ON,
Olivares-Corichi
IM,
Granados-Silvestre
MA,
Hernandez-Pando
R,
Ibarra-Rubio
ME. Garlic
ameliorates
gentamicin
nephrotoxicity:
relation
to
antioxidant enzymes. Free Radic Biol Med 2000; 29(7):
602-611. 4. Pedraza-Chaverri J, Maldonado PD, Medina-Campos
ON,
Olivares-Corichi
IM,
Granados-Silvestre
MA,
Hernandez-Pando
R,
Ibarra-Rubio
ME. Garlic
ameliorates
gentamicin
nephrotoxicity:
relation
to
antioxidant enzymes. Free Radic Biol Med 2000; 29(7):
602-611. DISCUSSION pertaining to claims relating to the content of this
article will be borne by the authors. Amin Al-
Doaiss did most of the laboratory work and wrote
the manuscript. Yazun Jarrar did the statistical
analysis, contributed to the interpretation of the
results,
and
writing
and
reviewing
the
manuscript. REFERENCES 1. Nagai J, Takano M. Molecular aspects of renal handling
of aminoglycosides and strategies for preventing the
nephrotoxicity. Drug Metab Pharmacokinet 2004; 19(3):
159-170. 2. Takahashi S, Xu C, Sakai T, Fujii K, Nakamura M. Infective endocarditis following urinary tract infection
caused by Globicatella sanguinis. IDCases 2018; 11:
18-21. Open Access This is an Open Access article that uses a fund-
ing model which does not charge readers or their
institutions for access and distributed under the
terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/
4.0) and the Budapest Open Access Initiative
(http://www.budapestopenaccessinitiative.org/rea
d), which permit unrestricted use, distribution,
and reproduction in any medium, provided the
original work is properly credited. Acknowledgement 5. Cuzzocrea S, Mazzon E, Dugo L, Serraino I, Di Paola R,
Britti D, De Sarro A, Pierpaoli S, Caputi A, Masini E, et
al. A role for superoxide in gentamicin-mediated
nephropathy in rats. Eur J Pharmacol 2002; 450(1): 67-
76. The authors are thankful to the Deanship of
Scientific Research and the Research Centre at
College of Science of King Saud University; for
supporting this research work. 6. Manikandan R, Beulaja M, Thiagarajan R, Priyadarsini A,
Saravanan R, Arumugam M. Ameliorative effects of
curcumin against renal injuries mediated by inducible
nitric oxide synthase and nuclear factor kappa B during
gentamicin-induced toxicity in Wistar rats. Eur J
Pharmacol 2011; 670(2-3): 578-585. Contribution of authors We declare that this work was done by the
authors named in this article, and all liabilities Trop J Pharm Res, July 2019; 18(7): 1441 Al-Doaiss & Jarrar 8. Nabavi SF, Nabavi SM, Moghaddam AH, Naqinezhad A,
Bigdellou R, Mohammadzadeh S. Protective effects of
Allium
paradoxum
against
gentamicin-induced
nephrotoxicity in mice. Food Funct 2012; 3(1): 28-29. 16. Klein EA, Thompson IM, Jr., Tangen CM, Crowley JJ,
Lucia MS, Goodman PJ, Minasian LM, Ford LG, Parnes
HL, Gaziano JM, et al. Vitamin E and the risk of prostate
cancer: the Selenium and Vitamin E Cancer Prevention
Trial (SELECT). JAMA 2011; 306(14): 1549-1556. 9. Lobo V, Patil A, Phatak A, Chandra N. Free radicals,
antioxidants and functional foods: Impact on human
health. Pharmacogn Rev 2010; 4(8): 118-126. 17. Rowsell HC. The Canadian Council on Animal Care--its
guidelines and policy directives: the veterinarian's
responsibility. Can J Vet Res 1991; 55(3): 205. 10. Kurutas EB. The importance of antioxidants which play
the role in cellular response against oxidative/nitrosative
stress: current state. Nutr J 2016; 15(1): 71. 18. Tavafi M, Ahmadvand H. Effect of rosmarinic acid on
inhibition of gentamicin induced nephrotoxicity in rats. Tissue Cell 2011; 43(6): 392-397. 11. Gonca S, Ceylan S, Yardimoglu M, Dalcik H, Yumbul Z,
Kokturk S, Filiz S. Protective effects of vitamin E and
selenium on the renal morphology in rats fed high-
cholesterol diets. Pathobiology 2000; 68(6): 258-263. 19. Kim SY, Moon A. Drug-induced nephrotoxicity and its
biomarkers. Biomol Ther (Seoul) 2012; 20(3): 268-272. 20. Abdel-Raheem IT, El-Sherbiny GA, Taye A. Green tea
ameliorates
renal
oxidative
damage
induced
by
gentamicin in rats. Pak J Pharm Sci 2010; 23(1): 21-28. 12. Monteiro DA, Rantin FT, Kalinin AL. The effects of
selenium on oxidative stress biomarkers
in the
freshwater characid fish matrinxa, Brycon cephalus
(Gunther,
1869)
exposed
to
organophosphate
insecticide Folisuper 600 BR (methyl parathion). Comp
Biochem Physiol C Toxicol Pharmacol 2009; 149(1): 40-
49. 21. Yaman I, Balikci E. Protective effects of nigella sativa
against gentamicin-induced nephrotoxicity in rats. Exp
Toxicol Pathol 2010; 62(2): 183-190. 22. Khan SA, Priyamvada S, Farooq N, Khan S, Khan MW,
Yusufi AN. Protective effect of green tea extract on
gentamicin-induced
nephrotoxicity
and
oxidative
damage in rat kidney. Pharmacol Res 2009; 59(4): 254-
262. 13. Karabulut-Bulan O, Bolkent S, Yanardag R, Bilgin-
Sokmen B. The role of vitamin C, vitamin E, and
selenium on cadmium-induced renal toxicity of rats. Trop J Pharm Res, July 2019; 18(7): 1442 Contribution of authors Drug Chem Toxicol 2008; 31(4): 413-426. 23. Kadkhodaee M, Khastar H, Faghihi M, Ghaznavi R,
Zahmatkesh M. Effects of co-supplementation of
vitamins E and C on gentamicin-induced nephrotoxicity
in rat. Exp Physiol 2005; 90(4): 571-576. 14. Aboul-Soud MA, Al-Othman AM, El-Desoky GE, Al-
Othman ZA, Yusuf K, Ahmad J, Al-Khedhairy AA. Hepatoprotective effects of vitamin E/selenium against
malathion-induced injuries on the antioxidant status and
apoptosis-related gene expression in rats. J Toxicol Sci
2011; 36(3): 285-296. 24. Lippman SM, Klein EA, Goodman PJ, Lucia MS,
Thompson IM, Ford LG, Parnes HL, Minasian LM,
Gaziano JM, Hartline JA, et al. Effect of selenium and
vitamin E on risk of prostate cancer and other cancers:
the Selenium and Vitamin E Cancer Prevention Trial
(SELECT). JAMA 2009; 301(1): 39-51. 15. Ramsammy LS, Josepovitz C, Ling KY, Lane BP,
Kaloyanides GJ. Failure of inhibition of lipid peroxidation
by vitamin E to protect against gentamicin nephrotoxicity
in the rat. Biochem Pharmacol 1987; 36(13): 2125-2132. Trop J Pharm Res, July 2019; 18(7): 1442
|
https://openalex.org/W3198020003
|
https://link.springer.com/content/pdf/10.1007/s11678-021-00657-6.pdf
|
English
| null |
Joint-preserving surgical treatment options for irreparable posterosuperior rotator cuff tear
|
Obere Extremität
| 2,021
|
cc-by
| 5,431
|
Keywords Keywords
Biomechanical phenomena · Arthroplasty, replacement, shoulder · Tendon transfer · Shoulder
prosthesis · Tenodesis fered, particularly in the joint-preserving
scenarios. Partial repair, superior capsular reconstruction, latissimus
dorsi, lower trapezius, or a balloon? Florian Grubhofer1
· Jon JP Warner2
1University Hospital Balgrist, University of Zurich, Zurich, Switzerland
2Harvard Shoulder Service, Massachusetts General Hospital, Harvard Medical School, Boston, USA Abstract Treatment of irreparable rotator cufftears in young active patients is challenging. A variety of therapeutic options are available. Only a few joint-preserving treatment
options show reliable improvements over a long-term follow-up period. However,
the treatment outcomes of joint preservation procedures are not comparable to
those of RTSA, as patients are typically younger and have higher expectations. It is
remarkable that most of the joint-preserving therapeutic options for irreparable rotator
cuffruptures lack long-term treatment results. This article highlights the indications,
technical aspects, and treatment outcomes of the most commonly performed joint-
preserving surgeries for irreparable rotator cuffrupture. Review Article Obere Extremität 2021 · 16:247–254
https://doi.org/10.1007/s11678-021-00657-6
Received: 3 May 2021
Accepted: 26 May 2021
Published online: 3 September 2021
© The Author(s) 2021 Obere Extremität 2021 · 16:247–254
https://doi.org/10.1007/s11678-021-00657-6
Received: 3 May 2021
Accepted: 26 May 2021
Published online: 3 September 2021
© The Author(s) 2021 Keywords
Biomechanical phenomena · Arthroplasty, replacement, shoulder · Tendon transfer · Shoulder
prosthesis · Tenodesis Introduction The joint-preserving surgical treatment
methods discussed in this article include: The authors strongly believe that the suc-
cess of treatment of irreparable postero-
superior rotator cufftear (IPRCT) depends
primarily on the indication for the respec-
tive treatment, and secondarily on the cor-
rect technical execution of the operation
per se. – arthroscopicpartialrepair(APR)+ biceps
tenodesis or tenotomy – superior capsular reconstruction (SCR) – tendon transfer (lower trapezius and
latissimus dorsi) – subacromial balloon implantation Different joint-preserving treatment
options exist, but an established thera-
peutic algorithm does not yet exist even
though shoulder societies like the Ameri-
can Shoulder and Elbow Surgeons (ASES)
are actively striving for it. For example,
the Neer Circle (elected association of
110 ASES members) tried to establish
a treatment algorithm based on expert
opinion using a Delphi process. It was
found that there was consensus for only
afewnon-joint-preservingscenarios—the
majority of the experts’ approaches dif- The treatment decision-making process
depends, from the authors’ point of view,
on the three following factors: JonJPWarneristheseniorauthor. 1. biological age 2. chief complaint (pain, weakness,
stiffness) 3. rotator cufftear (RCT) pattern accord-
ing to the classification of Collin et al. [7]; . Fig. 1 In this review, joint-preserving surgical
treatmentoptions arehighlighted interms Obere Extremität 4 · 2021 247 Review Article Supraspinatus
Infraspinatus
Superior
subscapularis
Inferior
subscapularis
Type A
Type B
Type C
Type E
Type D
a
b
Fig. 1 9 Descriptive classi-
fication of irreparable pos-
terosuperiorrotatorcuff
tears according to Collin
et al. [7]. (a)Schematic lat-
eral view on humerus with
anteriorSubscapularis ten-
don, superiorSupraspina-
tus tendon,posteriorIn-
fraspinatus andTeres mi-
nortendon, (b)Schematic
illustration of irreparable
tendon tears (illustratedin
red) Supraspinatus
Infraspinatus
Superior
subscapularis
Inferior
subscapularis
a Type B Fig. 1 9 Descriptive classi-
fication of irreparable pos-
terosuperiorrotatorcuff
tears according to Collin
et al. [7]. (a)Schematic lat-
eral view on humerus with
anteriorSubscapularis ten-
don, superiorSupraspina-
tus tendon,posteriorIn-
fraspinatus andTeres mi-
nortendon, (b)Schematic
illustration of irreparable
tendon tears (illustratedin
red) Fig. 1 9 Descriptive classi-
fication of irreparable pos-
terosuperiorrotatorcuff
tears according to Collin
et al. [7]. (a)Schematic lat-
eral view on humerus with
anteriorSubscapularis ten-
don, superiorSupraspina-
tus tendon,posteriorIn-
fraspinatus andTeres mi-
nortendon, (b)Schematic
illustration of irreparable
tendon tears (illustratedin
red) Superior
subscapularis
Inferior
subscapularis Type A Type B b a of biomechanics, surgical technique, and
treatment outcomes. In addition, a treat-
ment algorithm used in the authors’ clinics
is presented (. Fig. 2). Biomechanical considerations and
surgical technique Results The clinical results of APR in short- and
mid-term follow-up show significantly im-
proved shoulder function and patient sat-
isfaction [8, 20, 27, 33]. Interestingly, the
5-year outcomes of patients treated with
APR for irreparable massive rotator cuff
tear are comparable to the outcomes of
patients who underwent anatomic arthro-
scopic reconstructionof reparablemassive
RCTs [21]. The integrity of the teres minor
was found to be an important predictive
factor [1, 29]. Radiologically, re-rupture of
the partial repair was described in 49%
[23]. No clinical outcome differences were
seen between patients after APR versus
arthroscopically assisted latissimus dorsi
tendon transfer (LDT) during a short-term
follow-up period [1]. A randomized trial
demonstrated no significant difference in
clinical treatment outcomes 2 years after
APR vs. SCR in 41 patients [19]. The goal of arthroscopic partial repair is to
restore the force couple consisting of the
remaining subscapularis and infraspinatus
tendons [5]. Complete anatomic recon-
struction of the rotator cuffis no longer
possible because of the advanced retrac-
tion. The upper edge of the infraspinatus
tendon is reattached as far as possible to
the original insertion site. The biceps ten-
don is either tenodesized or tenodized. It is known and has been published by
Boileau et al. that treatment of the bi-
ceps alone significantly improves shoul-
der function and satisfaction in patients
with massive irreparable RCTs [3]. In pa-
tients with irreparable posterosuperior ro-
tator cufftears (IPRCT) and concomitant
shoulder stiffness, the treatment results
of tendon transfer are poor; accordingly,
from the authors’ point of view, shoul-
der stiffness is a relative contraindication
for tendon transfers or SCR. In young
patients with a painful and at the same
time stiffened shoulder, APR can be con-
sidered as a treatment option and should
be combined with arthroscopic capsular
release. Postoperative mobilization be-
gins passively from the first postopera-
tive day, with actively assisted range-of-
motion exercises from the 4th–6th post-
operative week. Gentle strengthening to
a maximum of 5kg begins between the
10th and 12th postoperative weeks. Full
weightbearing is allowed from the 16th
postoperative week. Results Arthroscopicpartialrepair+ biceps
tenodesis/tenotomy Indication according to the
treatment algorithm – Biological age: young patients with
low demands – Chief complaint: pain, pain combined
with stiffness – Pattern of irreparable RCT: A, C, D, E Strengths and weaknesses – + All-arthroscopic procedure, promis-
ing mid-term outcomes – – Weakness for external rotation
or flexion is better addressed with
a tendon transfer. Long-term data
lacking Superior capsular reconstruction Indication according to the
algorithm Surgical technique The procedure is performed arthroscop-
ically in a beach chair position. At the
authors’ institutions, a 3-mm dermal al-
lograft is used. After thorough subacro-
mial debridement and arthroscopic biceps
tenotomyortenodesis, partialrepairofthe
remaining rotator cuffis performed if pos-
sible. Thesuperior glenoid is debrided and
exposed to fix the allograft with two to
threeanchorsmediallybetweenthe10and
12 o’clockpositions. Fixation on the lateral
aspectofthegreatertuberosityisachieved
withadouble-rowsuturebridgetechnique
with the arm held at 45° of abduction. Indication according to the
algorithm Indication according to the
algorithm – Biological age: young patients with
low demands. – Chief complaint: pain – Pattern of irreparable RCT: A, isolated
irreparable supraspinatus tear – Pattern of irreparable RCT: A, isolated
irreparable supraspinatus tear Strengths and weaknesses Strengths and weaknesses – + All-arthroscopic procedure (if allo-
graft is used). – – Allo-/or autograft (fascia lata) neces-
sary, long-term outcome data pending, 248
Obere Extremität 4 · 2021 Diagnosis
Tear patternª
Surgery
Biological
age
Chief
Complaint
A, C, D
LDT
B
LTT
SCR
or
Ballon
Anterior LDT combined
with posterior LTT
Anterior LDT combined
with APR
APR
APR
with capsular release
RTSA
A, B, C, D, E
A, C, D, E
C, D, E
B
A,
isolated supraspinatus
E,
isolated irreparable
infra- and/or teres
minor tear
Stiffness
Pain
Weakness
Pain and dysfunction
Old and inactive
patient
Young and active
patient
Irreparable
posterosuperior
rotator cuff
tear
Fig. 2 8 Flowchart of the authors’ preferredtreatment algorithm of irreparableposterosuperiorrotatorcuff.LDT latissimus
dorsitendontransfer,LTTlowertrapeziustransfer,SCRsuprascapularreconstruction,APRarthroscopicpartialrepair,RTSAre-
verse total shoulderarthroplasty. aaccording to Collin Classification forirreparable rotatorcufftears Surgery Biological
age Pain Fig. 2 8 Flowchart of the authors’ preferredtreatment algorithm of irreparableposterosuperiorrotatorcuff.LDT latissimus
dorsitendontransfer,LTTlowertrapeziustransfer,SCRsuprascapularreconstruction,APRarthroscopicpartialrepair,RTSAre-
verse total shoulderarthroplasty. aaccording to Collin Classification forirreparable rotatorcufftears Postoperatively, patients are immobilized
in a 45-degree abduction pillow for a total
of 6 weeks, starting with passive range of
motion between 4 and 6 weeks and active
assistive range of motion between 6 and
8 weeks. Light weightbearing is allowed
after 10 to 12 weeks. Unlimited weight
bearing is allowed 16 weeks postopera-
tively. implant costs (allograft and multiple
anchors). an increase in their tendon tension and
thereby restoring the force couple [26]. – Risk factors for poor outcome: ir-
reparable posterior rotator cuffrupture
(infraspinatus and/or especially with
involvement of teres minor), surgeon’s
experience (<10). – Risk factors for poor outcome: ir-
reparable posterior rotator cuffrupture
(infraspinatus and/or especially with
involvement of teres minor), surgeon’s
experience (<10). Biomechanics According to SCR inventor T. Mihata, im-
plantation of an auto- or allograft mem-
brane that replaces the superior capsule
andtheirreparablerotatorcuffleadstoim-
proved vertical stability and thus reduces
subacromial impingement [25]. Further-
more, the implanted membrane acts as
an anchor for the remaining subscapu-
laris and infraspinatus tendons, leading to Surgical technique The technique is described very clearly
in the publication by Wagner et al. [14]. Here, the authors wish to point out the
mostimportantsteps and possiblesources
of error, which are crucial for the outcome
of the operation in their opinion. – – Auto- or allograft necessary, long-
term outcome data pending, par-
tially open procedure, implant cost
(allograft). Positioning. The procedure can be per-
formed in a beach chair or lateral decubi-
tus position. The positioning is crucial for
this surgery. Positioning and subsequent
draping must allow for good exposure of
the medial scapular border. A common
mistake is that the scapula is insufficiently
exposed. – Risk factors for poor outcome: dia-
betes mellitus →stiffness, deltoid
dysfunction, non-compliance. – Risk factors for poor outcome: dia-
betes mellitus →stiffness, deltoid
dysfunction, non-compliance. Indication according to the
algorithm Indication according to the
algorithm Indication according to the
algorithm – Biological age: young patients with
high demands Subsequently, this technique was also
used in patients with irreparable rotator
cuffrupture. Biomechanically, the force
vector of the inferior trapezius portion
was found to be similar to that of the in-
fraspinatus and teres minor muscles. An-
other advantage of the lower trapezius
muscle is its synergistic action with the
infraspinatus and teres minor, as the mus-
cle contracts during glenohumeral exter-
nal rotation. A disadvantage of the lower
trapezius muscle is the reduced excursion
of the muscle as well as the short tendon – Chief complaint: weakness; weakness
for external rotation> weakness for
flexion – Pattern of irreparable RCT: A, C, D, E or
isolated irreparable infraspinatus and
teres minor tear. Preparation of the graft channel. Ten-
sion- and kinking-free sliding for the auto/
allograft is mandatory. Often the muscle
fascia of the infraspinatus muscle causes
mechanical restrictions for the graft, and it
isthereforeimportantthatitbesufficiently
released. Results It should be
emphasized that numerous level III and IV
studies exist that show good to excellent
short- and mid-term results, even in pa-
tients with preoperative pseudoparalysis
[4, 6, 24]. Results Clinical outcomes from the authors’ clinic
(Massachusetts General Hospital) were
published in the Journal of Shoulder and
Elbow Surgery in 2019 [34]. The results
were exceedingly modest. In total, SCR
surgery was performed in 65 patients by
6 different fellowship-trained shoulder Obere Extremität 4 · 2021 249 Review Article length, which makes direct fixation to the
greater tuberosity impossible. Therefore,
interpositional tenodesis with an allograft
or autograft is necessary. surgeons within the period from Jan-
uary 2015 to November 2017; however,
31 patients had to be excluded as they
were not available for 12-month follow-
up. Of the 34 patients included, 65%
were dissatisfied with the treatment out-
come at a mean follow-up of 12 months. The 1- and 2-year failure-free survival
rates were 34 and 16%, respectively. The
1- and 2-year “free of reoperation rate”
was 64 and 44%, respectively. There was
no significant improvement in shoulder
function or pain with SCR surgery. Risk
factors for a poor treatment outcome
were the extent of fatty infiltration in
the infraspinatus muscles and the num-
ber of SCR operations performed by the
operating surgeon (<10). It should be
emphasized that numerous level III and IV
studies exist that show good to excellent
short- and mid-term results, even in pa-
tients with preoperative pseudoparalysis
[4, 6, 24]. latissimus dorsi transfer—especially
in obese patients, force vector of the
lower trapezius similar to posterior cuff
muscles, primarily synergistic muscle
(lower trapezius muscle contracts with
external rotation). Potential treatment
option in patients with an additional
irreparable subscapularis lesion (with
combined anterior latissimus dorsi
transfer; Elhassan B.; in submission
process, not yet published). surgeons within the period from Jan-
uary 2015 to November 2017; however,
31 patients had to be excluded as they
were not available for 12-month follow-
up. Of the 34 patients included, 65%
were dissatisfied with the treatment out-
come at a mean follow-up of 12 months. The 1- and 2-year failure-free survival
rates were 34 and 16%, respectively. The
1- and 2-year “free of reoperation rate”
was 64 and 44%, respectively. There was
no significant improvement in shoulder
function or pain with SCR surgery. Risk
factors for a poor treatment outcome
were the extent of fatty infiltration in
the infraspinatus muscles and the num-
ber of SCR operations performed by the
operating surgeon (<10). 250
Obere Extremität 4 · 2021 Biomechanics LTT was originally developed to treat pa-
tients with brachial plexus palsy [2, 11]. These patients suffer in particular from
a lack of external rotation due to fail-
ure of the external rotator muscles (in-
fraspinatus and teres minor). In addi-
tion, cervical nerve root 5 is frequently
involved, which causes dysfunction of the
deltoid muscle. Prior to development of
LTT, these patients with deltoid dysfunc-
tion were treated with LDT. A common
complication in these patients was pos-
terior (sub)dislocation, which led to the
search for an alternative tendon transfer
to the LDT [2, 11]. In this regard, LTT was
developed by Elhassan to address these
patients with less complications [13]. It
wasshownbiomechanicallythatLTT trans-
fer, leaving the latissimus dorsi tendon in-
tact, did not present posterior instability
problems in these patients with deltoid
dysfunction [12]. Nevertheless, the poor treatment re-
sults at the authors’ institution (Mas-
sachusetts General Hospital) have led to
a drastic reduction of the indication for
SCR, which is now only considered in
patients with strictly isolated supraspina-
tus tendon tears or type A lesions. The
majority of patients—especially those
with advanced fatty degeneration of the
posterosuperior rotator cuffmuscles (in-
fraspinatus and teres minor)—are treated
according to the treatment algorithm with
tendon transfer. Skinincision. Originally, an L-shaped skin
incision was described. Nowadays, an
oblique skin incision which starts medi-
ally 1cm medial to the scapular rim and
1cm inferior to the spina scapulae and
is guided 4–5cm laterally, parallel to the
course of the scapula spine, is preferred. A common source of error here is that
the skin incision is placed too far laterally. This can lead to difficult exposure of the
inferior trapezius muscle–tendon unit. Lower trapezius exposure. Removal of
the fat tissue pad that covers the fascia
of the lower trapezius muscle and ten-
donmakes exposureof thelower trapezius
easier. At the lateral margin of the lower
trapezius, the layer between the trapez-
ius tendon and infraspinatus fascia can be
developed by blunt dissection with the
finger. If this layer can be developed, har-
vest of the lower trapezius tendon is easy
to perform. Lower trapezius transfer Biomechanics Harvest. The latissimus tendon is wide
and long, and the teres major tendon is
short. The layer between the latissimus
tendon and muscle belly must be clearly
separated before tendon release. Tendon
release can be performed either openly
via the axilla or arthroscopically. In the study by Elhassan et al., a signifi-
cant improvement in function, pain, and
subjective shoulder score was observed in
32 of 33 patients (mean age 53 years) after
a mean follow-up duration of 47 months
[13]. The better the active and passive mo-
bilitypreoperatively, themorelikelyagood
treatment outcome can be assumed. Ac-
cordingly, from the authors’ point of view,
shoulder stiffness is a (relative) contraindi-
cation for tendon transfers. Another study
by Elhassan demonstrated significant im-
provement in shoulder function and pa-
tient satisfaction 12 months postopera-
tively in 90% of a total of 41 patients
[14]. True pseudoparalysis was present
in 46% (19 patients) preoperatively. In
18 of the 19 pseudoparalytic patients, the
pseudoparalysis resolved and a significant
improvement in shoulder function and pa-
tient satisfaction was achieved. The pioneering description of LDT for
chronic posterosuperior RCTs was pre-
sented by Gerber in 1988 [17]. The latissimus dorsi acts as a power-
ful internal rotator, adductor, and extensor
of the humerus in the glenohumeral joint. The force vector is significantly more verti-
cal than the lower trapezius portion due to
its natural course. However, the latissimus
dorsi muscle exhibits such good excursion
that the tendon can be inserted directly
at the superolateral facet of the greater
tuberosity. The transfer restores the force
couple of the rotator cuff, allowing cen-
tering of the humeral head during deltoid
activation. Muscular release. The latissimus muscle
has a very high excursion if the muscu-
lar release is performed adequately. The
muscle belly should be released posteri-
orly and far distally. Anteriorly, attention
must be paid to the nerve pedicle, which
enters the muscle anteriorly about 14cm
distal to the tendinous attachment. Tendon shuttle. The layer between the
deltoid muscle and the posterior cuffmus-
cles (infraspinatus and teres minor) should
be dissected wide open, both from in-
traarticularly and via the open approach
posteriorly to allow the tendon transfer to
glide freely. Strengths and weaknesses Strengths and weaknesses – + Addresses posterior cuffdeficiency
of infraspinatus AND teres minor,
technically less demanding than 250
Obere Extremität 4 · 2021 latissimus muscle, no graft needed, low
implant costs (2–3 anchors). com/share/a5eb4a01-32d6-4aac-a300-
3b099041e440/. latissimus muscle, no graft needed, low
implant costs (2–3 anchors). Arm position during graft–trapezius
tendon tenodesis. During tenodesis the
arm is placed in combined maximal ex-
ternal rotation and 60–90° of abduction. After tenodesis and postoperatively, it is
crucial that the arm be kept in maximum
external rotation and immobilized in an
external rotation brace. The tension cre-
ated on the back during internal rotation
of the arm is very high and could endan-
ger the tenodesis. Accordingly, it is also
very important to assume a high level of
brace-wearing compliance. com/share/a5eb4a01-32d6-4aac-a300-
3b099041e440/. implant costs (2 3 anchors). – – Technically challenging, treatment
outcome dependent on the integrity
of the teres minor and subscapularis
muscle, partially open procedure,
latissimus function out of phase
(internal rotator, vertical force vector). – Risk factors for poor outcome: shoulder
stiffness, irreparable subscapularis tear,
teres minor atrophy, pseudoparalysis,
high critical shoulder angle, previous
rotator cuffsurgeries. – – Technically challenging, treatment
outcome dependent on the integrity
of the teres minor and subscapularis
muscle, partially open procedure,
latissimus function out of phase
(internal rotator, vertical force vector). Herein, the authors focus on the pearls
and pitfalls of the technique: Positioning. In beach chair positioning,
an arm holder can be attached ipsilater-
ally. With maximum flexion and internal
rotation of the arm, the posterior axial
fold, which corresponds to the latissimus
dorsi muscle and tendon, is stretched to
the maximum. In this position, the skin
incision and harvest of the tendon from
the humerus should be performed. – Risk factors for poor outcome: shoulder
stiffness, irreparable subscapularis tear,
teres minor atrophy, pseudoparalysis,
high critical shoulder angle, previous
rotator cuffsurgeries. Surgical technique The procedure was first described as an
open procedure in a lateral decubitus po-
sition [15]. Nowadays, the majority of the
procedureisarthroscopicallyassisted, with
the patient in the beach chair position
and the tendon being harvested openly
through a skin incision along the posterior
axillaryfold[18]. Theall-arthroscopictech-
nique has also been described, although
it has not yet become widely performed
[9]. The advantages of the arthroscopi-
cally assisted approach are the ability to
supply the subscapularis tendon, perform
a partial repair of the posterosuperior ro-
tator cufftendons if possible, sparing of
the deltoid muscle, and less scarring. Indication according to the
algorithm Indication according to the
algorithm Fixation of the tendon. The anterome-
dial and anterolateral tagging sutures are
fixed anteriorly at the superolateral aspect
of the greater tuberosity via knotless an-
chors. The fixation is facilitated if the re-
spective sutures are shuttled out through
a separate anteromedial and anterolateral
portal. A third anchor might be needed at
theposterioraspectorthegreatertuberos-
ity to avoid windshield whipper effect of
the tendon. – Biological age: young patients with
high demands – Chief complaint: weakness; weakness
for flexion> weakness for external
rotation – Pattern of irreparable RCT: A, C, D Biomechanics A major strength of LDT as a treatment
option is that it is the only therapeutic op-
tion for the treatment of IPRCT for which
long-term results are available [10, 16]. In
the study by Gerber et al., with a mean
follow-up of 12 years (minimum follow-up
of 10 years), it was shown that shoulder
function(Constantscore%pre-vs. postop-
erative: 56vs80%)andpatientsatisfaction
(subjective shoulder value from preopera-
tive29topostoperative70%)of44patients
with 46 operated shoulders were sustain-
ably improved over this long period [16]. Mid-term results of the newer arthroscop-
ically assisted technique are also available
and do not differ significantly from the
open technique [32]. Long-term failure
rates range from 10% [10] to 14% [32]
and 30% [16]. The balloon spacer is made of biodegrad-
able copolymer (poly-lactide and ε-capro-
lactone) that is completely broken down
by the body within 1 year. The injected
physiological saline solution starts to de-
flate already after 3 months. The main
biomechanical effect propagated is the
avoidance or even revision of superior de-
centration of the humeral head [28]. Strengths and weaknesses There
are
several
descriptions
of
arthroscopically assisted LDT. A par-
ticularly illustrative presentation of the
single surgical steps can be viewed
on VuMedi. link: https://www.vumedi. – + Long-term data available showing
sustained improvement even after
12 years. Great excursion of the Postoperative rehabilitation. Patient
compliance is important and should be
assessed preoperatively. Postoperative
immobilization in a 30-degree abduction Obere Extremität 4 · 2021 251 Review Article pad for 6–8 weeks. Passive and active
assistive exercise therapy after 8 weeks,
gentle strengthening after 12 weeks,
and transition to full weightbearing after
16 weeks. the FDA and, therefore, the authors
have not treated any patients with
a subacromial balloon in Boston to
date; the experience at the Balgrist
is also very limited (<10 cases), with
mixed results. beit inconsistently, with implantation time
alone lasting an average of 10min (range
2–30min). A comparison study demonstrated no
statistically significant differences in pain
and clinical outcomes 1 year postopera-
tively between APR alone (n= 16) and APR
and balloon spacer (n= 16) patients [22]. Subacromial balloon Corresponding address
Florian Grubhofer, MD
University Hospital Balgrist, Universityof Zurich
Forchstrasse 340, 8008 Zurich, Switzerland
florian.grubhofer@balgrist.ch Indication according to the
algorithm Indication according to the
algorithm Practical conclusion – The majority of the authors’ younger
patients with irreparable posterosupe-
rior tears of the rotator cuffare treated
with tendon transfers in combination
with partial repair. – Onlyinveryfewselectedcases(isolated
irreparable superior rupture with pain
as the chief complaint) is SCR indicated. Another potential indication is protec-
tion of an arthroscopic rotator cuffrecon-
struction using the balloon spacer on top
of the repair to reduce the head in the
glenohumeral joint during the rotator cuff
reconstruction healing period [31]. – The subacromial balloon spacer has
just been approved in the USA, and in
Switzerland the insurance companies
are also unwilling to cover the costs. – The subacromial balloon spacer has
just been approved in the USA, and in
Switzerland the insurance companies
are also unwilling to cover the costs. Thus, there is not sufficient experience
to be able to make a statement about
its use. Thus, there is not sufficient experience
to be able to make a statement about
its use. Results – Finally, the evidence of the individual
therapy options is mostly based on
level IV and a few level III studies, with
short- to mid-term follow ups—with
the exception of the LTD (10 years
follow up). Level I and level II studies
with long-term follow-up are necessary
to provide clarity on the optimal
treatment strategy. – Finally, the evidence of the individual
therapy options is mostly based on
level IV and a few level III studies, with
short- to mid-term follow ups—with
the exception of the LTD (10 years
follow up). Level I and level II studies
with long-term follow-up are necessary
to provide clarity on the optimal
treatment strategy. The evidence of treatment outcomes is
weak, with only mostly level IV and few
level III studies showing good short- to
mid-term results but no superiority com-
pared to patients treated with APR [22]. In the only level I study by Verma N et al.,
no significant differences in clinical out-
comes and patient satisfaction were seen
betweentheballoonspacer group (n= 93)
and the partial repair group (n= 91). This
study was presented during the ASES an-
nual meeting in October 2020. The study
serves as an FDA approval study. There-
fore, the study patients were allowed to be
treated with a subacromial balloon spacer
in the USA. The approval process is still
pending, and it remains unclear whether
the balloon spacer will be used in the US. Overtheyears, thefollowingriskfactors
for poor outcome have been identified:
fatty atrophy of the teres minor muscle,
preoperative pseudoparalysis, excessively
high critical shoulder angle, irreparable
subscapularis tear, shoulder stiffness, and
previous rotator cuffprocedures. Indication according to the
algorithm Indication according to the
algorithm 252
Obere Extremität 4 · 2021 Florian Grubhofer, MD – Biological age: young patients with
low demands – Chief complaint: pain – Pattern of irreparable RCT: A, isolated
irreparable supraspinatus tear Funding. Open access funding provided by Univer-
sity of Zurich Funding. Open access funding provided by Univer-
sity of Zurich In a systematic review by Stewart RK
et al., a total of 10 level IV and 2 level III
studies withameanfollow-up of 23 (range
12–52) months were investigated [30]. It
was shown that the Constant score was
improved between 19 and 50 points by
Stewart et al. [30]. The complication rate
of 2.1% was relatively low and included
superficial and deep wound infection and
balloon spacer dislocation. In 3 of the
12 studies, surgical time was collected, al- Strengths and weaknesses References 1. Baverel LP, Bonnevialle N, Joudet T et al (2021)
Short-termoutcomesofarthroscopicpartialrepair
vs. latissimusdorsitendontransferinpatientswith
massive and partially repairable rotator cufftears. JShoulderElbowSurg30:282–289 1. Baverel LP, Bonnevialle N, Joudet T et al (2021)
Short-termoutcomesofarthroscopicpartialrepair
vs. latissimusdorsitendontransferinpatientswith
massive and partially repairable rotator cufftears. JShoulderElbowSurg30:282–289 12. Elhassan B, Bishop AT, Hartzler RU et al (2012)
Tendon transfer options about the shoulder in
patients with brachial plexus injury. J Bone Joint
SurgAm94:1391–1398 with massive rotator cufftears: a case-control
study. Musculoskelet Surg. https://doi.org/10. 1007/s12306-020-00649-9 2. Bertelli JA (2009) Lengthening of subscapularis
andtransferofthelowertrapeziusinthecorrection
ofrecurrentinternalrotationcontracturefollowing
obstetricbrachialplexuspalsy. JBoneJointSurgBr
91:943–948 23. Malahias MA, Kostretzis L, Chronopoulos E et al
(2019) Arthroscopic partial repair for massive
rotator cufftears: Does it work? A systematic
review.SportsMedOpen5:13 13. Elhassan BT, Wagner ER, Werthel JD (2016)
Outcomeoflowertrapeziustransfertoreconstruct
massiveirreparableposterior-superiorrotatorcuff
tear.JShoulderElbowSurg25:1346–1353 3. Boileau P, Baque F, Valerio L et al (2007) Isolated
arthroscopic biceps tenotomy or tenodesis
improves symptoms in patients with massive
irreparablerotatorcufftears. JBoneJointSurgAm
89:747–757 24. Mihata T, Lee TQ, Hasegawa A et al (2020)
Arthroscopic superior capsule reconstruction for
irreparablerotatorcufftears:comparisonofclinical
outcomeswithandwithoutsubscapularistear.Am
JSportsMed48:3429–3438 14. Elhassan BT, Sanchez-Sotelo J, Wagner ER (2020)
Outcomeofarthroscopicallyassistedlowertrapez-
ius transfer to reconstruct massive irreparable
posterior-superior rotator cufftears. J Shoulder
ElbowSurg29(10):2135–2142 4. Burkhart SS, Hartzler RU (2019) Superior capsular
reconstructionreversesprofoundpseudoparalysis
in patients with irreparable rotator cufftears and
minimalornoglenohumeralarthritis.Arthroscopy
35:22–28 25. Mihata T, McGarry MH, Pirolo JM et al (2012)
Superior capsule reconstruction to restore su-
perior stability in irreparable rotator cufftears:
abiomechanicalcadavericstudy. AmJSportsMed
40:2248–2255 15. Gerber C (1992) Latissimus dorsi transfer for the
treatment of irreparable tears of the rotator cuff. ClinOrthopRelatRes275:152–160 5. Burkhart SS, Nottage WM, Ogilvie-Harris DJ et al
(1994)Partialrepairofirreparablerotatorcufftears. Arthroscopy10:363–370 16. Gerber C, Rahm SA, Catanzaro S et al (2013)
Latissimus dorsi tendon transfer for treatment
of irreparable posterosuperior rotator cufftears:
long-term results at a minimum follow-up of ten
years.JBoneJointSurgAm95:1920–1926 26. Nimura A, Kato A, Yamaguchi K et al (2012)
The superior capsule of the shoulder joint
complements the insertion of the rotator cuff. JShoulderElbowSurg21:867–872 6. Burkhart SS, Pranckun JJ, Hartzler RU (2020) Su-
perior capsular reconstruction for the operatively
irreparable rotator cufftear: clinical outcomes are
maintained 2 years after surgery. Arthroscopy
36:373–380 17. Gerber C, Vinh TS, Hertel R et al (1988) Latissimus
dorsitransferforthetreatmentofmassivetearsof
the rotator cuff. A preliminary report. Clin Orthop
RelatRes232:51–61 27. PorcelliniG,CastagnaA,CesariEetal(2011)Partial
repair of irreparable supraspinatus tendon tears:
clinicalandradiographicevaluationsatlong-term
follow-up.JShoulderElbowSurg20:1170–1177 7. Collin P, Matsumura N, Ladermann A et al (2014)
Relationshipbetweenmassivechronicrotatorcuff
tear pattern and loss of active shoulder range of
motion.JShoulderElbowSurg23:1195–1202 18. Schlüsselwörter Schlüsselwörter
Biomechanische Phänomene · Schulterendoprothetik · Sehnentransfer · Schulterprothese ·
Tenodese Gelenkerhaltende chirurgische Therapieoptionen der
posterosuperioren Rotatorenmanschettenruptur. Teilrekonstruktion,
SCR, Latissimus Dorsi oder Inferiorer Trapezius Transfer oder Ballon? Open Access. ThisarticleislicensedunderaCreative
CommonsAttribution4.0InternationalLicense,which
permitsuse,sharing,adaptation,distributionandre-
productioninanymediumorformat,aslongasyou
giveappropriatecredittotheoriginalauthor(s)and
thesource,providealinktotheCreativeCommonsli-
cence,andindicateifchangesweremade. Theimages
orotherthirdpartymaterialinthisarticleareincluded
inthearticle’sCreativeCommonslicence,unlessin-
dicatedotherwiseinacreditlinetothematerial. If
materialisnotincludedinthearticle’sCreativeCom-
monslicenceandyourintendeduseisnotpermitted
bystatutoryregulationorexceedsthepermitteduse,
you willneedtoobtainpermissiondirectlyfromthe
copyrightholder. Toviewacopyofthislicence,visit
http://creativecommons.org/licenses/by/4.0/. Die Behandlung der irreparablen posterosuperioren Rotatorenmanschettenruptur
bei jungen, aktiven Patienten stellt eine Herausforderung dar. Es bestehen
verschiedene Therapieoptionen. Nur vereinzelte gelenkserhaltende Therapieoptionen
zeigen über einen langen Nachkontrollzeitraum verlässliche Verbesserungen der
Schulterfunktion und Patientenzufriedenheit. Die Behandlungsergebnisse der
gelenkserhaltenden Operationen sind jedoch nicht mit jenen der RTSA vergleichbar,
da die Patienten typischerweise jünger sind und höhere Ansprüche haben. Bemerkenswert ist, dass zu den meisten gelenkerhaltenden Eingriffen bei irreparablen
Rotatorenmanschettenrupturen Langzeittherapieergebnisse fehlen. In dieser
Übersichtsarbeit werden die am häufigsten vorgenommenen gelenkerhaltenden
Eingriffe hinsichtlich Indikationen, technischer Aspekte und Behandlungsresultaten
beleuchtet. Declarations – + Short operation time, easy to im-
plement surgically, all-arthroscopic
procedure. Conflict of interest. J.J.P.Warnerdeclaresthefol-
lowing: Stryker: Royaltyandconsultingonshoulder
implantsproductsforjointreplacement;Orthospace:
StockforcompanywhichproducestheInspaceBallon
(noconsultingorroyalty;thiscompanyhasbeensold
toStryker). F.Grubhoferdeclaresthathehasnoconflict
ofinterest. – – NotUS Food and DrugAdministration
(FDA) approved, no long-term data
available, implant costs. – The subacromial balloon spacer has
just yet been approved (June 2021) by 252
Obere Extremität 4 · 2021 Zusammenfassung Forthisarticlenostudieswithhumanparticipants
oranimalswereperformedbyanyoftheauthors. Allstudiescitedwereinaccordancewiththeethical
standardsindicatedineachcase. Forthisarticlenostudieswithhumanparticipants
oranimalswereperformedbyanyoftheauthors. Allstudiescitedwereinaccordancewiththeethical
standardsindicatedineachcase. Gelenkerhaltende chirurgische Therapieoptionen der
posterosuperioren Rotatorenmanschettenruptur. Teilrekonstruktion,
SCR, Latissimus Dorsi oder Inferiorer Trapezius Transfer oder Ballon? 254
Obere Extremität 4 · 2021 References Gervasi E, Causero A, Parodi PC et al (2007)
Arthroscopiclatissimusdorsitransfer.Arthroscopy
23(11):1243.e1–1243.e4 28. Savarese E, Romeo R (2012) New solution for
massive, irreparable rotator cufftears:
the
subacromial “biodegradable spacer”. Arthrosc
Tech1:e69–e74 19. GreinerS,KaeaebM,VossAetal(2021)Comparison
of superior capsular reconstruction and partial
infraspinatus repair: a matched-pair analysis of
irreparablerotatorcufftears. OrthopJSportsMed
9:2325967120984264 8. CuffDJ, Pupello DR, Santoni BG (2016) Partial
rotator cuffrepair and biceps tenotomy for the
treatment of patients with massive cufftears and
retained overhead elevation: midterm outcomes
with a minimum 5 years of follow-up. J Shoulder
ElbowSurg25:1803–1809 29. Shon MS, Koh KH, Lim TK et al (2015) Arthroscopic
partial repair of irreparable rotator cufftears:
preoperative factors associated with outcome
deterioration over 2 years. Am J Sports Med
43:1965–1975 20. Iagulli ND, Field LD, Hobgood ER et al (2012) Com-
parison of partial versus complete arthroscopic
repair of massive rotator cufftears. Am J Sports
Med40:1022–1026 30. Stewart RK, Kaplin L, Parada SA et al (2019)
Outcomes of subacromial balloon spacer implan-
tation for massive and irreparable rotator cuff
tears: a systematic review. Orthop J Sports Med
7:2325967119875717 9. Cutbush K, Peter NA, Hirpara K (2016) All-
arthroscopic latissimus dorsi transfer. Arthrosc
Tech5:e607–e613 21. JeongJY,KimSJ,YoonTHetal(2020)Arthroscopic
repair of large and massive rotator cufftears:
completerepairwithaggressivereleasecompared
withpartialrepairaloneataminimumfollow-upof
5years.JBoneJointSurgAm102:1248–1254 10. El-Azab HM, Rott O, Irlenbusch U (2015) Long-
term follow-up after latissimus dorsi transfer for
irreparable posterosuperior rotator cufftears. JBoneJointSurgAm97:462–469 31. Szollosy G, Rosso C, Fogerty S et al (2014)
Subacromial spacer placement for protection of
rotatorcuffrepair.ArthroscTech3:e605–e609 11. Elhassan B, Bishop A, Shin A et al (2010)
Shouldertendontransferoptionsforadultpatients
with brachial plexus injury. J Hand Surg Am
35:1211–1219 22. Malahias MA, Brilakis E, Avramidis G et al (2020)
Arthroscopic partial repair with versus without
biodegradable subacromial spacer for patients 32. Waltenspul M, Jochum B, Filli L et al (2021) Mid-
termresultsofarthroscopically-assistedlatissimus
dorsi transfer for irreparable posterosuperior Obere Extremität 4 · 2021 253 Review Article rotator cufftears. J Shoulder Elbow Surg. https://
doi.org/10.1016/j.jse.2021.03.149 rotator cufftears. J Shoulder Elbow Surg. https://
doi.org/10.1016/j.jse.2021.03.149 j j
33. Wellmann M, Lichtenberg S, Da Silva G et al
(2013) Results of arthroscopic partial repair of
large retracted rotator cufftears. Arthroscopy
29:1275–1282 33. Wellmann M, Lichtenberg S, Da Silva G et al
(2013) Results of arthroscopic partial repair of
large retracted rotator cufftears. Arthroscopy
29:1275–1282 34. Woodmass JM, Wagner ER, Borque KA et al (2019)
Superior capsule reconstruction using dermal
allograft: early outcomes and survival. 34. Woodmass JM, Wagner ER, Borque KA et al (2019)
Superior capsule reconstruction using dermal
allograft: early outcomes and survival. J Shoulder
ElbowSurg28:S100–S109 33. Wellmann M, Lichtenberg S, Da Silva G et al
(2013) Results of arthroscopic partial repair of
large retracted rotator cufftears. Arthroscopy
29:1275–1282 References J Shoulder
ElbowSurg28:S100–S109 254
Obere Extremität 4 · 2021 254
Obere Extremität 4 · 2021
|
https://openalex.org/W2995446947
|
https://hal.science/hal-01837517/document
|
English
| null |
Designing an argumentative decision-aiding tool for urban planning. AIPA : an interface between multicriteria decision aiding and argumentative frameworks
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,017
|
cc-by
| 7,568
|
To cite this version: Benjamin Delhomme, Franck Taillandier, Irène Abi-Zeid, Rallou Thomopoulos, Cédric Baudrit, et
al.. Designing an argumentative decision-aiding tool for urban planning. AIPA : an interface be-
tween multicriteria decision aiding and argumentative frameworks. Colloque OPDE 2017, Institut
National de Recherche Agronomique (INRA). UMR Innovation et Développement dans l’Agriculture
et l’Alimentation (0951)., Oct 2017, Montpellier, France. hal-01837517 Designing an argumentative decision-aiding tool for
urban planning. AIPA : an interface between
multicriteria decision aiding and argumentative
frameworks Benjamin Delhomme, Franck Taillandier, Irène Abi-Zeid, Rallou
Thomopoulos, Cédric Baudrit, Laurent Mora Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01837517 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 DESIGNING AN ARGUMENTATIVE DECISION-AIDING TOOL FOR URBAN
PLANNING
AIPA: AN INTERFACE BETWEEN MULTICRITERIA DECISION AIDING AND ARGUMENTATIVE FRAMEWORKS
Benjamin Delhomme (1), Franck Taillandier (1), Irène Abi-Zeid (2),
Rallou Thomopoulos (3), Cédric Baudrit (1), Laurent Mora (1)
(1) Univ. Bordeaux, I2M, UMR 5295, F-33400 Talence, France
(2) Department of Operations and Decision Systems, Université Laval, Québec, Canada
(3) INRA IATE Joint Research Unit / INRIA GraphIK, F-34060 Montpellier, France
Abstract:
Urban planning is an important issue for all cities. In order to meet the challenges of sustainable
urban planning, we propose a participative decision-support tool that allows stakeholders to engage
jointly in structuring a decision process, to identify alternatives, to construct criteria, to challenge
their relevance and to evaluate them. The novelty in our approach is the use of an argumentative
approach to support multicriteria decision aiding. The use of an argumentative framework allows
the stakeholders to formalize the decision problem by taking explicitly into account the diverse
opinions expressed and ensuring their traceability. Through the argumentative approach, our goal
is thus to enhance participatory decision making by organizing and formalizing debates between
stakeholders. To this effect, we propose AIPA, an interface the makes the transition between natural
language and abstract argumentation systems. Our aim is to place debates at the center of the
decision analysis process in order to facilitate the acceptance of the final decision by all parties. Keywords:
Decision-aiding, urban planning, argumentative approach, participative decision, multicriteria
decision analysis 1. INTRODUCTION Urban planning is an important issue for all cities. The question is how to develop or redevelop a
neighborhood, i.e. design buildings, green spaces and transportation infrastructures such as roads and
tramway tracks. Urban planning projects must take advantage of the current state and invent the city
of tomorrow while meeting the challenges of sustainable development. The many dimensions of
sustainable development include social (creating a social link, ensuring a good living environment for
the inhabitants, fulfilling spiritual needs, etc.), economic (limiting investment costs for the
municipality, limiting costs for residents, limiting maintenance costs, etc.), environmental (limiting
energy consumption, limiting impacts on wildlife, etc.), cultural (taking into account local
particularities), spatial (paying attention to the territorial distribution of people), and ethical (pursuing
fairness and equity principles). These different dimensions necessarily lead to contradictory objectives
that must be reconciled in a decision process. Therefore, designing an urban planning project from a
sustainable perspective while considering short, medium and long term impacts, is a quite complex
endeavor. Furthermore, sustainable development implies other dimensions, such as governance and
participation: sustainable development can only be achieved in consultation with the various
stakeholders involved in decision-making, ideally leading to a wide consensus; a development project
that generates strong opposition among local residents for example, could not be considered
sustainable. Consequently, the various stakeholders involved such as the mayor, city council, urban
planners, inhabitants, engineers, who may have different points of view and different objectives, must
and can be included in the process (Joerin et al., 2009; Rey-Valette et al., 2007). Many qualitative public participation approaches, based on dialogue to guide the actors to a final
decision in public decision-making, have been used for urban planning (O'Faircheallaigh, 2010; Evans
& Kotchetkova, 2009). These participatory approaches are interesting for many reasons: first, they
involve a wide range of actors in the decision process; second, they make it possible to reach a decision
closer to the values and concerns of each actor; and third they favor the final decision’s acceptance by
the actors, due to the transparency of the process. However, one of the main limitations reported on
these approaches concerns their lower level of formalization and reproducibility (Hutchel & Molet,
1986). Colloque OPDE 2017 Montpellier
« Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation have homogeneous preferences and are considered as a single decision-maker. Nonetheless, many
multicriteria methods can be, and have been used in a multi-actor context, such as MACBETH (Bana E
Costa, 2001; Marleau Donais et al., 2017) or ELECTRE (Oberti, 2004) for example, where a facilitator
conducts decision conferencing with several stakeholders. However, the focus is often not on the
discussion process and on the arguments presented but rather on the construction of a common
preference model through consensus. Consequently, it is difficult to explicitly integrate within the
mathematical frameworks of these methods, the richness of the discussions between the actors as
well as the problem’s complexity (Ministère de l'Ecologie, du Développement durable, des Transports
et du Logement: Paris, 2004). Moreover, the main challenge in using MCDA is undoubtedly the
structuring phase since decision problems are often quite difficult to deal with, and alternatives and
criteria are rarely readily available (Marttunen et al., 2017; Belton & Stewart, 2010; Franco and
Montibeller, 2010). Therefore, structuring and formalizing a decision problem to fit into a MCDA
framework can be difficult, notably in a multi-stakeholder context where the following pertinent
questions are justified at the end of the process: ● Are the criteria retained relevant for all the stakeholders? ● Are the alternatives constructed relevant for all the stakeholders? ● What does the common preference model represent? ● Where are the traces of the discussions pertaining to the previous questions? In addition, the final recommendation obtained by a MCDA method could be also be subject to
discussion: Is it really acceptable for all the stakeholders? Why was such a decision reached by the
MCDA method? Does it really reflect the decision makers preferences? To address these questions,
some argumentations technique for the explanation of MCDA results have been applied at a more
theoretical level (Amgoud et al., 2005; Labreuche, 2011). In addition, the final recommendation obtained by a MCDA method could be also be subject to
discussion: Is it really acceptable for all the stakeholders? Why was such a decision reached by the
MCDA method? Does it really reflect the decision makers preferences? To address these questions,
some argumentations technique for the explanation of MCDA results have been applied at a more
theoretical level (Amgoud et al., 2005; Labreuche, 2011). 1. INTRODUCTION The lack of formalization limits the traceability of the decision; someone who wishes to
understand the decision could, at best, refer to a report or a transcription of the discussions which is
time consuming and probably not sufficient to understand the final decision. Moreover, this kind of
approach does not generally provide information such as the global project ranking and the evaluation
table that could help understand the rationale behind the choices made (e.g. values considered,
project requirements, information available, consequences, etc.). To go beyond these limitations, the scientific literature contains numerous formal decision support
methods that have been used for urban planning, whether they are mono-criterion based such as
benefit/cost analysis or multicriteria based. MultiCriteria Decision Aiding (MCDA) has been around for
the last 50 years, resulting in a large number of methods and applications (Greco et al., 2016). However, these methods often do not advocate a participative approach and assume that stakeholders 2 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation Our main interest in developing AIPA is to assist the stakeholders in the structuring phase of the
decision problem and in the final discussion about the solution proposed by MCDA. This approach is
based on the dynamic modeling of arguments and on the explicit formalization of the opinions and
preferences of the actors in order to conduct a transparent decision process and to arrive to a jointly
constructed and acceptable decision. It is in line with the philosophy of computer-supported
collaborative decision making (Scheuer et al., 2010; Karacapilidis & Papadias, 2001). By helping to capture arguments during a discussion in natural language and to translate them into
formal arguments, AIPA is meant as a first step towards filling this gap. The formal arguments are then
used in an argumentation framework implemented in AIPA, where semantics are used to infer a subset
of collectively acceptable arguments, called extensions. Although applicable to any domain, the
intended application of this project is urban planning. The novelty of our approach resides in the use of an argumentative framework to take explicitly into
account the diverse opinions expressed. Our goal is to enhance participatory decision support in
general, and in urban planning in particular, through semi-automated argumentative and semantic
approaches that help in organizing and formalizing discussions between stakeholders. The proposed
tool is meant as an interface between natural language arguments and abstract argumentation
frameworks, in which a collectively acceptable a set of arguments is identified. This combination of
decision aiding and argumentative methods is in line with (Dix et al., 2009) and (Simari et al., 2011)
who raise the pertinent questions of: “What does an argumentation-theoretic approach add over and
above decision theory? How can one integrate argumentation tools with classical decision theory and
other existing models of decision making?”. Possible answers to these questions were proposed in
Ouerdane et al. (2010) as research avenues. The rest of this paper is organized as follows: in Section 2 we present AIPA and background concepts
and definitions relating to argumentation frameworks. In Section 3, we describe how AIPA can support
a MCDA process and illustrate its use using an example. We conclude in section 4 with future
perspectives. « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Notwithstanding the approach that is used, there are, to our knowledge, no tools available to help
organize the outcomes of discussions, in real time, in a way to ensure traceability, encourage more
discussion, and provide the rationale behind the decisions made in a multiple criteria setting. In order
to remedy this situation, particularly during the structuring phase of a MCDA process, we propose AIPA
(Argumentation Interface for Participative Approach), a decision support tool for multi-actor, multi-
criteria decision projects that allows us to formalize the natural language arguments exchanged, in
order to challenge their relevance and to evaluate them in an argumentation framework. Projects combining argumentation, MCDA and computational social choice have been successfully
used in other application domains, especially concerning the sustainability of agriculture, food and
environment systems, research (Bisquert et al., 2017; Thomopoulos et al., 2015). However, there are
still no tools to date that have succeeded in combining decision-aiding technique with participatory
argumentative approaches, making them operationally usable for urban planning. 3 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation An argumentation framework is an oriented graph where nodes are arguments and edges are attack
relations between arguments. In Dung’s framework, an argument and the attack relation are abstract
and can be differently instantiated and defined in different contexts (Walton, 2009). Quoting Dung
(1995): “an argument is an abstract entity whose role is solely determined by its relations to other
arguments. No special attention is paid to the internal structure of the arguments”. An argument can
for example be a set of statements composed of a conclusion and at least one premise, linked by an
inference or a logical relation (Breton & Gauthier, 2000). Attacking an argument could be achieved by
raising doubts about its acceptability through critical questions, by questioning its premises, or by
putting forward that the premises are not relevant to the conclusion. Another way to attack an
argument is by presenting an argument with an opposing conclusion. In this last case, when arguments
support different conclusions, an attack relation is be said to exist. One application of a value-based
argumentation framework to a real-life problem can be found in (Tremblay & Abi-Zeid, 2016) where a
recommendation regarding the development of a hydropower plant was analyzed a posteriori. In that
case, an argument was defined as a statement consisting of a premise and a conclusion, where a
conclusion was one of three alternatives: accept the project, refuse the project, or accept the project
with modifications. Although theoretically relevant, the application of Dung’s framework to real decisions is not
straightforward. One of the main challenges resides in the definition of an argument. As discussed
earlier, there is no general model to formalize a natural argument (i.e. an argument stated by a
stakeholder during a discussion in its primal form) in order to be used in an abstract argumentation
framework in a real decision-making context. In the words of Baroni & Giacomin (2009), « While the
word argument may recall several intuitive meanings, like the ones of “line of reasoning leading from
some premise to a conclusion” or of “utterance in a dispute”, abstract argument systems are not (even
implicitly or indirectly) bound to any of them: an abstract argument is not assumed to have any specific
structure but, roughly speaking, an argument is anything that may attack or be attacked by another
argument. 2. ARGUMENTATION IN AIPA In order to evaluate the collective acceptance of arguments and reach a decision, argumentation
frameworks introduced by Dung in his seminal paper (Dung, 1995) and the different frameworks
derived from it, such as Value-based AF (Bench-Capon, 2003), (Dunne et al., 2011), and (Amgoud &
Ben-Naim, 2013) may be used. Argumentation frameworks are abstract argumentation models of
argumentative discourse, used to confront conflicting arguments in order to arrive at a conclusion. Arguments are intuitively understood to be statements to support, contradict, or explain other
statements that could be decisions or opinions (Amgoud & Prade, 2009). In a decision-making context
of choice, an argument can support or reject a given option (Amgoud, 2009). An argument’s conclusion
can for example be that a given urban development project is rejected. 4 Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » » Furthermore, the notion of “attack between arguments” does not have a direct and
consistent connection with natural language. In addition, the formalization of argument as an oriented
graph can be difficult to understand by stakeholders. Indeed, the structure of the abstract argument
and the relation between them do not correspond to the intuitive understanding of what an argument
is. Furthermore, when the number of arguments and/or attacks is large, the graph becomes illegible
and difficult to interpret by stakeholders. In order to address the deficiencies described above, we developed AIPA as an interface between
natural language arguments made by several stakeholders during discussions and an argumentation
framework (Figure 1). AIPA is designed to: (a) consider different stakeholders, (b) consider
contradictory objectives/criteria, (c) be usable by any argumentation framework, (d) favor discussion,
(e) give immediate results to be used in the discussion (e.g., a list of criteria), (f) be easily understood
by non-experts in mathematics and computer science. It gives a concrete meaning to an abstract 5 5 Colloque OPDE 2017 Montpellier An argument a1 is acceptable with respect to a set S of arguments if and only if for all
arguments that attack a1, those arguments are attacked by at least an argument in S. Example 3. (Example 2 - continued) Let S denote the set containing only a3, a1 is acceptable with regard
to S since S attacks a2. S is then said to defend a1. Definition 3. A set of arguments S is admissible if and only if S is conflict-free and each argument in S is
acceptable with respect to S. The empty set (without arguments) is always admissible. Definition 3. A set of arguments S is admissible if and only if S is conflict-free and each argument in S is
acceptable with respect to S. The empty set (without arguments) is always admissible. Example 4. (Example 3 - continued) Let S denote the set containing a1 and a3, S is admissible since a1
and a3 are acceptable with regard to S. Example 4. (Example 3 - continued) Let S denote the set containing a1 and a3, S is admissible since a1
and a3 are acceptable with regard to S. A set of collectively acceptable arguments, according to a given semantic is called an extensions. Dung
defined different types of semantics that yield extensions (“preferred”, “stable”, “complete”, and
“grounded”) according to the properties expected from these extensions, i.e. those that should be
considered to make a decision. Definition 4. A set S is a preferred extension of an argumentation framework if and only if S is admissible
and maximal; a set S is maximal if S is not a subset of some other set. Definition 4. A set S is a preferred extension of an argumentation framework if and only if S is admissible
and maximal; a set S is maximal if S is not a subset of some other set. Example 5. (Example 4 - continued) The set 1 is admissible and it is not a subset of another set; it is
therefore a preferred extension. Example 5. (Example 4 - continued) The set 1 is admissible and it is not a subset of another set; it is
therefore a preferred extension. Definition 5. A set S of arguments is a stable extension if and only if S is conflict-free and S attacks every
argument that does not belong to it. Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » argument by automatically translating natural language argument them into arguments usable by an
argumentation framework. The aim is to allow stakeholders as end-users to argue in real-time with
simple tools without having to learn argumentation frameworks. Figure 1: Positioning of AIPA
2.1. Argumentation Frameworks – Definitions
We present below some definitions and example to help understand how a set of collectively
acceptable arguments can be reached based on Dung’s formalism. E
l 1 L
AF b
i
f
k (Fi
2)
i i
h
1
2
d Figure 1: Positioning of AIPA Figure 1: Positioning of AIPA Figure 1: Positioning of AIPA 2.1. Argumentation Frameworks – Definitions
We present below some definitions and example to help understand how a set of collectively
acceptable arguments can be reached based on Dung’s formalism. 2.1. Argumentation Frameworks – Definitions 2.1. Argumentation Frameworks – Definitions
We present below some definitions and example to help understand how a set of collectively
acceptable arguments can be reached based on Dung’s formalism. We present below some definitions and example to help understand how a set of collectively
acceptable arguments can be reached based on Dung’s formalism. Example 1. Let AF be an argumentation framework (Figure 2) containing three arguments: a1, a2 and
a3 with the following attack relation:
● a1 attacks a2
● a2 attacks a1
● a3 attacks a2
Figure 2: Attack graph Example 1. Let AF be an argumentation framework (Figure 2) containing three arguments: a1, a2 and
a3 with the following attack relation: Example 1. Let AF be an argumentation framework (Figure 2) containing three arguments: a1, a2 and
a3 with the following attack relation: Example 1. Let AF be an argumentation framework (Figure 2) containing three arguments: a1, a2 and
a3 with the following attack relation: a3 with the following attack relation:
● a1 attacks a2
● a2 attacks a1
● a3 attacks a2 Figure 2: Attack graph Figure 2: Attack graph 6 6 Colloque OPDE 2017 Montpellier Example 2 (Example 1 - continued). Since a1 and a3 that do not attack each other, the set S consisting
of a1 and a3 is conflict-free. Definition 2. « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Example 8. (Example 7 - continued) Since there is only one complete extension, the set S composed by
a1 and a3 is the grounded extension. Based on these extensions, Dung defines two inference rules in order to infer acceptable arguments:
credulous and skeptical. In credulous inferences, an argument is accepted if it belongs to at least one
preferred extension. In skeptical inferences, an argument is accepted if it belongs to the grounded
extension (all preferred extensions). A dispute is said to be resoluble when the preferred extension is
unique, since there is only one set of arguments capable of acceptance (Bench-Capon, 2003). Colloque OPDE 2017 Montpellier A stable extension is a preferred extension, the reciprocal is false. Example 6. The set S1 composed by a1 and a2 does not attack the argument a3; therefore, it is not a
stable extension. However, the set S composed by a1 and a3 is a stable extension. Definition 6. A set S is a complete extension if and only if S is admissible and each acceptable argument
with respect to S belongs to S. Example 7. The set S composed by a1 and a3 is an admissible set, and since every argument acceptable
with respect to S belongs to S, S is a complete extension. Definition 7. A set S is a grounded extension if it is the smallest complete extension (the one with the
minimum number of arguments). A grounded extension is unique and may be equal to the empty set. Definition 7. A set S is a grounded extension if it is the smallest complete extension (the one with the
minimum number of arguments). A grounded extension is unique and may be equal to the empty set. Definition 7. A set S is a grounded extension if it is the smallest complete extension (the one with the
minimum number of arguments). A grounded extension is unique and may be equal to the empty set. 7 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier 2.2. AIPA’s Implementation p
In AIPA, an argument is a concept representing a proposition (assertion) that can be either a Conclusion
or a Statement (Figure 3). A conclusion is a particular proposition pertaining to a given decision. For
instance, a conclusion could be ”The project A should be selected”, or “The value of project A on
criterion j is equal to Very High”. A statement is a proposition providing a justification why another
argument is supported or not. Because a statement concept is still too broad, we formed two sub-
concepts: StatementFor and StatementAgainst. For example, the StatementFor “The project A
encourages the use of public transportation” supports the conclusion ”The project A should be
selected” and the StatementAgainst “Project B is particularly deficient regarding waste management”
attacks the conclusion ”The project B should be selected”. A statement can be understood as a premise
for a conclusion. Furthermore, a conclusion called Cneg is always created: it is the complementary of
all the other conclusions. For instance, if two conclusions are “Select the project A” and “Select the
project B”, the conclusion Cneg corresponds to “Select neither A, neither B”. When a Statement that is a StatementAgainst has an about relation with a conclusion or another
statement, this relation is translated into an attack relation. A StatementFor with an about relation
with a given conclusion X, will have an attack relation with all other conclusions, that are mutually
exclusive with conclusion X. Two conclusions that are mutually exclusive will attack each other
respectively. Otherwise, they will have no attack relation in the resulting argument graph. 8 Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Figure 3: An argument model Figure 3: An argument model Figure 3: An argument model AIPA currently implements Dung’s framework and the skeptical inference is used to obtain a solution:
A solution is a subset of collectively acceptable arguments corresponding to the grounded extension. If no solution is found, meaning that the grounded extension is the empty set, the stakeholders have
then two possibilities: (a) add new argument and/or (b) add/refine/modify a conclusion. « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » to A in Dung’s framework. If a StatementFor S2 is made about a conclusion such as B, it will be
converted into an attack from S2 to every conclusion that shares the same topic (in this case date,
therefore attack of C and Cneg), except of the course for the conclusion that it supports (in this case
B). If B is deemed acceptable instead of C, then the two conclusions A and B will be deemed acceptable
(consensus) conclusions for the stakeholders involved. An argument is deemed acceptable if it belongs
to the grounded extension (solution). 2.2. AIPA’s Implementation Consider a case with three conclusions A, B and C: Consider a case with three conclusions A, B and C: ● A – The cost of this project should be a criterion ● B – The project should start in January ● C – The project should start in February In this example, B and C are in conflict regarding a date, hence B will attack C, C will attack B and both
of them will be attacked by and will attack a new Cneg conclusion “The project should not start in
January AND the project should not start in February”. On the other hand, the conclusion A does not
compete with other conclusion except its own negation “The cost of this project shouldn’t be a
criterion”, thus no attack will appear between A and the set {B,C}. As for the statements, if a
StatementAgainst S1 is made about a conclusion such as A, it will be translated into an attack from S1 9 Colloque OPDE 2017 Montpellier 3.1. MCDA Structuration process p
Discussions to help structure a multiple criteria decision problem consist mainly of three phases
assuming that the different projects (alternatives) have already been defined and cannot be modified
(Figure 5). In the first phase, the stakeholders construct the list of criteria. They can: (a) propose a new
conclusion or refine an existing one (b) propose a statement for or against a conclusion. For example,
for (a), if a stakeholder wished to add a new criterion “Cultural”, he adds a new conclusion “Use
Cultural criterion”. An example of a new statement (b) is: “Cultural is part of Social, it should not appear
as a new criterion but as part of the already existing Social criterion”. Each time, a stakeholder proposes
a new argument (statement or conclusion), AIPA provides the acceptable arguments (part of the
grounded extension). The proposed list of criteria is constantly updated and presented to the
stakeholders. We assume that project data regarding the different chosen criteria are available; for
instance, if the project cost is considered as a criterion, it is possible to define the cost of each project. Figure 5: A decision structuring model Figure 5: A decision structuring model When all the stakeholders are in agreement regarding the list of criteria or if no one wants to propose
new arguments, phase 2 can begin: it focuses on the criteria parameters (e.g. weight, thresholds, etc.). Globally, in this phase, the stakeholders have to propose conclusions enabling to compare criteria; for
instance: “Social should be privileged compared to economic criterion”. As in the previous phase, they
can add new conclusion as a refinement of old ones or add statements. Based on the criteria
preference and assuming that the evaluations of the alternatives on these criteria are possible, the
projects can be then ranked by a MCDA method. In the last phase, the stakeholders may directly use
the results of the MCDA method or add new statements (e.g. “The building of project A looks better
than those of the project B which is ranked first). The final word is always given to the different
stakeholders and must be the subject of a real discussion. The process is iterative. During the
discussion, any actor can propose new arguments which may impact the acceptable solution (e.g. list
of criteria, project ranking, etc.). 3. STRUCTURING A MCDA DECISION PROBLEM WITH AIPA One of the objectives in developing AIPA is to provide support to the structuring phase of a MCDA
process. Below, we illustrate with a simple example where AIPA can fit in the decision process and how
it can support the stakeholders involved. Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » 2.3 AIPA’s Interface The AIPA implementation allows the user to add an argument, to use an AF to make inferences in order
to define the acceptable arguments and to present the result to the user. A given user puts forward an
argument and specifies whether it is “for” or “against” another argument. Other users have the
possibility to invalidate this argument by proposing a new StatementAgainst against it in the form of
“This argument is not valid”. The results presented to users is the solution consisting of the lists of
acceptable arguments (grounded extension) and non-acceptable arguments (outside the grounded
extension) in the form of a table and an argumentation graph. These operations are almost
instantaneous, a necessary condition for use in real time by multiple users. AIPA is a web application written in Java. This allows concurrent use with a multi-platform support. Moreover, the computing process is done on the server side and there are many javascript graphic
libraries available that offer different views for the arguments. An example of the current
implementation is given in Figure 4. It is a first prototype currently at a very basic level and will be
improved in the future. Figure 4: Implementation of AIPA Figure 4: Implementation of AIPA 10 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » the traceability of the reasoning that lead to a preferred project; any participant can, at any moment,
read the arguments (close to natural language) and understand the choice of a conclusion. The two
first phases are also iterative. If the comparison of the criteria in phase 2 reveals the need for a new
criterion, it is always possible to return to phase 1. In practice, the different phases correspond to meetings between stakeholders. Each phase does not
necessarily correspond to one meeting; the number and duration of these meetings depend on the
type of project, the number of stakeholders, etc. 3.1. MCDA Structuration process The aim of this approach is to place the discussion at the center of
the process in order to facilitate the acceptance of the final decision by all parties. In addition, it keeps 11 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » 3.2. An application example In order to illustrate how AIPA can be used, we propose a simple example. Throughout this example,
we assume a discussion about an urban planning project pertaining to a parcel at Pessac near the tram
track with different stakeholders: the mayor (Mayor), the technical services managers (Tech), the
directors of a citizen association (Citizen) and a person responsible of a nature protection agency
(Nature). Five different development projects (projects A, B, C, D and E) are being considered. The first discussion is about the criteria that should be used, and any person involved in the process
can propose a conclusion. As Mayor puts forward the conclusion “The cost should be a criterion”,
Nature submits environmental and infrastructure aspects, respectively “surface of green area” and
“urban density” as criteria. This primary exchange gives the following list of conclusions: ● Mayor: C1 - “The cost should be a criterion.” ● Mayor: C1 - “The cost should be a criterion.” ● Nature: C2 - “The surface of green areas should be a criterion.” ● Nature: C3 - “The urban density should be a criterion.” According to these three conclusions, three Cneg conclusions are automatically generated: ● AIPA: C1Neg - “The cost should not be a criterion.” ● AIPA: C2Neg - “The surface of green areas should not be a criterion.” ● AIPA: C3Neg - “The urban density should not be a criterion.” Then, three premises are introduced by: hen, three premises are introduced by: ● Mayor: S1 - “Since we only have 2 million € for this project.” StatementFor -> C1. ● Nature: S2 - “Green areas reduce the heat island effect.” StatementFor -> C2. ● Tech: S3 - “Higher density cities are more sustainable than low density cities.” StatementFor - >
C3. At this stage, the three criteria are valid candidates (i.e. C1, C2 and C3 are acceptable according to the
chosen inference) but Citizen is not in agreement with S3 and makes the statement: At this stage, the three criteria are valid candidates (i.e. C1, C2 and C3 are acceptable according to the
chosen inference) but Citizen is not in agreement with S3 and makes the statement: ● Citizen: S4 - “High density buildings are not sustainable because they degrade the landscape
and the quality of life” StatementAgainst -> S3. 12 Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Since no-one is able to counter-argue Citizen by attacking S4, C3 is discarded. The criteria to be retained
(solution) are: Cost (C1) and Nature (C2). This series of statements is shown on the left of Figure 6 and
its translation (by AIPA) into an argument graph in Dung’s framework is seen on the right of the figure. Figure 6: Criteria related graphs Figure 6: Criteria related graphs Following the criteria discussion is the criteria importance, to be used for example in an ordinal type
MCDA method, and another instance is then created with no a priori conclusions. Assume that
stakeholders provide the following conclusions: ● Tech: - C1 - “Cost is as important as Surface of green areas.” ● Mayor: C2 - “Cost is more important than Surface of green areas.” ● Nature: C3 - “Cost is less important than Surface of green areas.” In this context, only one conclusion can be acceptable since they are all about the same topic (criteria
preference) and are thus mutually exclusive: “Cost” and “Surface of green areas” comparison. The
discussion results in different arguments (not given here), which yields C2 as the valid conclusion; i.e. 13 Colloque OPDE 2017 Montpellier Colloque OPDE 2017 Montpellier Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Cost is more important than Surface of green areas, as shown in Figure 7, we represent “Cost” with
“++” and “Surface of green areas” with “+”. Figure 7: Evaluation table Based on the evaluation table of Figure 7, project B appears as the best candidate. For simplicty’s sake,
it is actually a dominant solution. However, given another evaluation table, it could have been ranked
first based on an aggregation method. A new AIPA instance is made with two conclusions: Based on the evaluation table of Figure 7, project B appears as the best candidate. For simplicty’s sake,
it is actually a dominant solution. However, given another evaluation table, it could have been ranked
first based on an aggregation method. Colloque OPDE 2017 Montpellier
« Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Colloque OPDE 2017 Montpellier
« Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » A new AIPA instance is made with two conclusions: ● AIPA: C1 - “Project B should be selected.” ● AIPA: C1 - “Project B should be selected.” ● AIPA: C1 - “Project B should be selected.” ● AIPA: CNeg - “Another project should be selected.” AIPA: CNeg - “Another project should be selected.” A statementFor is automatically generated to support C1: ● AIPA: S1: “The Project B is the best according to the evaluation and aggregation results.”
StatementFor -> C1 If no-one has new arguments, the decision process ends here. However, Tech thinks that project E
should be retained since the security is better, and this element was not explicitly used as a criterion. Hence a new conclusion C2 is added as a new statement: ● Tech: C2 - “Projet E is better.” ● Tech: C2 - “Projet E is better.” ● Tech: C2 - “Projet E is better.” ● Tech: S1 - “Project E ensures a better security.” StatementFor -> C2 At this point, stakeholders can still provide counterarguments in order to attack or defend one of the
projects or to question a previous statement. This follow-on discussion allows the participants to
express their feelings and opinions on the projects, notably when they are difficult to formalize in a
criterion (e.g. judgement on aesthetic). Discussions continue until a common agreement is found. If no
consensus can be found with the help of the argumentation framework, the decision-maker(s) would
have to resolve the dispute on the basis of other methods such as voting for example. This Phase 3
enhances the discussion during the decision process. Our basic assumption is that the final choice
belongs to the stakeholders and not to the MCDA tool which role is to act as a support for reflection
and discussion. Obviously, if all the stakeholders are in agreement with the MCDA results, then Phase 3 becomes unnecessary. 14 BIBLIOGRAPHY Amgoud L., 2009. Argumentation for Decision Making In Simari G., Rahwan I. (eds) Argumentation in Artificial
Intelligence. Springer, Boston, MA : 301-320. Amgoud L., Ben-Naim, J., 2013. Ranking-based semantics for argumentation frameworks. Lecture
Computer Science, Vol 8078, 134–147. Amgoud L., Bonnefon J.F., Prade H., 2005. An Argumentation-Based Approach to Multiple Criteria
ECSQARU, Vol. 3571, 269-280. Bana E Costa C.A., 2001. The use of multi-criteria decision analysis to support the search for less conflict
options in a multi-actor context: case study. Journal of Multi-Criteria Decision Analysis, Vol 10, 111-125 Bana E Costa C.A., 2001. The use of multi-criteria decision analysis to support the search for less conflicting policy
options in a multi-actor context: case study. Journal of Multi-Criteria Decision Analysis, Vol 10, 111-125. Baroni P
Giacomin M
2009
Semantics of Abstract Argument Systems In Simari G
Rahwan I
(eds) Baroni P., Giacomin M., 2009. Semantics of Abstract Argument Systems In Simari G., Rahwan
Argumentation in Artificial Intelligence. Springer, Boston, MA : 25-44. Belton V., Stewart T., 2010. Problem structuring and multiple criteria decision analysis In Trends in multiple
criteria decision analysis. Ehrgott, Matthias ; Figueira, José; Greco, Salvatore. Springer : 209-239 Bench-Capon T., 2003. Persuasion in Practical Argument Using Value-based Argumentation Frameworks. Journal
of Logic and Computation, Vol 13(3), 429-448. Bisquert P., Croitoru M., Dupin de Saint-Cyr F., Hecham A., 2017. Formalizing Cognitive Acceptance of Arguments:
Durum Wheat Selection Interdisciplinary Study. Minds Mach., Vol 27(1), 233-252. Dix J., Parsons S., Prakken H., Simari G., 2009. Research challenges for argumentation. Computer Science -
Research and Development, Vol 23(1), 27-34. Donais F.M., Abi-Zeid I., Lavoie R., 2017. Building a Shared Model for Multi-criteria Group Decision Making. Lecture Notes in Business Information Processing, Vol 293, 175-186. Dung P.M., (1995). On the acceptability of arguments and its fundamental role in nonmonotonic reasoning, logic
programming and n-person games. Artificial Intelligence, Vol 77(2), 321-357. Dunne P.E., Hunter A., McBurney P., Parsons S., Wooldridge M., 2011. Weighted argument systems: Basic
definitions, algorithms, and complexity results. Artificial Intelligence, Vol 175, 457–486. Evans R.,Kotchetkova I., 2009. Qualitative research and deliberative methods: promise or peril? Qualitative
Research, 2009. Vol 9(5), 625-643. Franco L.A., Montibeller G., 2010. Problem structuring for multicriteria decision analysis interventions. Wiley
Encyclopedia of Operations Research and Management Science. Hutchel A., Molet H., 1986. Rational modelling in understanding and aiding human decision-making: About two
case studies. European Journal of Operational Research, Vol 24(1), 178-186. Colloque OPDE 2017 Montpellier « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » 4. CONCLUSION In this paper, we proposed an innovative approach that allows to: (a) use argumentation in a
participative decision problem through a new model called AIPA and (b) couple argumentation with
multicriteria decision analysis in order to support the problem structuring phase. AIPA is a model
implemented in a computer tool that acts as an interface between natural language arguments and
abstract argumentation frameworks. One of the main advantages of AIPA is that it does not alter the
natural discussion structure. Another advantage is that it keeps a trace of the discussions in order to
help justify choices that have been made thereby maximizing transparency. Our goal is to apply AIPA
to support participative urban planning. Work is ongoing to develop a domain specific ontology to
facilitate the use of AIPA in urban planning projects. To our knowledge, this research project is quite novel and our approach is unique in urban planning. However, several points must to be improved and further developed: We are currently developing a
decision making ontology that can automatically check the consistency of arguments and transform
them into the underlying AIPA arguments model. Future work includes the implementation of the
transition from AIPA to MCDA. Furthermore, we plan to use our tool in the next year to support
participative city planning in Bordeaux Metropole. This real-life experience will be valuable and will
provide us with many avenues for improving our tool. The feedback will allow us to assess the practical
benefits and drawbacks of introducing formal argumentative approaches in multi-actor, multicriteria
decision making. 15 BIBLIOGRAPHY Joerin F., Desthieux G., Beuze S.B., Nembrini A., 2009. Participatory diagnosis in urban planning: Proposal for a
learning process based on geographical information. Journal of Environmental Management, Vol 90(6), 2002-
2011. Karacapilidis N., Papadias D., 2001. Computer supported argumentation and collaborative decision making: the
HERMES system. Information systems, Vol 26(4), 259-277. Labreuche C., 2011. A general framework for explaining the results of a multi-attribute preference model. Artificial Intelligence, Vol 175(7-8), 1410-1448. Marttunen M., Lienert J., Belton V., 2017. Structuring problems for Multi-Criteria Decision Analysis in practice: A
literature review of method combinations. European Journal of Operational Research, Vol. 263 (1), 1-17. Ministère de l'Ecologie, du Développement durable, des Transports et du Logement: Paris, (2004). Modèles (les),
obstacles et enjeux de la critique. ENPC, LATTS, Juin 2004, 95 p. 16 « Concevoir, adapter, évaluer des dispositifs pour faciliter et étendre la participation » Oberti P., 2004. Décision publique et recherche procédurale : illustration d’une démarche multicritère à la
localisation participative d’un parc éolien en région corse In Journées de l’Association Française de Science
Economique. Ouerdane W., Maudet N., Tsoukias A., 2010. Argumentation theory and decision aiding In Trends in multiple
criteria decision analysis. Ehrgott, Matthias ; Figueira, José; Greco, Salvatore. Springer : 177-208. O'Faircheallaigh C., 2010. Public participation and environmental impact assessment: Purposes, implications, and
lessons for public policy making. Environmental Impact Assessment Review, Vol 30(1), 19-27. O'Faircheallaigh C., 2010. Public participation and environmental impact assessment: Purposes, implications, and
lessons for public policy making. Environmental Impact Assessment Review, Vol 30(1), 19-27. Rey-Valette H., Damart S., Roussel S., 2007. A multicriteria participation-based methodology for selecting
sustainable development indicators: an incentive tool for concerted decision making beyond the diagnosis
framework. International Journal of Sustainable Development, Vol 10.1-2, 122-138. Scheuer O., Loll F., Pinkwart N., McLaren B.M., 2010. Computer-supported argumentation: A review of the state
of the art. International Journal of Computer-Supported Collaborative Learning, Vol 5(1), 43-102. Simari G.R., 2011. A Brief Overview of Research in Argumentation Systems In Scalable Uncertainty Management:
5th International Conference, SUM 2011, Dayton, OH, USA, October 10-13, 2011. Proceedings, S. Benferhat and
J. Grant, Editors. 2011, Springer Berlin Heidelberg: Berlin : 81-95. Thomopoulos R., Croitoru M., Tamani N., 2015. Decision support for agri-food chains: A reverse engineering
argumentation-based approach. Ecological Informatics, Vol 26, Part 2, 182-191. Tremblay J., Abi-Zeid I., 2016. Value-based argumentation for policy decision analysis: methodology and an
exploratory case study of a hydroelectric project in Québec. Annals of Operations Research, Vol 236(1), 233-253. 17 View publication stats
View publication stats
|
https://openalex.org/W2808898846
|
https://curis.ku.dk/ws/files/209352770/1_s2.0_S0370269318304982_main.pdf
|
English
| null |
Azimuthally-differential pion femtoscopy relative to the third harmonic event plane in Pb–Pb collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.gif" overflow="scroll"><mml:msqrt><mml:mrow><mml:msub><mml:mrow><mml:mi>s</mml:mi></mml:mrow><mml:mrow><mml:mi mathvariant="normal">NN</mml:mi></mml:mrow></mml:msub></mml:mrow></mml:msqrt><mml:mo>=</mml:mo><mml:mn>2.76</mml:mn><mml:mspace width="0.25em" /><mml:mtext>TeV</mml:mtext></mml:math>
|
Physics letters. B
| 2,018
|
cc-by
| 13,480
|
university of copenhagen university of copenhagen
Azimuthally-differential pion femtoscopy relative to the third harmonic event plane in
Pb-Pbcollisions at root(NN)-N-S=2.76TeV
Acharya, S.; Acosta, F.T.; Adamova, D.; Adolfsson, J; Aggarwal, MM.; Rinella, G.A.; Agnello,
Maria; Agrawal, N.; Ahammed, Z.; Ahn, S.U.; Aiola, S.; Akindinov, A.; Al-Turany, M.; Alam, S.;
Albuquerque, DSD; Aleksandrov, D.; Alessandro, B; Molina, Rafael A.; Ali, Yusuf; Alici, A.;
Alkin, A.; Alme, J.; Alt, T.; Bearden, Ian; Bilandzic, Ante; bsm989, bsm989; Gajdosova,
Katarina; Bourjau, Christian Alexander; Gaardhøje, Jens Jørgen; Nielsen, Børge Svane;
Thoresen, Freja; Zhou, You; Christensen, Christian Holm; Chojnacki, Marek; Ozelin De Lima
Pimentel, Lais
Published in: university of copenhagen Download date: 24. Oct. 2024 Citation for published version (APA):
Acharya, S., Acosta, F. T., Adamova, D., Adolfsson, J., Aggarwal, MM., Rinella, G. A., Agnello, M., Agrawal, N.,
Ahammed, Z., Ahn, S. U., Aiola, S., Akindinov, A., Al-Turany, M., Alam, S., Albuquerque, DSD., Aleksandrov, D.,
Alessandro, B., Molina, R. A., Ali, Y., ... Ozelin De Lima Pimentel, L. (2018). Azimuthally-differential pion
femtoscopy relative to the third harmonic event plane in Pb-Pbcollisions at root(NN)-N-S=2.76TeV. Physics
Letters B, 785, 320-331. https://doi.org/10.1016/j.physletb.2018.06.042 1. Introduction in particle emission [6], the particles emitted at a particular an-
gle relative to the flow plane carry information about the source
as seen from that corresponding direction; these correlations also
lead to the HBT radii to be sensitive to the collective velocity fields,
from which information about the dynamics of the system evolu-
tion can be extracted. Heavy-ion collisions at LHC energies create a hot and dense
medium known as the quark–gluon plasma (QGP) [1]. The QGP
fireball first expands, cools, and then freezes out into a collec-
tion of final-state hadrons. Correlations among the particles carry
information about the space–time extent of the emitting source,
and are imprinted on the final-state spectra due to a quantum-
mechanical interference effect [2]. Commonly known as intensity
or Hanbury–Brown–Twiss (HBT) interferometry, the correlation of
two identical particles at small relative momentum, is an effective
tool to study the space–time (“femtoscopic”) structure of the emit-
ting source in relativistic heavy-ion collisions [3]. The initial state
of a heavy-ion collision is characterized by spatial anisotropies
that lead to anisotropies in pressure gradients, and consequently
to azimuthal anisotropies in final particle distributions, commonly
called anisotropic flow. Anisotropic flow is usually characterized
by a Fourier decomposition of the particle azimuthal distribution
and quantified by the flow coefficients vn and the corresponding
symmetry plane angles n [4]. Elliptic flow is quantified by the
second flow harmonic coefficient v2, whereas triangular flow [5]
is quantified by v3. Due to the position–momentum correlations Azimuthally-differential femtoscopic measurements can be per-
formed relative to the direction of different harmonics event
planes [7,8]. The measurements of the HBT radii with respect
to the first harmonic event plane (directed flow) at the AGS [9]
revealed that the source was tilted relative to the beam direc-
tion [10]. The HBT radii variations relative to the second har-
monic event plane angle (2) provide information on the pion
source elliptic eccentricity at freeze-out. The recent ALICE mea-
surements [11] indicate that due to the strong in-plane expansion
the final-state source elliptic eccentricity is more than a factor
2–3 smaller compared to the initial-state. a r t i c l e
i n f o Article history:
Received 5 April 2018
Received in revised form 18 June 2018
Accepted 19 June 2018
Available online 22 June 2018
Editor: L. Rolandi Azimuthally-differential femtoscopic measurements, being sensitive to spatio-temporal characteristics of
the source as well as to the collective velocity fields at freeze out, provide very important information on
the nature and dynamics of the system evolution. While the HBT radii oscillations relative to the second
harmonic event plane measured recently reflect mostly the spatial geometry of the source, model studies
have shown that the HBT radii oscillations relative to the third harmonic event plane are predominantly
defined by the velocity fields. In this Letter, we present the first results on azimuthally-differential
pion femtoscopy relative to the third harmonic event plane as a function of the pion pair transverse
momentum kT for different collision centralities in Pb–Pb collisions at √sNN = 2.76 TeV. We find that the
Rside and Rout radii, which characterize the pion source size in the directions perpendicular and parallel
to the pion transverse momentum, oscillate in phase relative to the third harmonic event plane, similar
to the results from 3+1D hydrodynamical calculations. The observed radii oscillations unambiguously
signal a collective expansion and anisotropy in the velocity fields. A comparison of the measured radii
oscillations with the Blast-Wave model calculations indicate that the initial state triangularity is washed-
out at freeze out. © 2018 European Organization for Nuclear Research. Published by Elsevier B.V. This is an open access
article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. ⋆E-mail address: alice -publications @cern .ch.
well as the system geometrical shape can be
by azimuthally differential femtoscopic mea
https://doi.org/10.1016/j.physletb.2018.06.042
0370-2693/© 2018 European Organization for Nuclear Research. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. Azimuthally-differential pion femtoscopy relative to the third
harmonic event plane in Pb–Pb collisions at √sNN = 2.76 TeV .ALICE Collaboration ⋆ Azimuthally-differential pion femtoscopy relative to the third harmonic event plane in
Pb-Pbcollisions at root(NN)-N-S=2.76TeV Acharya, S.; Acosta, F.T.; Adamova, D.; Adolfsson, J; Aggarwal, MM.; Rinella, G.A.; Agnello,
Maria; Agrawal, N.; Ahammed, Z.; Ahn, S.U.; Aiola, S.; Akindinov, A.; Al-Turany, M.; Alam, S. Albuquerque, DSD; Aleksandrov, D.; Alessandro, B; Molina, Rafael A.; Ali, Yusuf; Alici, A.;
Alkin, A.; Alme, J.; Alt, T.; Bearden, Ian; Bilandzic, Ante; bsm989, bsm989; Gajdosova,
Katarina; Bourjau, Christian Alexander; Gaardhøje, Jens Jørgen; Nielsen, Børge Svane;
Thoresen, Freja; Zhou, You; Christensen, Christian Holm; Chojnacki, Marek; Ozelin De Lima
Pimentel, Lais
Published in:
Physics Letters B Document license:
CC BY Citation for published version (APA):
Acharya, S., Acosta, F. T., Adamova, D., Adolfsson, J., Aggarwal, MM., Rinella, G. A., Agnello, M., Agrawal, N.,
Ahammed, Z., Ahn, S. U., Aiola, S., Akindinov, A., Al-Turany, M., Alam, S., Albuquerque, DSD., Aleksandrov, D.,
Alessandro, B., Molina, R. A., Ali, Y., ... Ozelin De Lima Pimentel, L. (2018). Azimuthally-differential pion
femtoscopy relative to the third harmonic event plane in Pb-Pbcollisions at root(NN)-N-S=2.76TeV. Physics
Letters B, 785, 320-331. https://doi.org/10.1016/j.physletb.2018.06.042 Download date: 24. Oct. 2024 Physics Letters B 785 (2018) 320–331 https://doi.org/10.1016/j.physletb.2018.06.042
0370-2693/© 2018 European Organization for Nuclear Research. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. ⋆E-mail address: alice -publications @cern .ch. 2. Data analysis The analysis was performed over the data sample recorded in
2011 during the second Pb–Pb running period at the LHC. Ap-
proximately 2 million minimum bias events, 29.2 million central
trigger events, and 34.1 million semi-central trigger events were
used. The minimum bias, semi-central, and central triggers used
all require a signal in both V0 detectors [17]. The V0 detector, also
used for the centrality determination [18], is a small angle detector
of scintillator arrays covering pseudorapidity ranges 2.8 < η < 5.1
and −3.7 < η < −1.7 for a collision vertex occurring at the cen-
ter of the ALICE detector. The results of this analysis are reported
for collision centrality classes expressed as ranges of the fraction
of the inelastic Pb–Pb cross section: 0–5%, 5–10%, 10–20%, 20–30%,
30–40%, and 40–50%. Events with the primary event vertex along
the beam direction |V z| < 8 cm were used in this analysis to en-
sure a uniform pseudorapidity acceptance. A detailed description
of the ALICE detector can be found in [19,20]. The Time Projection
Chamber (TPC) has full azimuthal coverage and allows charged-
particle track reconstruction in the pseudorapidity range |η| < 0.8,
as well as particle identification via the specific ionization energy
loss dE/dx associated with each track. In addition to the TPC, the
Time-Of-Flight (TOF) detector was used for identification of parti-
cles with transverse momentum pT > 0.5 GeV/c. To isolate the Bose–Einstein contribution in the correlation
function, effects due to final-state Coulomb repulsion must be
taken into account. For that, the Bowler–Sinyukov fitting proce-
dure [26,27] was used in which the Coulomb weight is only ap-
plied to the fraction of pairs (λ) that participate in the Bose–
Einstein correlation. In this approach, the correlation function is
fitted by p
/
The TPC has 18 sectors covering full azimuth with 159 pad rows
radially placed in each sector. Tracks with at least 80 space points
in the TPC were used in this analysis. Tracks compatible with a de-
cay in flight (kink topology) were rejected. The track quality was
determined by the χ 2 of the Kalman filter fit to the reconstructed
TPC clusters [21]. The χ 2 per degree of freedom was required to
be less than 4. 1. Introduction While the HBT radii
modulations relative to 2 are defined mostly by the source ge-
ometry, the azimuthal dependence of the HBT radii relative to the
third harmonic event plane (3) originate predominantly in the
anisotropies of the collective velocity fields – for a triangular, but
static source the radii do not exhibit any oscillations [12]. Models
studies [13,14] show that the anisotropy in expansion velocity as
well as the system geometrical shape can be strongly constrained
by azimuthally differential femtoscopic measurements relative to ALICE Collaboration / Physics Letters B 785 (2018) 320–331 321 3. The HBT radii oscillations relative to the third harmonic event
plane have been first observed in Au–Au collisions at RHIC energy
by the PHENIX Collaboration [15]. Unfortunately, due to large un-
certainties these measurements did not allow to obtain detailed
information on the origin of the observed oscillations. background distribution is built by using the mixed-event tech-
nique [22] in which pairs are made out of particles from three
different events with similar centrality (less than 2% difference),
event-plane angle (less than 6◦difference), and event vertex po-
sition along the beam direction (less than 4 cm difference). Both
the A(q) and B(q) distributions were measured differentially with
respect to the third harmonic event-plane angle EP,3. Note, that
measurements relative to EP,3 will smear any contribution from
elliptic flow as the elliptic and triangular event planes are un-
correlated [23]. The third harmonic event-plane angle EP,3 was
determined using TPC tracks. To avoid auto-correlation each event
was split into two subevents (−0.8 < η < 0 and 0 < η < 0.8). Pairs
were chosen from one subevent and the third harmonic event-
plane angle EP,3 was estimated using the particles from the other
subevent, and vice-versa, with the event plane resolution deter-
mined from the correlations between the event planes determined
in different subevents [4]. Requiring a minimum value in the two-
track separation parameters ϕ∗= |ϕ∗
1 −ϕ∗
2| and η = |η1 −η2|
reduces two-track reconstruction effects such as track splitting or
track merging. The quantity ϕ∗is defined in this analysis as the
azimuthal angle of the track in the laboratory frame at the radial
position of 1.6 m inside the TPC. Splitting is the effect when one
track is reconstructed as two tracks, and merging is the effect of
two tracks being reconstructed as one. 1. Introduction Also, to reduce the split-
ting effect, pairs that share more than 5% of the TPC clusters were
removed from the analysis. It is observed that at large relative mo-
mentum the correlation function is a constant, and the background
pair distribution is normalized such that this constant is equal to
unity. The analysis was performed for different collision centralities
in several ranges of kT, the magnitude of the pion-pair transverse
momentum kT = (pT,1 + pT,2)/2, and in bins of ϕ = ϕpair −EP,3,
where ϕpair is the pair azimuthal angle. The Bertsch–Pratt [3,24]
out–side–long coordinate system was used with the long direc-
tion pointing along the beam axis, out along the transverse pair
momentum, and side being perpendicular to the other two. The
three-dimensional correlation function was analyzed in the Longi-
tudinally Co-Moving System (LCMS) [25], in which the total longi-
tudinal momentum of the pair is zero, p1,L = −p2,L. In this Letter, the first azimuthally-differential femtoscopic mea-
surement relative to the third harmonic event plane in Pb–Pb
collisions at √sNN = 2.76 TeV from the ALICE experiment are pre-
sented. We compare our results to the toy-model calculations
from [13] to get an insight on the role of the anisotropies in the
velocity fields and the system shape. In addition, we compare our
results to a 3 + 1D hydrodynamical calculations [14] and a Blast-
Wave Model [16] for a quantitative characterization of the final
source shape. 2. Data analysis The difference in the re-
sults from using different pair cuts rather than the default pair cuts
were included in the systematic uncertainties (1–4%). For different
kT and centrality ranges, different fitting ranges of correlation func-
tion were used as the width of the correlation function depends
on kT and centrality range. The difference in the results from using
different fit ranges are due to the contamination of electrons in the
particle identification and the non-perfect Gaussian source (1–3%). We also studied the difference in the results by using positive and
negative pion pairs separately as well as data obtained with two
opposite magnetic field polarities of the ALICE L3 magnet. They
have been analyzed separately and a small difference in the results
(less than 3%) has been also accounted for in the systematic uncer-
tainty. The total systematic uncertainties were obtained by adding
in quadrature the contributions from all various sources mentioned
above. The systematic uncertainty associated with the event plane
determination is negligible compared to other systematic uncer-
tainties; the procedure for the reaction plane resolution correction
of the results is described in the next section. R2
μ(ϕ) = R2
μ,0 + 2R2
μ,3 cos(3ϕ)
(μ = out, side, long),
R2
os(ϕ) = R2
os,0 + 2R2
os,3 sin(3ϕ). (4) (4) Fitting the radii’s azimuthal dependence with the functional
forms of Eq. (4) allows us to extract the average radii and the
amplitudes of oscillations. The χ 2 per number of degree of free-
dom is 0.3–1.8 depending on kT and centrality range. The results
for the average radii R2
out,0, R2
side,0, and R2
os,0 were found to be
consistent with those reported previously in [11] in azimuthally
inclusive analysis. The extracted amplitudes of oscillations have to
be corrected for the finite event plane resolution. There exist sev-
eral methods for such a correction [30], which produce consistent
results [31] well within uncertainties of this analysis. The results
shown below have been obtained with the simplest method first
used by the E895 Collaboration [9], in which the amplitude of os-
cillation is divided by the event plane resolution. In this analysis
the event plane resolution correction factor is about 0.6–0.7, de-
pending on centrality. Fig. 2 shows the oscillation parameters R2
out,3, R2
side,3, R2
long,3,
and R2
os,3 for different centrality and kT ranges. 2. Data analysis All radii oscil-
lations exhibit weak centrality dependence, likely reflecting the
weak centrality dependence of the triangular flow itself. The kT
dependence is different for different radii oscillations: while the
magnitudes of R2
out,3 and R2
os,3 are smallest for the smallest kT
range, it is opposite for R2
side,3 (and, possibly for R2
long,3), where
the oscillations become stronger. The parameter R2
long,3 is consis-
tent with zero within the systematic uncertainties while R2
os,3 is
positive for all centralities and kT ranges except for the lowest kT
range 0.2–0.3 GeV/c. Note that R2
out,3 and R2
side,3 are negative for 2. Data analysis For primary track selection, only trajectories passing
within 3.2 cm from the primary vertex in the longitudinal direc-
tion and 2.4 cm in the transverse direction were used. Based on
the specific ionization energy loss in the TPC gas compared with
the corresponding Bethe–Bloch curve, and the time of flight in
TOF, a probability for each track to be a pion, kaon, proton, or
electron was determined. Particles for which the pion probabil-
ity was the largest were used in this analysis. This resulted in an
overall purity above 95%, with small contamination from electrons
in the region where the dE/dx for the two particle types over-
lap. Pions were selected in the pseudorapidity range |η| < 0.8 and
0.15 < pT < 1.5 GeV/c. C(q,ϕ) = N[(1 −λ) + λK(q)(1 + G(q,ϕ))],
(2) (2) where N is the normalization factor. The function G(q, ϕ) de-
scribes the Bose–Einstein correlations and K(q) is the Coulomb
part of the two-pion wave function integrated over a source func-
tion corresponding to G(q). In this analysis the Gaussian form of
G(q, ϕ) [28] was used G(q,ϕ) = exp
−q2
outR2
out(ϕ) −q2
sideR2
side(ϕ)
−q2
longR2
long(ϕ) −2qoutqsideR2
os(ϕ)
−2qsideqlongR2
sl(ϕ) −2qoutqlongR2
ol(ϕ)
,
(3) (3) The correlation function C(q) was calculated as C(q) = A(q)
B(q) ,
(1) where the parameters Rout, Rside, and Rlong are traditionally called
HBT radii in the out, side, and long directions. The cross-terms R2
os,
R2
sl, and R2
ol describe the correlation in the out-side, side-long, and
out-long directions, respectively. C(q) = A(q)
B(q) , (1) where q = p1 −p2 is the relative momentum of two pions, A(q)
is the distribution of particle pairs from the same event, and B(q)
is the background distribution of uncorrelated particle pairs. The The systematic uncertainties on the extracted radii, discussed
below, vary in kT and centrality. They include uncertainties related 322 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 Fig. 1. The azimuthal dependence of R2
out, R2
side, and R2
long as a function of ϕ = ϕpair −3 for centrality percentile 20–30% and four different kT ranges. Solid lines represent
the fit to the functional forms of Eq. (4). The shaded bands show the systematic uncertainty. Fig. 1. 2. Data analysis The azimuthal dependence of R2
out, R2
side, and R2
long as a function of ϕ = ϕpair −3 for centrality percentile 20–30% and four different kT ranges. Solid lines represent
the fit to the functional forms of Eq. (4). The shaded bands show the systematic uncertainty. Fig. 1. The azimuthal dependence of R2
out, R2
side, and R2
long as a function of ϕ = ϕpair −3 for centrality percentile 20–30% and fo
the fit to the functional forms of Eq. (4). The shaded bands show the systematic uncertainty. to the source symmetry in longitudinal direction, and are not fur-
ther investigated. The curves represent the fits to the data using
the functions [12]: to the tracking efficiency and track quality, momentum resolu-
tion, different values for pair cuts (ϕ∗and η), and correlation
function fit ranges [29]. Similarly to the azimuthally inclusive anal-
ysis [29], different pair cuts were used, with the default values
chosen based on a Monte Carlo study. The difference in the re-
sults from using different pair cuts rather than the default pair cuts
were included in the systematic uncertainties (1–4%). For different
kT and centrality ranges, different fitting ranges of correlation func-
tion were used as the width of the correlation function depends
on kT and centrality range. The difference in the results from using
different fit ranges are due to the contamination of electrons in the
particle identification and the non-perfect Gaussian source (1–3%). We also studied the difference in the results by using positive and
negative pion pairs separately as well as data obtained with two
opposite magnetic field polarities of the ALICE L3 magnet. They
have been analyzed separately and a small difference in the results
(less than 3%) has been also accounted for in the systematic uncer-
tainty. The total systematic uncertainties were obtained by adding
in quadrature the contributions from all various sources mentioned
above. The systematic uncertainty associated with the event plane
determination is negligible compared to other systematic uncer-
tainties; the procedure for the reaction plane resolution correction
of the results is described in the next section. to the tracking efficiency and track quality, momentum resolu-
tion, different values for pair cuts (ϕ∗and η), and correlation
function fit ranges [29]. Similarly to the azimuthally inclusive anal-
ysis [29], different pair cuts were used, with the default values
chosen based on a Monte Carlo study. 3. Results Fig. 1 presents the dependence of R2
out, R2
side, and R2
long on the
pion emission angle relative to the third harmonic event plane
for centrality 20–30% and different kT ranges. Note that R2
out and
R2
side exhibit in-phase oscillations (for a quantitative analysis, see
below). Within the uncertainties of the measurement, R2
long oscil-
lations, if any, are insignificant. Oscillations of R2
ol and R2
sl radii
(not shown) are found to be consistent with zero, as expected due ALICE Collaboration / Physics Letters B 785 (2018) 320–331 323 Fig. 2. The amplitudes of radii oscillations R2
out,3, R2
side,3, R2
long,3, and R2
os,3 versus centrality percentiles for four kT ranges. Square brackets indicate systematic uncertainties.
∞
Fig. 2. The amplitudes of radii oscillations R2
out,3, R2
side,3, R2
long,3, and R2
os,3 versus centrality percentiles for four kT ranges. Square brackets indicate systematic uncertainties. Fig. 2. The amplitudes of radii oscillations R2
out,3, R2
side,3, R2
long,3, and R2
os,3 versus centrality percentiles for four kT ranges. Square b R(φ) = R0
1 −
∞
n=2
an cos(n(φ −n))
,
(5) all centralities and kT ranges. In the toy model simulations [13]
such phases of radii oscillations were observed only in the so-
called “flow anisotropy dominated case” (a circular source with the
radial expansion velocity including the third harmonic modulation)
and not for “geometry dominated” case (triangular shape source
with radial expansion velocity proportional to radial distance from
the center, with corners having largest expansion velocity). (5) where n’s denote the orientations of the n-th order symmetry
planes. The amplitudes an and the phases n are model parame-
ters. The magnitude of the transverse expansion velocity is param-
eterized as vt = tanhρ, where the transverse rapidity ρ [13,16] is ,
g
g
p
y)
Fig. 3 shows the relative amplitudes of radius oscillations
R2
out,3/R2
side,0, R2
side,3/R2
side,0, and R2
os,3/R2
side,0. Similar to the pre-
vious analyses and theoretical calculations [14] we report all the
radii oscillations relative to the side radius the least affected by
the emission time duration. There exist no obvious centrality de-
pendence. As the average radii decrease with increasing kT, the
kT dependence of relative oscillation amplitudes appear much
stronger for “out” and “out-side” radii, while “side” radius rela-
tive amplitude exhibits no kT dependence with the uncertainties. 3. Results The shaded bands in Fig. 3 indicate the results of 3+1D hydro-
dynamical calculations [14]. These calculations assume constant
shear viscosity to entropy ratio η/s = 0.08 and bulk viscosity that
is nonzero in the hadronic phase ζ/s = 0.04, and the initial density
from a Glauber Monte Carlo model. The parameters of the model,
were tuned to reproduce the measured charged particle spectra,
the elliptic and triangular flow. We find that the relative ampli-
tudes R2
side,3/R2
side,0 agree with these results rather well, while the
relative amplitudes R2
out,3/R2
side,0 and R2
os,2/R2
side,0 agree only qual-
itatively. According to the 3+1D hydrodynamical calculations, the
negative signs of R2
side,3 and R2
out,3 parameters are an indication
that the initial triangularity has been washed-out or even reversed
at freeze-out due to triangular flow [14]. ρ(˜r,φb) = ρ0 ˜r
1 +
∞
n=2
2ρn cos(n(φb −n))
. (6) (6) Here ˜r = r/R(φ), and φb(φ) is the transverse boost direction as-
sumed to be perpendicular to the surface of constant ˜r. The re-
sults of this model presented below were obtained assuming a
kinetic freeze-out temperature of 120 MeV, and maximum ex-
pansion rapidity ρ0 = 0.8, tuned to describe single particle spec-
tra. Fig. 4 shows the relative amplitudes of the radius oscillations
R2
out,3/R2
out,0, and R2
side,3/R2
side,0 as a function of Blast-wave model
third-order parameters, spatial anisotropy a3 and transverse flow
anisotropy ρ3. Thin dashed lines represent the lines of constant
relative amplitudes, with numbers next to lines indicating the rel-
ative amplitude values. Thick dashed lines show the ALICE results
for R2
out,3/R2
out,0 and R2
side,3/R2
side,0 with the thickness of the lines
indicating the uncertainties. The intersection of the two dashed
lines corresponds to a3 and ρ3 parameters consistent with ALICE
measurements. The ALICE data and the Blast-Wave model calcula-
tions correspond to pairs with kT = 0.6 GeV/c and the centrality
range 5–10%. The comparison have been also performed for other
centralities and the corresponding Blast-Wave model parameters
have been deduced. Fig. 5 presents the final source spatial and
transverse flow anisotropies for different centrality ranges from
matching the ALICE data with the Blast-Wave model calculations. The contours correspond to one sigma uncertainty as derived from To investigate further on the final source shape, we compare
our results to the Blast-Wave model calculations [16]. 3. Results The thick lines show the corresponding ALICE results, with width of the lines rep-
resenting the experimental uncertainties. (For interpretation of the colors in the
figure(s), the reader is referred to the web version of this article.)
Fig. 5. Blast-Wave model [16] source parameters, final spatial (a3) and transverse
flow (ρ3) anisotropies, for different centrality ranges, as obtained from the fit to AL-
ICE radii oscillation parameters. The contours represent the one sigma uncertainty. negative values of the final source anisotropy would be interpreted
as that the triangular orientation at the initial-state is reversed at
freeze out. 4. Summary Fig. 5. Blast-Wave model [16] source parameters, final spatial (a3) and transverse
flow (ρ3) anisotropies, for different centrality ranges, as obtained from the fit to AL-
ICE radii oscillation parameters. The contours represent the one sigma uncertainty. Fig. 4. The relative amplitudes of the radius oscillations R2
out,3/R2
out,0,
and
R2
side,3/R2
side,0
on the third-order anisotropies in space (a3) and trans-
verse flow (ρ3) for the centrality range 5–10% and kT = 0.6 GeV/c from the
Blast-Wave model [16]. The thin dashed lines show the lines of a constant rela-
tive amplitude, in magenta for R2
out,3/R2
out,0 and in dark yellow for R2
side,3/R2
side,0. The thick lines show the corresponding ALICE results, with width of the lines rep-
resenting the experimental uncertainties. (For interpretation of the colors in the
figure(s), the reader is referred to the web version of this article.) negative values of the final source anisotropy would be interpreted
as that the triangular orientation at the initial-state is reversed at
freeze out. 3. Results In that
model, the spatial geometry of the pion source at freeze-out is pa-
rameterized by ALICE Collaboration / Physics Letters B 785 (2018) 320–331 324 Fig. 3. Amplitudes of the relative radii oscillations R2
out,3/R2
side,0, R2
side,3/R2
side,0, and R2
os,2/R2
side,0 versus centrality for four kT ranges. Square brackets indicate systematic
uncertainties. The shaded bands are the 3+1D hydrodynamical calculations [14] and the width of the bands represent the uncertainties in the model calculations. Fig. 3. Amplitudes of the relative radii oscillations R2
out,3/R2
side,0, R2
side,3/R2
side,0, and R2
os,2/R2
side,0 versus centrality for four kT ranges. Square brackets indicate systematic
uncertainties. The shaded bands are the 3+1D hydrodynamical calculations [14] and the width of the bands represent the uncertainties in the model calculations. Fig. 3. Amplitudes of the relative radii oscillations R2
out,3/R2
side,0, R2
side,3/R2
side,0, and R2
os,2/R2
side,0 versus centrality for four kT r
uncertainties. The shaded bands are the 3+1D hydrodynamical calculations [14] and the width of the bands represent the uncerta Fig. 5. Blast-Wave model [16] source parameters, final spatial (a3) and transverse
flow (ρ3) anisotropies, for different centrality ranges, as obtained from the fit to AL-
ICE radii oscillation parameters. The contours represent the one sigma uncertainty. Fig. 4. The relative amplitudes of the radius oscillations R2
out,3/R2
out,0,
and
R2
side,3/R2
side,0
on the third-order anisotropies in space (a3) and trans-
verse flow (ρ3) for the centrality range 5–10% and kT = 0.6 GeV/c from the
Blast-Wave model [16]. The thin dashed lines show the lines of a constant rela-
tive amplitude, in magenta for R2
out,3/R2
out,0 and in dark yellow for R2
side,3/R2
side,0. The thick lines show the corresponding ALICE results, with width of the lines rep-
resenting the experimental uncertainties. (For interpretation of the colors in the
figure(s), the reader is referred to the web version of this article.) Fig. 4. The relative amplitudes of the radius oscillations R2
out,3/R2
out,0,
and
R2
side,3/R2
side,0
on the third-order anisotropies in space (a3) and trans-
verse flow (ρ3) for the centrality range 5–10% and kT = 0.6 GeV/c from the
Blast-Wave model [16]. The thin dashed lines show the lines of a constant rela-
tive amplitude, in magenta for R2
out,3/R2
out,0 and in dark yellow for R2
side,3/R2
side,0. 4. Summary tive Science and Technology, Nagasaki Institute of Applied Science
(IIST), Japan Society for the Promotion of Science (JSPS) KAKENHI
and Japanese Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan; Consejo Nacional de Ciencia (CONA-
CYT) y Tecnología, through Fondo de Cooperación Internacional en
Ciencia y Tecnología (FONCICYT) and Dirección General de Asuntos
del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie
voor Wetenschappelijk Onderzoek (NWO), Netherlands; The Re-
search Council of Norway, Norway; Commission on Science and
Technology for Sustainable Development in the South (COMSATS),
Pakistan; Pontificia Universidad Católica del Perú, Peru; Ministry of
Science and Higher Education and National Science Centre, Poland;
Korea Institute of Science and Technology Information and National
Research Foundation of Korea (NRF), Republic of Korea; Ministry of
Education and Scientific Research, Institute of Atomic Physics and
Romanian National Agency for Science, Technology and Innovation,
Romania; Joint Institute for Nuclear Research (JINR), Ministry of
Education and Science of the Russian Federation and National Re-
search Centre Kurchatov Institute, Russia; Ministry of Education,
Science, Research and Sport of the Slovak Republic, Slovakia; Na-
tional Research Foundation of South Africa, South Africa; Centro de
Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaen-
ergía, Cuba and Centro de Investigaciones Energéticas, Medioambi-
entales y Tecnológicas (CIEMAT), Spain; Swedish Research Council
(VR) and Knut and Alice Wallenberg Foundation (KAW), Sweden;
European Organization for Nuclear Research, Switzerland; National
Science and Technology Development Agency (NSDTA), Suranaree
University of Technology (SUT) and Office of the Higher Educa-
tion Commission under NRU project of Thailand, Thailand; Turkish
Atomic Energy Agency (TAEK), Turkey; National Academy of Sci-
ences of Ukraine, Ukraine; Science and Technology Facilities Coun-
cil (STFC), United Kingdom; National Science Foundation of the
United States of America (NSF) and U.S. Department of Energy, Of-
fice of Nuclear Physics (DOE NP), United States of America. of the HBT radii unambiguously indicate a collective expansion of
the system and anisotropy in collective velocity fields at freeze-out. Clear in-phase oscillations of Rout and Rside, with both R2
out,3 and
R2
side,3 parameters (as defined in Eq. (4)) being negative, have been
observed for all centralities and kT ranges. According to model cal-
culations [13] the observed Rout and Rside in-phase oscillations are
characteristics of the source with strong triangular flow and close
to zero spatial anisotropy. 4. Summary This conclusion is further confirmed by a
detailed comparison of our results with the Blast-Wave model cal-
culations [16], from which the parameters of the source, the spatial
anisotropy and modulations in the radial expansion velocity, have
been derived, with spatial triangular anisotropy being more than
an order of magnitude smaller than the typical initial anisotropy
values. The oscillation amplitudes exhibit weak centrality depen-
dence, and in general decrease with decreasing kT except for R2
side,3
which on opposite is the largest in the smallest kT bin. The results
of the 3+1D hydrodynamic calculations [14] are in a good qual-
itative agreement with our measurements but, quantitatively, the
model predicts a stronger dependence of R2
out,3 oscillations on kT
than observed in the data. Acknowledgements We thank J. Cimerman and B. Tomasik for providing us with the
results of the Blast-Model calculations [16]. The ALICE Collaboration would like to thank all its engineers
and technicians for their invaluable contributions to the construc-
tion of the experiment and the CERN accelerator teams for the out-
standing performance of the LHC complex. The ALICE Collaboration
gratefully acknowledges the resources and support provided by
all Grid centres and the Worldwide LHC Computing Grid (WLCG)
collaboration. The ALICE Collaboration acknowledges the follow-
ing funding agencies for their support in building and running
the ALICE detector: A.I. Alikhanyan National Science Laboratory
(Yerevan Physics Institute) Foundation (ANSL), State Committee
of Science and World Federation of Scientists (WFS), Armenia;
Austrian Academy of Sciences and Nationalstiftung für Forschung,
Technologie und Entwicklung, Austria; Ministry of Communica-
tions and High Technologies, National Nuclear Research Center,
Azerbaijan; Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Universidade Federal do Rio Grande do Sul
(UFRGS), Financiadora de Estudos e Projetos (Finep) and Fundação
de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Brazil;
Ministry of Science & Technology of China (MSTC), National Natu-
ral Science Foundation of China (NSFC) and Ministry of Education
of China (MOEC), China; Ministry of Science and Education, Croa-
tia; Ministry of Education, Youth and Sports of the Czech Republic,
Czech Republic; The Danish Council for Independent Research –
Natural Sciences, the Carlsberg Foundation and Danish National Re-
search Foundation (DNRF), Denmark; Helsinki Institute of Physics
(HIP), Finland; Commissariat à l’Énergie Atomique (CEA) and Insti-
tut National de Physique Nucléaire et de Physique des Particules
(IN2P3) and Centre National de la Recherche Scientifique (CNRS),
France; Bundesministerium für Bildung, Wissenschaft, Forschung
und Technologie (BMBF) and GSI Helmholtzzentrum für Schwe-
rionenforschung GmbH, Germany; General Secretariat for Research
and Technology, Ministry of Education, Research and Religions,
Greece; National Research, Development and Innovation Office,
Hungary; Department of Atomic Energy, Government of India
(DAE), Department of Science and Technology, Government of India
(DST), University Grants Commission, Government of India (UGC)
and Council of Scientific and Industrial Research (CSIR), India; In-
donesian Institute of Science, Indonesia; Centro Fermi – Museo
Storico della Fisica e Centro Studi e Ricerche Enrico Fermi and Isti-
tuto Nazionale di Fisica Nucleare (INFN), Italy; Institute for Innova- 4. Summary We have reported a measurement of two-pion azimuthally-
differential femtoscopy relative to the third harmonic event plane
in Pb–Pb collisions at √sNN = 2.76 TeV. The observed oscillations the fit of the model to the data. It is observed that the final source
anisotropy is close to zero, significantly smaller than the initial tri-
angular eccentricities that are typically of the order of 0.2–0.3. The ALICE Collaboration / Physics Letters B 785 (2018) 320–331 325 tive Science and Technology, Nagasaki Institute of Applied Science
(IIST), Japan Society for the Promotion of Science (JSPS) KAKENHI
and Japanese Ministry of Education, Culture, Sports, Science and
Technology (MEXT), Japan; Consejo Nacional de Ciencia (CONA-
CYT) y Tecnología, through Fondo de Cooperación Internacional en
Ciencia y Tecnología (FONCICYT) and Dirección General de Asuntos
del Personal Academico (DGAPA), Mexico; Nederlandse Organisatie
voor Wetenschappelijk Onderzoek (NWO), Netherlands; The Re-
search Council of Norway, Norway; Commission on Science and
Technology for Sustainable Development in the South (COMSATS),
Pakistan; Pontificia Universidad Católica del Perú, Peru; Ministry of
Science and Higher Education and National Science Centre, Poland;
Korea Institute of Science and Technology Information and National
Research Foundation of Korea (NRF), Republic of Korea; Ministry of
Education and Scientific Research, Institute of Atomic Physics and
Romanian National Agency for Science, Technology and Innovation,
Romania; Joint Institute for Nuclear Research (JINR), Ministry of
Education and Science of the Russian Federation and National Re-
search Centre Kurchatov Institute, Russia; Ministry of Education,
Science, Research and Sport of the Slovak Republic, Slovakia; Na-
tional Research Foundation of South Africa, South Africa; Centro de
Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Cubaen-
ergía, Cuba and Centro de Investigaciones Energéticas, Medioambi-
entales y Tecnológicas (CIEMAT), Spain; Swedish Research Council
(VR) and Knut and Alice Wallenberg Foundation (KAW), Sweden;
European Organization for Nuclear Research, Switzerland; National
Science and Technology Development Agency (NSDTA), Suranaree
University of Technology (SUT) and Office of the Higher Educa-
tion Commission under NRU project of Thailand, Thailand; Turkish
Atomic Energy Agency (TAEK), Turkey; National Academy of Sci-
ences of Ukraine, Ukraine; Science and Technology Facilities Coun-
cil (STFC), United Kingdom; National Science Foundation of the
United States of America (NSF) and U.S. Department of Energy, Of-
fice of Nuclear Physics (DOE NP), United States of America. References [1] B. Muller, J. Schukraft, B. Wyslouch, First results from Pb + Pb collisions at the
LHC, Annu. Rev. Nucl. Part. Sci. 62 (2012) 361–386, arXiv:1202 .3233 [hep -ex]. [2] G. Goldhaber, S. Goldhaber, W. Lee, A. Pais, Influence of Bose–Einstein statistics
on the anti-proton proton annihilation process, Phys. Rev. 120 (1960) 300–312. [3] G. Bertsch, M. Gong, M. Tohyama, Pion interferometry in ultrarelativistic heavy-
ion collisions, Phys. Rev. C 37 (1988) 1896–1900. [4] A.M. Poskanzer, S.A. Voloshin, Methods for analyzing anisotropic flow in rel-
ativistic nuclear collisions, Phys. Rev. C 58 (1998) 1671–1678, arXiv:nucl -ex /
9805001 [nucl -ex]. [5] B. Alver, G. Roland, Collision geometry fluctuations and triangular flow in
heavy-ion collisions, Phys. Rev. C 81 (2010) 054905, arXiv:1003 .0194 [nucl -th],
Erratum: Phys. Rev. C 82 (039903) (2010). [6] M.G. Bowler, Bose–Einstein symmetrization: coherence and chaos: with partic-
ular application to e+e−annihilation, Z. Phys. C 29 (1985) 617. [7] S.A. Voloshin, W.E. Cleland, HBT analysis of anisotropic transverse flow, Phys. Rev. C 53 (1996) 896–900, arXiv:nucl -th /9509025 [nucl -th]. [8] S.A. Voloshin, W.E. Cleland, Anisotropic transverse flow and the HBT correlation
function, Phys. Rev. C 54 (1996) 3212–3217, arXiv:nucl -th /9606033 [nucl -th]. [9] E895 Collaboration, M.A. Lisa, et al., Azimuthal dependence of pion interferom-
etry at the AGS, Phys. Lett. B 496 (2000) 1–8, arXiv:nucl -ex /0007022 [nucl -ex]. [10] M.A. Lisa, U.W. Heinz, U.A. Wiedemann, Tilted pion sources from azimuthally
sensitive HBT interferometry, Phys. Lett. B 489 (2000) 287–292, arXiv:nucl -th /
0003022 [nucl -th]. [11] ALICE Collaboration, D. Adamova, et al., Azimuthally differential pion fem-
toscopy in Pb–Pb collisions at √sNN = 2.76 TeV, Phys. Rev. Lett. 118 (22) (2017)
222301, arXiv:1702 .01612 [nucl -ex]. [12] S.A. Voloshin, Femtoscopy of the system shape fluctuations in heavy ion colli-
sions, J. Phys. G 38 (2011) 124097, arXiv:1106 .5830 [nucl -th]. [13] C.J. Plumberg, C. Shen, U.W. Heinz, Hanbury–Brown–Twiss interferometry rela-
tive to the triangular flow plane in heavy-ion collisions, Phys. Rev. C 88 (2013)
044914, arXiv:1306 .1485 [nucl -th]. [14] P. Bozek, Azimuthally sensitive femtoscopy in event-by-event hydrodynamics,
Phys. Rev. C 89 (2014) 044904, arXiv:1401.4894 [nucl -th]. ALICE Collaboration / Physics Letters B 785 (2018) 320–331 326 [23] ALICE Collaboration, K. Aamodt, et al., Higher harmonic anisotropic flow mea-
surements of charged particles in Pb–Pb collisions at √sNN = 2.76 TeV, Phys. Rev. Lett. 107 (2011) 032301, arXiv:1105 .3865 [nucl -ex]. References [15] PHENIX Collaboration, A. Adare, et al., Azimuthal-angle dependence of charged-
pion-interferometry measurements with respect to second- and third-order
event planes in Au+Au collisions at √sNN = 200 GeV, Phys. Rev. Lett. 112
(2014) 222301, arXiv:1401.7680 [nucl -ex]. [24] S. Pratt, Pion interferometry of quark–gluon plasma, Phys. Rev. D 33 (1986)
1314–1327. [16] J. Cimerman, B. Tomasik, M. Csanad, S. Lokos, Higher-order anisotropies in
the Blast-Wave model – disentangling flow and density field anisotropies, Eur. Phys. J. A 53 (8) (2017) 161, arXiv:1702 .01735 [nucl -th]. [25] S. Pratt, T. Csörg˝o, Structure of the peak in Bose–Einstein correlations, Conf. Proc. C 9106175 (1991) 75–90. [26] M.G. Bowler, Bose–Einstein correlations in quark initiated jets, Part. World 2
(1991) 1–6. [17] ALICE Collaboration, E. Abbas, et al., Performance of the ALICE VZERO system,
JINST 8 (2013) P10016, arXiv:1306 .3130 [nucl -ex]. [27] Y. Sinyukov, R. Lednicky, S. Akkelin, J. Pluta, B. Erazmus, Coulomb corrections
for interferometry analysis of expanding hadron systems, Phys. Lett. B 432
(1998) 248–257. [18] ALICE Collaboration, K. Aamodt, et al., Centrality dependence of the
charged-particle multiplicity density at mid-rapidity in Pb–Pb collisions at
√sNN
=
2.76 TeV, Phys. Rev. Lett. 106 (2011) 032301, arXiv:1012 .1657
[nucl -ex]. [28] S. Pratt, T. Csörg˝o, J. Zimányi, Detailed predictions for two-pion correlations in
ultrarelativistic heavy-ion collisions, Phys. Rev. C 42 (Dec 1990) 2646–2652. [19] ALICE Collaboration, K. Aamodt, et al., The ALICE experiment at the CERN LHC,
JINST 3 (2008) S08002. [29] ALICE Collaboration, J. Adam, et al., Centrality dependence of pion freeze-out
radii in Pb–Pb collisions at √sNN = 2.76 TeV, Phys. Rev. C 93 (2016) 024905,
arXiv:1507.06842 [nucl -ex]. [20] ALICE Collaboration, B. Abelev, et al., Performance of the ALICE experiment at
the CERN LHC, Int. J. Mod. Phys. A 29 (2014) 1430044, arXiv:1402 .4476 [nucl -
ex]. [30] M.A. Lisa, S. Pratt, R. Soltz, U. Wiedemann, Femtoscopy in relativistic heavy ion
collisions, Annu. Rev. Nucl. Part. Sci. 55 (2005) 357–402, arXiv:nucl -ex /0505014
[nucl -ex]. [21] J. Alme, et al., The ALICE TPC, a large 3-dimensional tracking device with fast
readout for ultra-high multiplicity events, Nucl. Instrum. Methods A 622 (2010)
316–367, arXiv:1001.1950 [physics .ins -det]. [31] STAR Collaboration, L. Adamczyk, et al., Beam-energy-dependent two-pion in-
terferometry and the freeze-out eccentricity of pions measured in heavy ion
collisions at the STAR detector, Phys. Rev. C 92 (2015) 014904, arXiv:1403 .4972
[nucl -ex]. [22] G. References Kopylov, Like particle correlations as a tool to study the multiple production
mechanism, Phys. Lett. B 50 (1974) 472–474. ALICE Collaboration S. Acharya 139, F.T. Acosta 22, D. Adamová 94, J. Adolfsson 81, M.M. Aggarwal 98, G. Aglieri Rinella 36,
M. Agnello 33, N. Agrawal 48, Z. Ahammed 139, S.U. Ahn 77, S. Aiola 144, A. Akindinov 64, M. Al-Turany 104,
S.N. Alam 139, D.S.D. Albuquerque 120, D. Aleksandrov 88, B. Alessandro 58, R. Alfaro Molina 72, Y. Ali 16,
A. Alici 11,53,29, A. Alkin 3, J. Alme 24, T. Alt 69, L. Altenkamper 24, I. Altsybeev 138, C. Andrei 47,
D. Andreou 36, H.A. Andrews 108, A. Andronic 104, M. Angeletti 36, V. Anguelov 102, C. Anson 17,
T. Antiˇci´c 105, F. Antinori 56, P. Antonioli 53, N. Apadula 80, L. Aphecetche 112, H. Appelshäuser 69,
S. Arcelli 29, R. Arnaldi 58, O.W. Arnold 103,115, I.C. Arsene 23, M. Arslandok 102, B. Audurier 112,
A. Augustinus 36, R. Averbeck 104, M.D. Azmi 18, A. Badalà 55, Y.W. Baek 60,76, S. Bagnasco 58,
R. Bailhache 69, R. Bala 99, A. Baldisseri 135, M. Ball 43, R.C. Baral 86, A.M. Barbano 28, R. Barbera 30,
F. Barile 52, L. Barioglio 28, G.G. Barnaföldi 143, L.S. Barnby 93, V. Barret 132, P. Bartalini 7, K. Barth 36,
E. Bartsch 69, N. Bastid 132, S. Basu 141, G. Batigne 112, B. Batyunya 75, P.C. Batzing 23, J.L. Bazo Alba 109,
I.G. Bearden 89, H. Beck 102, C. Bedda 63, N.K. Behera 60, I. Belikov 134, F. Bellini 36,29, H. Bello Martinez 2,
R. Bellwied 124, L.G.E. Beltran 118, V. Belyaev 92, G. Bencedi 143, S. Beole 28, A. Bercuci 47, Y. Berdnikov 96,
D. Berenyi 143, R.A. Bertens 128, D. Berzano 58,36, L. Betev 36, P.P. Bhaduri 139, A. Bhasin 99, I.R. Bhat 99,
B. Bhattacharjee 42, J. Bhom 116, A. Bianchi 28, L. Bianchi 124, N. Bianchi 51, J. Bielˇcík 38, J. Bielˇcíková 94,
A. Bilandzic 103,115, G. Biro 143, R. Biswas 4, S. Biswas 4, J.T. Blair 117, D. Blau 88, C. Blume 69, G. Boca 136,
F. Bock 36, A. Bogdanov 92, L. Boldizsár 143, M. Bombara 39, G. Bonomi 137, M. Bonora 36, H. Borel 135,
A. Borissov 142,20, M. Borri 126, E. Botta 28, C. Bourjau 89, L. Bratrud 69, P. Braun-Munzinger 104,
M. Bregant 119, T.A. Broker 69, M. Broz 38, E.J. Brucken 44, E. Bruna 58, G.E. Bruno 36,35, D. Budnikov 106,
H. Buesching 69, S. Bufalino 33, P. Buhler 111, P. Buncic 36, O. ALICE Collaboration Busch 131, Z. Buthelezi 73, J.B. Butt 16,
J.T. Buxton 19, J. Cabala 114, D. Caffarri 36,90, H. Caines 144, A. Caliva 104, E. Calvo Villar 109, R.S. Camacho 2,
P. Camerini 27, A.A. Capon 111, F. Carena 36, W. Carena 36, F. Carnesecchi 11,29, J. Castillo Castellanos 135,
A.J. Castro 128, E.A.R. Casula 54, C. Ceballos Sanchez 9, S. Chandra 139, B. Chang 125, W. Chang 7,
S. Chapeland 36, M. Chartier 126, S. Chattopadhyay 139, S. Chattopadhyay 107, A. Chauvin 115,103,
C. Cheshkov 133, B. Cheynis 133, V. Chibante Barroso 36, D.D. Chinellato 120, S. Cho 60, P. Chochula 36,
S. Choudhury 139, T. Chowdhury 132, P. Christakoglou 90, C.H. Christensen 89, P. Christiansen 81,
T. Chujo 131, S.U. Chung 20, C. Cicalo 54, L. Cifarelli 11,29, F. Cindolo 53, J. Cleymans 123, F. Colamaria 52,35,
D. Colella 36,52,65, A. Collu 80, M. Colocci 29, M. Concas 58,ii, G. Conesa Balbastre 79, Z. Conesa del Valle 61,
J.G. Contreras 38, T.M. Cormier 95, Y. Corrales Morales 58, P. Cortese 34, M.R. Cosentino 121, F. Costa 36,
S. Costanza 136, J. Crkovská 61, P. Crochet 132, E. Cuautle 70, L. Cunqueiro 95,142, T. Dahms 115,103,
A. Dainese 56, M.C. Danisch 102, A. Danu 68, D. Das 107, I. Das 107, S. Das 4, A. Dash 86, S. Dash 48, S. De 49,
A. De Caro 32, G. de Cataldo 52, C. de Conti 119, J. de Cuveland 40, A. De Falco 26, D. De Gruttola 11,32,
N. De Marco 58, S. De Pasquale 32, R.D. De Souza 120, H.F. Degenhardt 119, A. Deisting 104,102, A. Deloff 85, S. Acharya 139, F.T. Acosta 22, D. Adamová 94, J. Adolfsson 81, M.M. Aggarwal 98, G. Aglieri Rinella 36,
M. Agnello 33, N. Agrawal 48, Z. Ahammed 139, S.U. Ahn 77, S. Aiola 144, A. Akindinov 64, M. Al-Turany 104,
S.N. Alam 139, D.S.D. Albuquerque 120, D. Aleksandrov 88, B. Alessandro 58, R. Alfaro Molina 72, Y. Ali 16,
A. Alici 11,53,29, A. Alkin 3, J. Alme 24, T. Alt 69, L. Altenkamper 24, I. Altsybeev 138, C. Andrei 47,
D. Andreou 36, H.A. Andrews 108, A. Andronic 104, M. Angeletti 36, V. Anguelov 102, C. Anson 17,
T. Antiˇci´c 105, F. Antinori 56, P. Antonioli 53, N. Apadula 80, L. Aphecetche 112, H. Appelshäuser 69,
S. Arcelli 29, R. Arnaldi 58, O.W. Arnold 103,115, I.C. Arsene 23, M. Arslandok 102, B. ALICE Collaboration Audurier 112,
A. Augustinus 36, R. Averbeck 104, M.D. Azmi 18, A. Badalà 55, Y.W. Baek 60,76, S. Bagnasco 58,
R. Bailhache 69, R. Bala 99, A. Baldisseri 135, M. Ball 43, R.C. Baral 86, A.M. Barbano 28, R. Barbera 30,
F. Barile 52, L. Barioglio 28, G.G. Barnaföldi 143, L.S. Barnby 93, V. Barret 132, P. Bartalini 7, K. Barth 36,
E. Bartsch 69, N. Bastid 132, S. Basu 141, G. Batigne 112, B. Batyunya 75, P.C. Batzing 23, J.L. Bazo Alba 109,
I.G. Bearden 89, H. Beck 102, C. Bedda 63, N.K. Behera 60, I. Belikov 134, F. Bellini 36,29, H. Bello Martinez 2,
R. Bellwied 124, L.G.E. Beltran 118, V. Belyaev 92, G. Bencedi 143, S. Beole 28, A. Bercuci 47, Y. Berdnikov 96,
D. Berenyi 143, R.A. Bertens 128, D. Berzano 58,36, L. Betev 36, P.P. Bhaduri 139, A. Bhasin 99, I.R. Bhat 99,
B. Bhattacharjee 42, J. Bhom 116, A. Bianchi 28, L. Bianchi 124, N. Bianchi 51, J. Bielˇcík 38, J. Bielˇcíková 94,
A. Bilandzic 103,115, G. Biro 143, R. Biswas 4, S. Biswas 4, J.T. Blair 117, D. Blau 88, C. Blume 69, G. Boca 136,
F. Bock 36, A. Bogdanov 92, L. Boldizsár 143, M. Bombara 39, G. Bonomi 137, M. Bonora 36, H. Borel 135,
A. Borissov 142,20, M. Borri 126, E. Botta 28, C. Bourjau 89, L. Bratrud 69, P. Braun-Munzinger 104, S. Choudhury 139, T. Chowdhury 132, P. Christakoglou 90, C.H. Christensen 89, P. Christiansen 81,
T. Chujo 131, S.U. Chung 20, C. Cicalo 54, L. Cifarelli 11,29, F. Cindolo 53, J. Cleymans 123, F. Colamaria 52,35,
D. Colella 36,52,65, A. Collu 80, M. Colocci 29, M. Concas 58,ii, G. Conesa Balbastre 79, Z. Conesa del Valle 61,
J.G. Contreras 38, T.M. Cormier 95, Y. Corrales Morales 58, P. Cortese 34, M.R. Cosentino 121, F. Costa 36,
S. Costanza 136, J. Crkovská 61, P. Crochet 132, E. Cuautle 70, L. Cunqueiro 95,142, T. Dahms 115,103,
A. Dainese 56, M.C. Danisch 102, A. Danu 68, D. Das 107, I. Das 107, S. Das 4, A. Dash 86, S. Dash 48, S. De 49,
A. De Caro 32, G. de Cataldo 52, C. de Conti 119, J. de Cuveland 40, A. De Falco 26, D. De Gruttola 11,32,
N. De Marco 58, S. De Pasquale 32, R.D. De Souza 120, H.F. Degenhardt 119, A. Deisting 104,102, A. ALICE Collaboration Deloff 85, ALICE Collaboration / Physics Letters B 785 (2018) 320–331
32
S. Delsanto 28, C. Deplano 90, P. Dhankher 48, D. Di Bari 35, A. Di Mauro 36, B. Di Ruzza 56, R.A. Diaz 9,
T. Dietel 123, P. Dillenseger 69, Y. Ding 7, R. Divià 36, Ø. Djuvsland 24, A. Dobrin 36,
D. Domenicis Gimenez 119, B. Dönigus 69, O. Dordic 23, L.V.R. Doremalen 63, A.K. Dubey 139, A. Dubla 104,
L. Ducroux 133, S. Dudi 98, A.K. Duggal 98, M. Dukhishyam 86, P. Dupieux 132, R.J. Ehlers 144, D. Elia 52,
E. Endress 109, H. Engel 74, E. Epple 144, B. Erazmus 112, F. Erhardt 97, M.R. Ersdal 24, B. Espagnon 61,
G. Eulisse 36, J. Eum 20, D. Evans 108, S. Evdokimov 91, L. Fabbietti 103,115, M. Faggin 31, J. Faivre 79,
A. Fantoni 51, M. Fasel 95, L. Feldkamp 142, A. Feliciello 58, G. Feofilov 138, A. Fernández Téllez 2,
A. Ferretti 28, A. Festanti 31,36, V.J.G. Feuillard 135,132, J. Figiel 116, M.A.S. Figueredo 119, S. Filchagin 106,
D. Finogeev 62, F.M. Fionda 24, M. Floris 36, S. Foertsch 73, P. Foka 104, S. Fokin 88, E. Fragiacomo 59,
A. Francescon 36, A. Francisco 112, U. Frankenfeld 104, G.G. Fronze 28, U. Fuchs 36, C. Furget 79, A. Furs 62,
M. Fusco Girard 32, J.J. Gaardhøje 89, M. Gagliardi 28, A.M. Gago 109, K. Gajdosova 89, M. Gallio 28,
C.D. Galvan 118, P. Ganoti 84, C. Garabatos 104, E. Garcia-Solis 12, K. Garg 30, C. Gargiulo 36, P. Gasik 115,103,
E.F. Gauger 117, M.B. Gay Ducati 71, M. Germain 112, J. Ghosh 107, P. Ghosh 139, S.K. Ghosh 4, P. Gianotti 51,
P. Giubellino 104,58, P. Giubilato 31, P. Glässel 102, D.M. Goméz Coral 72, A. Gomez Ramirez 74,
V. Gonzalez 104, P. González-Zamora 2, S. Gorbunov 40, L. Görlich 116, S. Gotovac 127, V. Grabski 72,
L.K. Graczykowski 140, K.L. Graham 108, L. Greiner 80, A. Grelli 63, C. Grigoras 36, V. Grigoriev 92,
A. Grigoryan 1, S. Grigoryan 75, J.M. Gronefeld 104, F. Grosa 33, J.F. Grosse-Oetringhaus 36, R. Grosso 104,
R. Guernane 79, B. Guerzoni 29, M. Guittiere 112, K. Gulbrandsen 89, T. Gunji 130, A. Gupta 99, R. Gupta 99,
I.B. Guzman 2, R. Haake 36, M.K. Habib 104, C. Hadjidakis 61, H. Hamagaki 82, G. Hamar 143, J.C. Hamon 134,
M.R. Haque 63, J.W. Harris 144, A. Harton 12, H. ALICE Collaboration Hassan 79, D. Hatzifotiadou 53,11, S. Hayashi 130,
S.T. Heckel 69, E. Hellbär 69, H. Helstrup 37, A. Herghelegiu 47, E.G. Hernandez 2, G. Herrera Corral 10,
F. Herrmann 142, K.F. Hetland 37, T.E. Hilden 44, H. Hillemanns 36, C. Hills 126, B. Hippolyte 134,
B. Hohlweger 103, D. Horak 38, S. Hornung 104, R. Hosokawa 131,79, P. Hristov 36, C. Hughes 128, P. Huhn 69,
T.J. Humanic 19, H. Hushnud 107, N. Hussain 42, T. Hussain 18, D. Hutter 40, D.S. Hwang 21, J.P. Iddon 126,
S.A. Iga Buitron 70, R. Ilkaev 106, M. Inaba 131, M. Ippolitov 88, M.S. Islam 107, M. Ivanov 104, V. Ivanov 96,
V. Izucheev 91, B. Jacak 80, N. Jacazio 29, P.M. Jacobs 80, M.B. Jadhav 48, S. Jadlovska 114, J. Jadlovsky 114,
S. Jaelani 63, C. Jahnke 115,119, M.J. Jakubowska 140, M.A. Janik 140, P.H.S.Y. Jayarathna 124, C. Jena 86,
M. Jercic 97, R.T. Jimenez Bustamante 104, P.G. Jones 108, A. Jusko 108, P. Kalinak 65, A. Kalweit 36,
J.H. Kang 145, V. Kaplin 92, S. Kar 139,7, A. Karasu Uysal 78, O. Karavichev 62, T. Karavicheva 62,
L. Karayan 102,104, P. Karczmarczyk 36, E. Karpechev 62, U. Kebschull 74, R. Keidel 46, D.L.D. Keijdener 63,
M. Keil 36, B. Ketzer 43, Z. Khabanova 90, S. Khan 18, S.A. Khan 139, A. Khanzadeev 96, Y. Kharlov 91,
A. Khatun 18, A. Khuntia 49, M.M. Kielbowicz 116, B. Kileng 37, B. Kim 131, D. Kim 145, D.J. Kim 125,
E.J. Kim 14, H. Kim 145, J.S. Kim 41, J. Kim 102, M. Kim 60, S. Kim 21, T. Kim 145, T. Kim 145, S. Kirsch 40,
I. Kisel 40, S. Kiselev 64, A. Kisiel 140, G. Kiss 143, J.L. Klay 6, C. Klein 69, J. Klein 36,58, C. Klein-Bösing 142,
S. Klewin 102, A. Kluge 36, M.L. Knichel 36,102, A.G. Knospe 124, C. Kobdaj 113, M. Kofarago 143,
M.K. Köhler 102, T. Kollegger 104, V. Kondratiev 138, N. Kondratyeva 92, E. Kondratyuk 91, A. Konevskikh 62,
M. Konyushikhin 141, O. Kovalenko 85, V. Kovalenko 138, M. Kowalski 116, I. Králik 65, A. Kravˇcáková 39,
L. Kreis 104, M. Krivda 108,65, F. Krizek 94, M. Krüger 69, E. Kryshen 96, M. Krzewicki 40, A.M. Kubera 19,
V. Kuˇcera 60,94, C. Kuhn 134, P.G. Kuijer 90, J. Kumar 48, L. Kumar 98, S. Kumar 48, S. Kundu 86,
P. Kurashvili 85, A. Kurepin 62, A.B. ALICE Collaboration Kurepin 62, A. Kuryakin 106, S. Kushpil 94, M.J. Kweon 60, Y. Kwon 145,
S.L. La Pointe 40, P. La Rocca 30, C. Lagana Fernandes 119, Y.S. Lai 80, I. Lakomov 36, R. Langoy 122,
K. Lapidus 144, C. Lara 74, A. Lardeux 23, P. Larionov 51, A. Lattuca 28, E. Laudi 36, R. Lavicka 38, R. Lea 27,
L. Leardini 102, S. Lee 145, F. Lehas 90, S. Lehner 111, J. Lehrbach 40, R.C. Lemmon 93, E. Leogrande 63,
I. León Monzón 118, P. Lévai 143, X. Li 13, X.L. Li 7, J. Lien 122, R. Lietava 108, B. Lim 20, S. Lindal 23,
V. Lindenstruth 40, S.W. Lindsay 126, C. Lippmann 104, M.A. Lisa 19, V. Litichevskyi 44, A. Liu 80,
H.M. Ljunggren 81, W.J. Llope 141, D.F. Lodato 63, V. Loginov 92, C. Loizides 95,80, P. Loncar 127, X. Lopez 132,
E. López Torres 9, A. Lowe 143, P. Luettig 69, J.R. Luhder 142, M. Lunardon 31, G. Luparello 59, M. Lupi 36,
A. Maevskaya 62, M. Mager 36, S.M. Mahmood 23, A. Maire 134, R.D. Majka 144, M. Malaev 96,
L. Malinina 75,iii, D. Mal’Kevich 64, P. Malzacher 104, A. Mamonov 106, V. Manko 88, F. Manso 132,
V. Manzari 52, Y. Mao 7, M. Marchisone 129,73,133, J. Mareš 67, G.V. Margagliotti 27, A. Margotti 53,
J. Margutti 63, A. Marín 104, C. Markert 117, M. Marquard 69, N.A. Martin 104, P. Martinengo 36,
M.I. Martínez 2, G. Martínez García 112, M. Martinez Pedreira 36, S. Masciocchi 104, M. Masera 28, ALICE Collaboration / Physics Letters B 785 (2018) 320–331 327 T. Dietel 123, P. Dillenseger 69, Y. Ding 7, R. Divià 36, Ø. Djuvsland 24, A. Dobrin 36,
119
69
23
63 328
ALICE Collaboration / Physics Letters B 785 (2018) 320–331
A. Masoni 54, L. Massacrier 61, E. Masson 112, A. Mastroserio 52, A.M. Mathis 103,115, P.F.T. Matuoka 119,
A. Matyja 128, C. Mayer 116, M. Mazzilli 35, M.A. Mazzoni 57, F. Meddi 25, Y. Melikyan 92,
A. Menchaca-Rocha 72, J. Mercado Pérez 102, M. Meres 15, C.S. Meza 109, S. Mhlanga 123, Y. Miake 131,
L. Micheletti 28, M.M. Mieskolainen 44, D.L. Mihaylov 103, K. Mikhaylov 64,75, A. Mischke 63, A.N. Mishra 70,
D. Mi´skowiec 104, J. Mitra 139, C.M. Mitu 68, N. Mohammadi 36,63, A.P. Mohanty 63, B. Mohanty 86,
M. Mohisin Khan 18,iv, D.A. Moreira De Godoy 142, L.A.P. ALICE Collaboration Moreno 2, S. Moretto 31, A. Morreale 112,
A. Morsch 36, V. Muccifora 51, E. Mudnic 127, D. Mühlheim 142, S. Muhuri 139, M. Mukherjee 4,
J.D. Mulligan 144, M.G. Munhoz 119, K. Münning 43, M.I.A. Munoz 80, R.H. Munzer 69, H. Murakami 130,
S. Murray 73, L. Musa 36, J. Musinsky 65, C.J. Myers 124, J.W. Myrcha 140, B. Naik 48, R. Nair 85, B.K. Nandi 48,
R. Nania 53,11, E. Nappi 52, A. Narayan 48, M.U. Naru 16, H. Natal da Luz 119, C. Nattrass 128, S.R. Navarro 2,
K. Nayak 86, R. Nayak 48, T.K. Nayak 139, S. Nazarenko 106, R.A. Negrao De Oliveira 69,36, L. Nellen 70,
S.V. Nesbo 37, G. Neskovic 40, F. Ng 124, M. Nicassio 104, J. Niedziela 140,36, B.S. Nielsen 89, S. Nikolaev 88,
S. Nikulin 88, V. Nikulin 96, F. Noferini 11,53, P. Nomokonov 75, G. Nooren 63, J.C.C. Noris 2, J. Norman 79,126,
A. Nyanin 88, J. Nystrand 24, H. Oeschler 20,102,i, H. Oh 145, A. Ohlson 102, L. Olah 143, J. Oleniacz 140,
A.C. Oliveira Da Silva 119, M.H. Oliver 144, J. Onderwaater 104, C. Oppedisano 58, R. Orava 44, M. Oravec 114,
A. Ortiz Velasquez 70, A. Oskarsson 81, J. Otwinowski 116, K. Oyama 82, Y. Pachmayer 102, V. Pacik 89,
D. Pagano 137, G. Pai´c 70, P. Palni 7, J. Pan 141, A.K. Pandey 48, S. Panebianco 135, V. Papikyan 1, P. Pareek 49,
J. Park 60, S. Parmar 98, A. Passfeld 142, S.P. Pathak 124, R.N. Patra 139, B. Paul 58, H. Pei 7, T. Peitzmann 63,
X. Peng 7, L.G. Pereira 71, H. Pereira Da Costa 135, D. Peresunko 92,88, E. Perez Lezama 69, V. Peskov 69,
Y. Pestov 5, V. Petráˇcek 38, M. Petrovici 47, C. Petta 30, R.P. Pezzi 71, S. Piano 59, M. Pikna 15, P. Pillot 112,
L.O.D.L. Pimentel 89, O. Pinazza 53,36, L. Pinsky 124, S. Pisano 51, D.B. Piyarathna 124, M. Płosko ´n 80,
M. Planinic 97, F. Pliquett 69, J. Pluta 140, S. Pochybova 143, P.L.M. Podesta-Lerma 118, M.G. Poghosyan 95,
B. Polichtchouk 91, N. Poljak 97, W. Poonsawat 113, A. Pop 47, H. Poppenborg 142,
S. Porteboeuf-Houssais 132, V. Pozdniakov 75, S.K. Prasad 4, R. Preghenella 53, F. Prino 58, C.A. Pruneau 141,
I. Pshenichnov 62, M. Puccio 28, V. Punin 106, J. Putschke 141, S. Raha 4, S. Rajput 99, J. Rak 125,
A. ALICE Collaboration Rakotozafindrabe 135, L. Ramello 34, F. Rami 134, D.B. Rana 124, R. Raniwala 100, S. Raniwala 100,
S.S. Räsänen 44, B.T. Rascanu 69, D. Rathee 98, V. Ratza 43, I. Ravasenga 33, K.F. Read 128,95, K. Redlich 85,v,
A. Rehman 24, P. Reichelt 69, F. Reidt 36, X. Ren 7, R. Renfordt 69, A. Reshetin 62, J.-P. Revol 11, K. Reygers 102,
V. Riabov 96, T. Richert 63,81, M. Richter 23, P. Riedler 36, W. Riegler 36, F. Riggi 30, C. Ristea 68,
M. Rodríguez Cahuantzi 2, K. Røed 23, R. Rogalev 91, E. Rogochaya 75, D. Rohr 36, D. Röhrich 24,
P.S. Rokita 140, F. Ronchetti 51, E.D. Rosas 70, K. Roslon 140, P. Rosnet 132, A. Rossi 56,31, A. Rotondi 136,
F. Roukoutakis 84, C. Roy 134, P. Roy 107, O.V. Rueda 70, R. Rui 27, B. Rumyantsev 75, A. Rustamov 87,
E. Ryabinkin 88, Y. Ryabov 96, A. Rybicki 116, S. Saarinen 44, S. Sadhu 139, S. Sadovsky 91, K. Šafaˇrík 36,
S.K. Saha 139, B. Sahoo 48, P. Sahoo 49, R. Sahoo 49, S. Sahoo 66, P.K. Sahu 66, J. Saini 139, S. Sakai 131,
M.A. Saleh 141, S. Sambyal 99, V. Samsonov 96,92, A. Sandoval 72, A. Sarkar 73, D. Sarkar 139, N. Sarkar 139,
P. Sarma 42, M.H.P. Sas 63, E. Scapparone 53, F. Scarlassara 31, B. Schaefer 95, H.S. Scheid 69, C. Schiaua 47,
R. Schicker 102, C. Schmidt 104, H.R. Schmidt 101, M.O. Schmidt 102, M. Schmidt 101, N.V. Schmidt 95,69,
J. Schukraft 36, Y. Schutz 36,134, K. Schwarz 104, K. Schweda 104, G. Scioli 29, E. Scomparin 58, M. Šefˇcík 39,
J.E. Seger 17, Y. Sekiguchi 130, D. Sekihata 45, I. Selyuzhenkov 92,104, K. Senosi 73, S. Senyukov 134,
E. Serradilla 72, P. Sett 48, A. Sevcenco 68, A. Shabanov 62, A. Shabetai 112, R. Shahoyan 36, W. Shaikh 107,
A. Shangaraev 91, A. Sharma 98, A. Sharma 99, N. Sharma 98, A.I. Sheikh 139, K. Shigaki 45, M. Shimomura 83,
S. Shirinkin 64, Q. Shou 110,7, K. Shtejer 28, Y. Sibiriak 88, S. Siddhanta 54, K.M. Sielewicz 36,
T. Siemiarczuk 85, S. Silaeva 88, D. Silvermyr 81, G. Simatovic 90, G. Simonetti 36,103, R. Singaraju 139,
R. Singh 86, V. Singhal 139, T. Sinha 107, B. Sitar 15, M. Sitta 34, T.B. Skaali 23, M. Slupecki 125, N. Smirnov 144,
R.J.M. Snellings 63, T.W. Snellman 125, J. Song 20, F. ALICE Collaboration Soramel 31, S. Sorensen 128, F. Sozzi 104,
I. Sputowska 116, J. Stachel 102, I. Stan 68, P. Stankus 95, E. Stenlund 81, D. Stocco 112, M.M. Storetvedt 37,
P. Strmen 15, A.A.P. Suaide 119, T. Sugitate 45, C. Suire 61, M. Suleymanov 16, M. Suljic 36,27, R. Sultanov 64,
M. Šumbera 94, S. Sumowidagdo 50, K. Suzuki 111, S. Swain 66, A. Szabo 15, I. Szarka 15, U. Tabassam 16,
J. Takahashi 120, G.J. Tambave 24, N. Tanaka 131, M. Tarhini 61,112, M. Tariq 18, M.G. Tarzila 47, A. Tauro 36,
G. Tejeda Muñoz 2, A. Telesca 36, C. Terrevoli 31, B. Teyssier 133, D. Thakur 49, S. Thakur 139, D. Thomas 117,
F. Thoresen 89, R. Tieulent 133, A. Tikhonov 62, A.R. Timmins 124, A. Toia 69, N. Topilskaya 62, M. Toppi 51,
S.R. Torres 118, S. Tripathy 49, S. Trogolo 28, G. Trombetta 35, L. Tropp 39, V. Trubnikov 3, W.H. Trzaska 125, ALICE Collaboration / Physics Letters B 785 (2018) 320–331 328 A. Masoni 54, L. Massacrier 61, E. Masson 112, A. Mastroserio 52, A.M. Mathis 103,115, P.F.T. Matuoka 119,
128
116
35
57
25
92 yer 116, M. Mazzilli 35, M.A. Mazzoni 57, F. Meddi 25, Y. Melikyan 92,
72
102
15
109
123 A. Menchaca-Rocha 72, J. Mercado Pérez 102, M. Meres 15, C.S. Meza 109, S. Mhlanga 123, Y. Miake 131,
L. Micheletti 28, M.M. Mieskolainen 44, D.L. Mihaylov 103, K. Mikhaylov 64,75, A. Mischke 63, A.N. Mishra 70,
D. Mi´skowiec 104, J. Mitra 139, C.M. Mitu 68, N. Mohammadi 36,63, A.P. Mohanty 63, B. Mohanty 86,
M. Mohisin Khan 18,iv, D.A. Moreira De Godoy 142, L.A.P. Moreno 2, S. Moretto 31, A. Morreale 112,
A. Morsch 36, V. Muccifora 51, E. Mudnic 127, D. Mühlheim 142, S. Muhuri 139, M. Mukherjee 4,
J.D. Mulligan 144, M.G. Munhoz 119, K. Münning 43, M.I.A. Munoz 80, R.H. Munzer 69, H. Murakami 130,
S. Murray 73, L. Musa 36, J. Musinsky 65, C.J. Myers 124, J.W. Myrcha 140, B. Naik 48, R. Nair 85, B.K. Nandi 48,
R. Nania 53,11, E. Nappi 52, A. Narayan 48, M.U. Naru 16, H. Natal da Luz 119, C. Nattrass 128, S.R. Navarro 2,
K. Nayak 86, R. Nayak 48, T.K. Nayak 139, S. Nazarenko 106, R.A. Negrao De Oliveira 69,36, L. Nellen 70,
S.V. Nesbo 37, G. Neskovic 40, F. Ng 124, M. Nicassio 104, J. ALICE Collaboration Niedziela 140,36, B.S. Nielsen 89, S. Nikolaev 88,
S. Nikulin 88, V. Nikulin 96, F. Noferini 11,53, P. Nomokonov 75, G. Nooren 63, J.C.C. Noris 2, J. Norman 79,126,
A. Nyanin 88, J. Nystrand 24, H. Oeschler 20,102,i, H. Oh 145, A. Ohlson 102, L. Olah 143, J. Oleniacz 140,
A.C. Oliveira Da Silva 119, M.H. Oliver 144, J. Onderwaater 104, C. Oppedisano 58, R. Orava 44, M. Oravec 114,
A. Ortiz Velasquez 70, A. Oskarsson 81, J. Otwinowski 116, K. Oyama 82, Y. Pachmayer 102, V. Pacik 89,
D. Pagano 137, G. Pai´c 70, P. Palni 7, J. Pan 141, A.K. Pandey 48, S. Panebianco 135, V. Papikyan 1, P. Pareek 49,
J. Park 60, S. Parmar 98, A. Passfeld 142, S.P. Pathak 124, R.N. Patra 139, B. Paul 58, H. Pei 7, T. Peitzmann 63,
X. Peng 7, L.G. Pereira 71, H. Pereira Da Costa 135, D. Peresunko 92,88, E. Perez Lezama 69, V. Peskov 69,
Y. Pestov 5, V. Petráˇcek 38, M. Petrovici 47, C. Petta 30, R.P. Pezzi 71, S. Piano 59, M. Pikna 15, P. Pillot 112,
L.O.D.L. Pimentel 89, O. Pinazza 53,36, L. Pinsky 124, S. Pisano 51, D.B. Piyarathna 124, M. Płosko ´n 80,
M. Planinic 97, F. Pliquett 69, J. Pluta 140, S. Pochybova 143, P.L.M. Podesta-Lerma 118, M.G. Poghosyan 95,
B. Polichtchouk 91, N. Poljak 97, W. Poonsawat 113, A. Pop 47, H. Poppenborg 142,
132
75
4
53
58
141 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 329 T.P. Trzcinski 140, B.A. Trzeciak 63, T. Tsuji 130, A. Tumkin 106, R. Turrisi 56, T.S. Tveter 23, K. Ullaland 24,
E.N. Umaka 124, A. Uras 133, G.L. Usai 26, A. Utrobicic 97, M. Vala 114, J. Van Der Maarel 63,
J.W. Van Hoorne 36, M. van Leeuwen 63, T. Vanat 94, P. Vande Vyvre 36, D. Varga 143, A. Vargas 2,
M. Vargyas 125, R. Varma 48, M. Vasileiou 84, A. Vasiliev 88, A. Vauthier 79, O. Vázquez Doce 115,103,
V. Vechernin 138, A.M. Veen 63, A. Velure 24, E. Vercellin 28, S. Vergara Limón 2, L. Vermunt 63, R. Vernet 8,
R. Vértesi 143, L. Vickovic 127, J. Viinikainen 125, Z. Vilakazi 129, O. Villalobos Baillie 108, A. Villatoro Tello 2,
A. Vinogradov 88, L. Vinogradov 138, T. Virgili 32, V. Vislavicius 81, A. Vodopyanov 75, M.A. Völkl 101,
K. Voloshin 64, S.A. Voloshin 141, G. ALICE Collaboration Volpe 35, B. von Haller 36, I. Vorobyev 115,103, D. Voscek 114,
D. Vranic 36,104, J. Vrláková 39, B. Wagner 24, H. Wang 63, M. Wang 7, Y. Watanabe 130,131, M. Weber 111,
S.G. Weber 104, A. Wegrzynek 36, D.F. Weiser 102, S.C. Wenzel 36, J.P. Wessels 142, U. Westerhoff 142,
A.M. Whitehead 123, J. Wiechula 69, J. Wikne 23, G. Wilk 85, J. Wilkinson 53, G.A. Willems 36,142,
M.C.S. Williams 53, E. Willsher 108, B. Windelband 102, W.E. Witt 128, R. Xu 7, S. Yalcin 78, K. Yamakawa 45,
P. Yang 7, S. Yano 45, Z. Yin 7, H. Yokoyama 131,79, I.-K. Yoo 20, J.H. Yoon 60, V. Yurchenko 3, V. Zaccolo 58,
A. Zaman 16, C. Zampolli 36, H.J.C. Zanoli 119, N. Zardoshti 108, A. Zarochentsev 138, P. Závada 67,
N. Zaviyalov 106, H. Zbroszczyk 140, M. Zhalov 96, H. Zhang 7, X. Zhang 7, Y. Zhang 7, Z. Zhang 132,7,
C. Zhao 23, N. Zhigareva 64, D. Zhou 7, Y. Zhou 89, Z. Zhou 24, H. Zhu 7, J. Zhu 7, Y. Zhu 7, A. Zichichi 29,11,
M.B. Zimmermann 36, G. Zinovjev 3, J. Zmeskal 111, S. Zou 7 1 A.I. Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia
2 1 A.I. ALICE Collaboration Alikhanyan National Science Laboratory (Yerevan Physics Institute) Foundation, Yerevan, Armenia 2 Benemérita Universidad Autónoma de Puebla, Puebla, Mexico 2 Benemérita Universidad Autónoma de Puebla, Puebla, Mexico 3 Bogolyubov Institute for Theoretical Physics, National Academy of 4 Bose Institute, Department of Physics and Centre for Astroparticl 4 Bose Institute, Department of Physics and Centre for Astroparticle Physics and Space Science (CAPSS), Kolkata, India
5 5 Budker Institute for Nuclear Physics, Novosibirsk, Russia 6 California Polytechnic State University, San Luis Obispo, CA, Uni 7 Central China Normal University, Wuhan, China 8 Centre de Calcul de l’IN2P3, Villeurbanne, Lyon, France 9 Centro de Aplicaciones Tecnológicas y Desarrollo Nuclear (CEADEN), Havana, Cuba p
g
y
(
)
10 Centro de Investigación y de Estudios Avanzados (CINVESTAV), Mexico City and Mérida, Mexico g
y
(
)
y
Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi”, Rome 11 Centro Fermi – Museo Storico della Fisica e Centro Studi e Ricerche “Enrico Fermi”, Rome, Italy Centro Fermi – Museo Storico della Fisica e Centro Studi e Ricerche “Enr 12 Chicago State University, Chicago, IL, United States 13 China Institute of Atomic Energy, Beijing, China 13 China Institute of Atomic Energy, Beijing, China 14 Chonbuk National University, Jeonju, Republic of Korea 14 Chonbuk National University, Jeonju, Republic of Korea 15 Comenius University Bratislava, Faculty of Mathematics, Physics and Informatics, Bratislava, Slovakia
16 15 Comenius University Bratislava, Faculty of Mathematics, Physi 16 COMSATS Institute of Information Technology (CIIT), Islamabad, Pakistan 17 Creighton University, Omaha, NE, United States 17 Creighton University, Omaha, NE, United States 17 Creighton University, Omaha, NE, United States 18 Department of Physics, Aligarh Muslim University, Aligarh, India 19 Department of Physics, Ohio State University, Columbus, OH, United States 20 Department of Physics, Pusan National University, Pusan, Republic of Korea 21 Department of Physics, Sejong University, Seoul, Republic of Korea 23 Department of Physics, University of Oslo, Oslo, Norway 24 Department of Physics and Technology, University of Ber 24 Department of Physics and Technology, University of Bergen, Bergen, Norway 27 Dipartimento di Fisica dell’Università and Sezione INFN, Trieste, Italy 28 Dipartimento di Fisica dell’Università and Sezione INFN, Turin, Italy 29 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Bologna, Italy 29 Dipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Bologna, Italy ipartimento di Fisica e Astronomia dell’Università and Sezione INFN, Cat 32 Dipartimento di Fisica ‘E.R. ALICE Collaboration Caianiello’ dell’Università and Gruppo Collegato INFN, Salerno, Italy 32 Dipartimento di Fisica ‘E.R. Caianiello’ dell’Università and Gruppo Collegato INFN, Salerno, Italy 33 Dipartimento DISAT del Politecnico and Sezione INFN, Turin, Italy 34 Dipartimento di Scienze e Innovazione Tecnologica dell’Università 35 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy 35 Dipartimento Interateneo di Fisica ‘M. Merlin’ and Sezione INFN, Bari, Italy 36 European Organization for Nuclear Research (CERN), Geneva, Switzerland 37 Faculty of Engineering and Science, Western Norway University of Ap 38 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic
39
Š 38 Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Pra 39 Faculty of Science, P.J. Šafárik University, Košice, Slovakia y f
,
J
f
y,
,
40 Frankfurt Institute for Advanced Studies, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany
41 41 Gangneung-Wonju National University, Gangneung, Republic of Korea 41 Gangneung-Wonju National University, Gangneung, Republic of Korea
42 42 Gauhati University, Department of Physics, Guwahati, India 42 Gauhati University, Department of Physics, Guwahati, India y
p
f
y
43 Helmholtz-Institut für Strahlen- und Kernphysik, Rheinische Friedrich-Wilhelms-Universität Bonn, Bonn, Germany 43 Helmholtz-Institut für Strahlen- und Kernphysik, Rh 44 Helsinki Institute of Physics (HIP), Helsinki, Finland 44 Helsinki Institute of Physics (HIP), Helsinki, Finland 45 Hiroshima University, Hiroshima, Japan ALICE Collaboration / Physics Letters B 785 (2018) 320–331 45 Hiroshima University, Hiroshima, Japan y
J p
46 Hochschule Worms, Zentrum für Technologietransfer und Telekommunikation (ZTT), Worms, Germany 46 Hochschule Worms, Zentrum für Technologietransfer und Telekommunikation (ZTT), Worms, Germany 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 47 Horia Hulubei National Institute of Physics and Nuclear Engineerin 47 Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania 48 Indian Institute of Technology Bombay (IIT), Mumbai 48 Indian Institute of Technology Bombay (IIT), Mumbai, India 49 Indian Institute of Technology Indore, Indore, India 49 Indian Institute of Technology Indore, Indore, India 50 Indonesian Institute of Sciences, Jakarta, Indonesia 50 Indonesian Institute of Sciences, Jakarta, Indonesia 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 51 INFN, Laboratori Nazionali di Frascati, Frascati, Italy 52 INFN, Sezione di Bari, Bari, Italy 53 INFN, Sezione di Bologna, Bologna, Italy ALICE Collaboration / Physics Letters B 785 (2018) 320–331 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 330 54 INFN, Sezione di Cagliari, Cagliari, Italy 55 INFN, Sezione di Catania, Catania, Italy 56 INFN, Sezione di Padova, Padova, Italy 57 INFN, Sezione di Roma, Rome, Italy 58 INFN, Sezione di Torino, Turin, Italy 59 INFN, Sezione di Trieste, Trieste, Italy 60 Inha University, Incheon, Republic of Korea NO), Institut National de Physique Nucléaire et de Physique des Particules (IN2P3/CNRS), Université de Paris-Sud, Université Paris-Saclay, Orsay,
S i
M
R
i Inha University, Incheon, Republic of Korea
61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des Particules (IN2P3/CNRS), Université de Paris-Sud, Université Paris-Saclay, Orsay,
France
62 Institute for Nuclear Research Academy of Sciences Moscow Russia y
p
f
61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des Particules (IN2P3/CNRS), Universi
France 61 Institut de Physique Nucléaire d’Orsay (IPNO), Institut National de Physique Nucléaire et de Physique des
France 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 62 Institute for Nuclear Research, Academy of Sciences, Moscow, Russia 63 Institute for Subatomic Physics, Utrecht University/Nikhef, Utrecht, N 64 Institute for Theoretical and Experimental Physics, Moscow, Russia 65 Institute of Experimental Physics, Slovak Academy of Sciences, Košic 65 Institute of Experimental Physics, Slovak Academy of Sciences, Košice, Slo 66 Institute of Physics, Bhubaneswar, India f
y
67 Institute of Physics of the Czech Academy of Sciences, Prague, Czech Republic 67 Institute of Physics of the Czech Academy of Sciences, Pr 68 Institute of Space Science (ISS), Bucharest, Romania 68 Institute of Space Science (ISS), Bucharest, Romania f p
(
)
69 Institut für Kernphysik, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany 69 Institut für Kernphysik, Johann Wolfgang Goethe-Un 69 Institut für Kernphysik, Johann Wolfgang Goethe-Universität Frankfurt, Frankfurt, Germany f
p y
J
fg
g
f
f
y
70 Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Mexico City, Mexico 70 Instituto de Ciencias Nucleares, Universidad Nacional Autónoma de México, Mexico City, Mexic 71 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegr y
71 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazi y
71 Instituto de Física, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil 72 Instituto de Física, Universidad Nacional Autónoma de México, Mexico City, Mexico 72 Instituto de Física, Universidad Nacional Autónoma de México, Mexico City, Mexico 73 iThemba LABS, National Research Foundation, Somerset West, South Africa 73 iThemba LABS, National Research Foundation, Somerset West, South Africa f
74 Johann-Wolfgang-Goethe Universität Frankfurt, Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany 74 Johann-Wolfgang-Goethe Universität Frankfurt, Institut für 74 Johann-Wolfgang-Goethe Universität Frankfurt, Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany
75 74 Johann-Wolfgang-Goethe Universität Frankfurt, Institut für Informatik, Fachbereich Informatik und Mathematik, Frankfurt, Germany
75 75 Joint Institute for Nuclear Research (JINR), Dubna, Russia 76 Konkuk University, Seoul, Republic of Korea 77 Korea Institute of Science and Technology Info 77 Korea Institute of Science and Technology Information, Daejeon 77 Korea Institute of Science and Technology Information, Daejeon, Republic of Korea 78 KTO Karatay University, Konya, Turkey 79 Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS-IN2P3, Grenoble, France
80 80 Lawrence Berkeley National Laboratory, Berkeley, CA, United State 80 Lawrence Berkeley National Laboratory, Berkeley, CA, United States 81 Lund University, Department of Physics, Division of Particle Physics, Lund, Sweden
82 81 Lund University, Department of Physics, Division of Particle Physics, Lund, Sweden 81 Lund University, Department of Physics, Division of Particle Phy 82 Nagasaki Institute of Applied Science, Nagasaki, Japa 82 Nagasaki Institute of Applied Science, Nagasaki, Japan 83 Nara Women’s University (NWU), Nara, Japan y (
),
, J p
84 National and Kapodistrian University of Athens, School of Science, Department of Physics, Athens, Greece
8 y (
)
J p
84 National and Kapodistrian University of Athens, School of Science, Department of Physics, Athens, Greece 84 National and Kapodistrian University of Athens, 84 National and Kapodistrian University of Athens, School of Science, Depa 85 National Centre for Nuclear Research, Warsaw, Polan f
86 National Institute of Science Education and Research, HBNI, Jatni, India f
86 National Institute of Science Education and Research 86 National Institute of Science Education and Research, HBNI, Jatni, India 87 National Nuclear Research Center, Baku, Azerbaijan 88 National Research Centre Kurchatov Institute, Moscow, Russia 88 National Research Centre Kurchatov Institute, Moscow, Russia 89 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 89 Niels Bohr Institute, University of Copenhagen, Copenhagen, Denmark 90 Nikhef, National Institute for Subatomic Physics, Amsterdam, Netherlands 91 NRC “Kurchatov Institute” IHEP, Protvino, Russia 92 NRNU Moscow Engineering Physics Institute, Moscow, Russia 92 NRNU Moscow Engineering Physics Institute, Moscow, Russia 93 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 93 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, United Kingdom 93 Nuclear Physics Group, STFC Daresbury Laboratory, Daresbury, 94 Nuclear Physics Institute of the Czech Academy of Sciences, ˇRež 95 Oak Ridge National Laboratory, Oak Ridge, TN, United States
96 95 Oak Ridge National Laboratory, Oak Ridge, TN, United States 96 Petersburg Nuclear Physics Institute, Gatchina, Russi 97 Physics Department, Faculty of Science, University of Zagreb, Zagreb 98 Physics Department, Panjab University, Chandigarh, India 99 Physics Department, University of Jammu, Jammu, India 100 Physics Department, University of Rajasthan, Jaipur, India 101 Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany 101 Physikalisches Institut, Eberhard-Karls-Universität Tübingen, Tübingen, Germany 102 Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany 02 Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidel 103 Physik Department, Technische Universität München, Munich, Germany 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, German
105 Rudjer Boškovi´c Institute, Zagreb, Croatia 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwe 104 Research Division and ExtreMe Matter Institute EMMI, GSI H 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmho 104 Research Division and ExtreMe Matter Institute EMMI, GSI Helmholtzzentrum für Schwerionenforschung GmbH, Darmstadt, Germany
105 Rudjer Boškovi´c Institute, Zagreb, Croatia 105 Rudjer Boškovi´c Institute, Zagreb, Croatia 105 Rudjer Boškovi´c Institute, Zagreb, Croatia 106 Russian Federal Nuclear Center (VNIIEF), Sarov, Russia 107 Saha Institute of Nuclear Physics, Kolkata, India f
y
108 School of Physics and Astronomy, University of Birmingham, Birmi 108 School of Physics and Astronomy, University of Birmingham, Birmingham, United Kingdom f
y
y
y f
g
g
g
109 Sección Física, Departamento de Ciencias, Pontificia Universidad Católica del Perú, Lima, Peru
110 109 Sección Física, Departamento de Ciencias, Pontificia Universidad Católica del Perú, Lima, Peru 110 Shanghai Institute of Applied Physics, Shanghai, China 111 Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 111 Stefan Meyer Institut für Subatomare Physik (SMI), Vienna, Austria 112 SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 112 SUBATECH, IMT Atlantique, Université de Nantes, CNRS-IN2P3, Nantes, France 113 Suranaree University of Technology, Nakhon Ratchasima, Thailand 114 Technical University of Košice, Košice, Slovakia 115 Technische Universität München, Excellence Cluster ‘Universe’, Munich, Germany 115 Technische Universität München, Excellence Clu 115 Technische Universität München, Excellence Cluster ‘Universe’, Munich, Germany 116 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland
117 116 The Henryk Niewodniczanski Institute of Nuclear Physics, Polish Academy of Sciences, Cracow, Poland
117 116 The Henryk Niewodniczanski Institute of Nuclear Physics, Pol 117 The University of Texas at Austin, Austin, TX, United States 118 Universidad Autónoma de Sinaloa, Culiacán, Mexico 119 Universidade de São Paulo (USP), São Paulo, Brazil 120 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 120 Universidade Estadual de Campinas (UNICAMP), Campinas, Brazil 120 Universidade Estadual de Campinas (UNICAMP), C 121 Universidade Federal do ABC, Santo Andre, Brazil 122 University College of Southeast Norway, Tonsberg, Norway 123 University of Cape Town, Cape Town, South Africa 124 University of Houston, Houston, TX, United States 125 University of Jyväskylä, Jyväskylä, Finland 126 University of Liverpool, Liverpool, United Kingdom y f
p
p
g
127 University of Split, Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, Split, Croatia
128
f
ll
d 127 University of Split, Faculty of Electrical Engineering, Mechanical Engineering and Naval Architecture, Split, Croatia
128 127 University of Split, Faculty of Electrical Engineering, Mechanica 128 University of Tennessee, Knoxville, TN, United States 129 University of the Witwatersrand, Johannesburg, South Africa 129 University of the Witwatersrand, Johannesburg, South Africa 130 University of Tokyo, Tokyo, Japan 131 University of Tsukuba, Tsukuba, Japan 331 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 ALICE Collaboration / Physics Letters B 785 (2018) 320–331 132 Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France 3 Université de Lyon, Université Lyon 1, CNRS/IN2P3, IPN-Lyon, Villeurba y
y
/
y
y
134 Université de Strasbourg, CNRS, IPHC UMR 7178, F-67000 Strasbourg, France 135 Université Paris-Saclay Centre d’Études de Saclay (CEA), IRFU, Department de Physique Nucléaire (DPhN), Saclay, France
136 136 Università degli Studi di Pavia, Pavia, Italy 137 Università di Brescia, Brescia, Italy ,
,
y
138 V. Fock Institute for Physics, St. Petersburg State University, St. Petersburg, Russia
139 139 Variable Energy Cyclotron Centre, Kolkata, India 140 Warsaw University of Technology, Warsaw, Poland y
y,
,
,
142 Westfälische Wilhelms-Universität Münster, Institut für Kernphysik, Münster, Germany
143 143 Wigner Research Centre for Physics, Hungarian Academy of Sciences, Budapest, Hungary
144 144 Yale University, New Haven, CT, United States ii Dipartimento DET del Politecnico di Torino, Turin, Italy. iii M.V. Lomonosov Moscow State University, D.V. Skobeltsyn Institute of Nuclear, Physics, Moscow, Russia. i v Department of Applied Physics, Aligarh Muslim University, Aliga v Institute of Theoretical Physics, University of Wroclaw, Poland.
|
https://openalex.org/W2118120355
|
https://refubium.fu-berlin.de/bitstream/fub188/15045/1/1471-2458-14-871.pdf
|
English
| null |
Berlin evaluates school tobacco prevention - BEST prevention: study design and methodology
|
BMC public health
| 2,014
|
cc-by
| 6,973
|
* Correspondence: falk.mueller-riemenschneider@charite.de
1Institute for Social Medicine, Epidemiology and Health Economics,
Charité Universitätsmedizin, Luisenstrasse 57, 10117 Berlin, Germany
2Saw Swee Hock School of Public Health, National University of Singapore,
16 Medical Drive, Singapore 117597, Singapore
Full list of author information is available at the end of the article © 2014 Müller-Riemenschneider et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated. Abstract Background: The hazardous health effects of smoking are established, but there remains a need to evaluate
existing smoking prevention strategies and to increase their effectiveness in adolescents. Strategies focusing on
parental attitudes and rule setting have been identified as a potentially effective approach. The present manuscript
describes objectives, study design and methodology of the BEST Prevention study. Methods/design: BEST Prevention is a three-armed cluster randomized-controlled trial among 7th grade (11–16
years) students in Berlin, Germany. Schools were enrolled between 2010 and 2011 and allocated using a centralized
randomization list into 1) a student smoking prevention intervention (visit to an established interactive circuit), 2)
the same intervention plus a parent intervention, and 3) a control group (visit to an established exercise and
nutrition interactive circuit). Students were assessed at baseline, 12 and 24 months via self-report, as well as via
carbon monoxide and cotinine in saliva at the 24 month follow-up. Statistical analyses uses multi-level regression
models with cluster effects (school and class within school) based on the intention to treat population. Here we
report descriptive baseline characteristics of recruited schools, and schools classes. Two schools from the control group
dropped out after allocation. Hence, 47 secondary schools from all 12 districts of the city, including 161 school classes
and 3023 students are participating in the study. Of those, 2801 students completed the baseline assessment. Discussion: The present manuscript provides details on the study design and methodology of a large school-based
smoking prevention trial in a metropolitan area in Germany. Findings from this study will yield important insight into
the long-term effectiveness of specific smoking prevention strategies, also in disadvantaged population groups. Trial registration: NCT01306552 (January 2011). Keywords: Smoking prevention, Adolescents, Parents, Randomized-controlled trial high which is cause for concern, given the numerous det-
rimental health effects of smoking [8]. Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Open Access Berlin evaluates school tobacco prevention - BEST
prevention: study design and methodology Falk Müller-Riemenschneider1,2*, Lilian Krist1, Christin Bürger1, Nanette Ströbele-Benschop3, Stephanie Roll1,
Nina Rieckmann4, Jacqueline Müller-Nordhorn4 and Stefan N Willich1 Background The hazardous health effects of smoking and second hand
smoke are well known. Although smoking rates in many
industrialized countries have declined over past decades,
absolute number of smokers is increasing and so is the ab-
solute mortality attributable to smoking [1-3]. Tobacco
use among children and adolescents in many industrial-
ized countries has decreased in recent years, including
Germany [4-7]. However, compared to other western
countries, smoking rates in Germany are still relatively The majority of adult smokers initiate this unhealthy
behavior during adolescence, and almost every adult
who smokes started smoking before the age of 26 [9]. Tar-
geting children and adolescents is therefore the most ap-
propriate approach to prevent smoking initiation in the
first place. School-based prevention strategies have trad-
itionally been an important approach to smoking preven-
tion in children and youths. Particular advantages are that
schools offer an almost universal reach of children and
youths. In addition, educational strategies fit mutually with
schools’ role [10]. However, the effectiveness of such strat-
egies has been mixed and systematic reviews reported Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Page 2 of 10 limited evidence of the long-term effectiveness of school-
based smoking prevention strategies [10,11]. being from families with migrant background in a large
metropolitan area. The present manuscript describes objec-
tives, study design and methodology of the BEST Preven-
tion study. Parents have a strong influence on their children’s
smoking behavior in various ways. For instance, children
of parents who smoke are more likely to smoke [12]. Moreover, parental anti-smoking attitudes and rules
have shown to be associated with children’s smoking be-
havior, irrespective of their own smoking behavior
[13-15]. Given the at best modest long-term effectiveness
of behavioral smoking prevention strategies [10]. and the
important role of parents in shaping children’s smoking
behavior, parental and family-based intervention strategies
have been added to student centered school-based smok-
ing prevention strategies. However, only a limited number
of methodologically rigorous studies have investigated the
effectiveness of family or parental approaches to smoking
prevention and while some reported favorable outcomes,
others reported less positive findings [16-19]. Recent sys-
tematic reviews of the evidence have suggested that stud-
ies incorporating parental or family components could
indeed beneficially influence smoking behavior in children
and adolescents [11,20]. Methods/design
Objectives The overall objective of this school-based intervention
study is to compare the effectiveness of different smoking
prevention strategies among 7th grade students. More spe-
cifically this study has three primary objectives: To investigate the effectiveness of a combined
student-parent intervention to reduce the prevalence
of regular smoking (defined as smoking at least one
cigarette per week) after two years compared to a
control group. To investigate the effectiveness of a combined
student-parent intervention to reduce the prevalence
of regular smoking after two years compared to a
student alone intervention. To investigate the effectiveness of a student
intervention to reduce the prevalence of regular
smoking after two years compared to a control group. Important secondary objectives include: Important secondary objectives include: In Germany, a considerable number of smoking pre-
vention efforts and programs targeted at children and
adolescents are available. However, the majority of these
activities have never been rigorously evaluated for their
efficacy and effectiveness. This is highlighted in systematic
reviews of smoking prevention strategies that identified
few methodologically rigorous studies from Germany
[10,11]. In addition, there seem to be considerable regional
disparities in smoking behavior [21]. Especially in the
former eastern parts of Germany and metropolitan areas,
such as Berlin, smoking rates tend to be substantially
higher compared to average smoking rates. At present, the
reasons for these disparities are poorly understood. However, to reduce inequalities in risk taking behavior
among adolescents and to subsequently reduce inequal-
ities in health, efforts to target these disadvantaged pop-
ulations will have to be strengthened. Hence, generally
there remains a need to continue developing more ef-
fective smoking prevention approaches and to evaluate
the effectiveness of individual intervention components,
particularly in disadvantaged population groups. To investigate the effectiveness of the interventions
with regard to other measures of smoking
prevalence (e.g. lifetime smoking prevalence, current
smoking status, 12 months prevalence) and in
relation to 1 year outcomes To investigate smoking status on a subsample of
students using objective measures of smoking
behavior (carbon monoxide [CO], cotinine in saliva)
and the relation between objective and self-reported
smoking measures To assess the acceptability of the program (e.g. Background In addition to a general paucity of
relevant studies these reviews emphasized the fact that the
additional effectiveness of parental or family interventions
on top of a student targeted smoking prevention strategies
was rarely investigated [11,20]. Methods/design
Objectives percentage of school principals that agree to support
the program, percentage of parents who participate in
the parental component, percentage of students
participating in the project) To assess whether possible intervention effects are
moderated by other factors, specifically demographic
characteristics (age, gender, individual and
neighborhood socio-economic status), type of school
(Gymnasium, integrated secondary school), smoking
status of friends and family members The Berlin Evaluates School Tobacco Prevention -
BEST Prevention study was designed to address import-
ant research needs. It aims to investigate the long-term
comparative effectiveness of a school-based intervention
versus a school-based plus parental intervention strategy. The BEST Prevention study targets adolescents in Berlin, a
diverse population group with a relatively high proportion Intervention 1 - student smoking prevention circuit
Wi hi
h
fi
h
l
h
l
l
f Intervention 1
student smoking prevention circuit
Within the first school year school-classes of schools
randomized to intervention 1 visited a hands-on smok-
ing prevention circuit with their school class. The cir-
cuit (“Rauchst Du noch oder lebst Du schon?” [“Still
smoking or already living?”]) was developed and is of-
fered by KARUNA e.V. a non-governmental, non-profit
organization for children and youths in need, with the
support of the Berlin senate for health and social affairs
The aim of the circuit is to inform students about the
harmful consequences of tobacco use, to strengthen
self-responsibility and self-confidence and to enter into
a dialogue with the students. The design of the circuit,
the practical approach, and the youth-friendly presenta-
tion aim to facilitate the development of a positive non-
smoking image among students. In addition, the circuit
aims to convey smoking-related preventive knowledge
using a game-based approach, including competitions
and activities in an age-appropriate and engaging way. Trained moderators lead through six interactive stations
of the circuit and an informative billboard (s. Table 1). Overall, completion takes approximately 2 hours. Stu-
dents learn about the harmful effects of smoking, toxic
ingredients of cigarettes, differences between smokers
and non-smokers in terms of their health status (athero-
sclerosis prevalence), loss of smell, breathing capacity
and premature aging. The framework for the six sta-
tions includes an introduction by the moderator in the
form of a presentation (about 20 min) and a final Study population This study includes 7th grade students from secondary
schools throughout Berlin. All 214 secondary schools in
the city state of Berlin were approached in 2010. Permis-
sion from the senate of education and research had been Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Page 3 of 10 randomization lists and allocation was concealed from
participating schools. randomization lists and allocation was concealed from
participating schools. obtained. Subsequently, school principals and contact
teachers in charge of health promotion and smoking
prevention at schools from all districts of the city were
informed, where possible through workshops about the
project and its goals. Secondary schools were subse-
quently invited to participate in the BEST-Prevention
study and were asked to indicate the number of classes
participating. All schools that wished to participate pro-
vided a letter of interest signed by the school principle. This letter also indicated which and how many 7th grade
classes were going to participate in the study. Schools
and students were enrolled in the study if the following
selection criteria were met. Intervention 1: Intervention 1:
student smoking prevention circuit student smoking prevention circuit Intervention 2: Intervention 2:
student smoking prevention circuit plus parent
intervention Control group:
student nutrition and physical activity circuit
(without smoking prevention) Given the nature of the intervention, schools and
study participants are not blinded to the assigned inter-
vention. However, data analysis of follow-up outcomes
will be blinded with regard to the intervention group. Student inclusion criteria
th Female or male in 7th grade Attends one of the participating schools Intellectual and physical ability to make an informed
decision about study participation Approval from the Charité-Universitätsmedizin Berlin
institutional review board was obtained and separate
signed written informed assent was required from par-
ticipating students as well as consent from at least one
parent/caregiver. Participant information and consent
forms were distributed during school classes prior to a
second appointment at schools to perform baseline data
collection. School inclusion criteria – Participating schools must have 7th grade classes
and should not offer an extensive smoking
prevention program for their students that includes
parental involvement Data collection for each student is conducted at three
different time points, including baseline (at the beginning
of the 7th grade school year), follow-up 1 (12 month) and
follow-up 2 (24 month) (Figure 1). Prior to baseline data
collection one additional visit was performed in all school
classes in order to inform students about the study and to
distribute participant information and consent forms. – Participating schools must agree not to use the
student smoking prevention intervention for the
duration of the study in case of being randomized to
the control group – Participating schools must agree to have a parents’
night where trained health coaches introduce and
discuss the topic of smoking prevention in youth,
in case of being randomized to the combined
student-parent intervention Study design This study is a three-armed parallel cluster randomized
controlled trial among secondary schools with 7th grade
classes. As the unit of randomization schools were ran-
domly
assigned
using
a
1:1:1
ratio
and
a
blocked
randomization with variable block length, stratified by
school type (Gymnasium vs. integrated secondary school)
to one of three intervention groups. The randomization
sequence was generated using central computer generated Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Page 4 of 10 Figure 1 Study design: three-armed cluster randomized-controlled trial. Figure 1 Study design: three-armed cluster randomized-controlled trial. aging tool of one student in the group in order to show
aging effects of smoking. The photo of the respective
student will be taken and manipulated (by April® Face
Aging Software) to show the student’s appearance in
20 years in two versions (if he/she had/had not
smoked). In addition, school classes are enrolled in a
Berlin wide competition. In each grade the class with
the most points wins the KARUNA - Champion
Award. discussion of “unbelievable” facts from the world of to-
bacco, e.g. from politics and media. (http://www.kar-
una-prevents.de/index.php). School classes complete the smoking prevention cir-
cuit in small groups of 3–5 participants. Each station
includes a quiz or a competition to be completed. Based on the results groups receive points and the total
number of points identifies the winning group. The
prize of the winning group is participation in a virtual Table 1 Description of interactive stations of the student smoking prevention circuit
Interactive station
Content
Activity
1. Addiction
• Education and awareness in relation to
the development of nicotine addiction
16 cards showing each a person and a statement have to
be allocated to 4 steps symbolizing the stages of addiction:
• Stages of the development of nicotine/cigarette
addiction: Interest →Trial →Habituation →Addiction
Students get points for each correct allocation
2. Knowledge
• Facts/information on tobacco smoke/cigarettes:
statistics, dangers, health effects, addiction etc. Students answer multiple choice questions on a computer
3. Aroma
• Sensory experience: Recognition of different odors
Students have to recognize 8 odors and allocate them
to diverse advertisements (e.g. cigarettes, cars, perfumes)
4. Breath
• Sensory experience: Breathing sounds of
smokers and non-smokers (and a lion)
Students listen to breathing sounds over a headphone
and have to allocate the sounds to a list of answers
5. Secondary outcomes include Other measures of smoking behavior (e.g. lifetime
prevalence, 30 day prevalence, 12 months
prevalence, number of cigarettes, cannabis use) Other health behaviors (e.g. alcohol, nutrition,
physical activity) Parental attitudes, rules and smoking behavior Cotinine and CO measurements on a random
subsample of students Current facts about smoking behavior in
adolescents, including smoking initiation During all three assessment points (baseline, 12 month
and 24 month) relevant outcomes are assessed using
self-administered questionnaires during school classes
from all study participants. Two to three trained mem-
bers of the study team are available during data collec-
tion and are responsible for all aspects of the data
collection process within school classes. At each school,
contact teachers supported the implementation of the data
collection and intervention implementation. A query man-
agement was implemented by the research team to track
data collection and intervention implementation at par-
ticipating schools. To coordinate the assessments at
schools, contact teachers were approached and reminded
multiple times and eg. via email, phone, and through the
principal in order to reduce the number of drop-outs at
the cluster level (school and school class). Students who
had provided complete written informed consent but were
not available during data collection in schools received
mailed questionnaires together with free return envelopes
for completion at home. adolescents, including smoking initiation Evidence regarding parental influence on adolescent
smoking Facts about smoking cessation in youths The evidence regarding parental attitudes towards
smoking, parental smoking behavior and parental
rules towards smoking behavior Discussion with parents The second part of the parental intervention consists of
mailed follow-up including informational materials during
the second year of the study (about 8 months after the
parent’s night). This follow-up contact re-emphasized in-
formation and strategies taught during the event. Study design Toxin Memory
• Relevant toxins in cigarettes and tobacco smoke
Students have to identify pairs of memory cards by allocating
a toxic ingredient of cigarettes to the product where it is
normally used, e.g.: Arsenic - rodent control; plumb –
battery; naphthalene - insecticides)
6. Arterio-sclerosis
• Development of atherosclerosis and its consequences
Students have to pump water through two water tubes. One is normal, the other one constricted to show differences
in the circulatory system of smokers and non-smokers. • Blood flow in non-smokers and in
long-term smokers with arteriosclerosis
Followed by quiz. Information billboard
• Physical appearance of smokers
As part of this billboard students try to recognize
smokers/non-smokers by physical appearance. Table 1 Description of interactive stations of the student smoking prevention circuit
Interactive station
Content
Activity Followed by quiz. As part of this billboard students try to recognize
smokers/non-smokers by physical appearance. Page 5 of 10 Page 5 of 10 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Primary outcome School-classes of schools randomized to the student-
parent intervention group also visited the same smoking
prevention circuit. In addition, an intervention for chil-
dren’s parents is offered. The parent intervention takes
place during the first year of the study and consists of two
parts. First, during the first routine parent’s night, parents
take part in an educational program about smoking pre-
vention in children and youths. The intervention is based
upon the program “Eltern stärken” [strengthening parents]
and is provided by trained health coaches and consists of a
30 minute presentation. The program follows a normative
approach and provides parents with knowledge and skills
to address smoking behavior in adolescents and their
children. Key topics addressed are: The primary outcome is the proportion of regular smokers
(smoking at least one cigarette per week) assessed by self-
report at 24 months (final follow-up). Intervention II - student plus parent smoking prevention
intervention Outcomes and data collection Sample size determination
h
l
f ll In addition to self-reported outcomes in students, the
main outcome, smoking behavior, is assessed objectively
at the final follow-up. Assessments are conducted in the
class-room during questionnaire assessments. Measure-
ments are conducted and handled in such a way that
findings are not visible to other students or the teacher. Objective outcome assessments include CO measure-
ments from exhaled air and saliva based cotinine mea-
surements. Assessments are conducted on a random
subsample of students from 15 schools. Students from 6
schools (2 in each intervention group) undergo CO mea-
surements. The CO content [22] is measured in ppm (part
per million) using the Bedfont Smokerlyzer Micro +. Smoking behavior according to this tool is classified as fol-
lows: 0–4 non-smoker, 5–6 dangerous exposure, 7–10
smoker, 11+ heavy smoker. Another subsample of stu-
dents from 6 schools (2 in each intervention group) pro-
vides saliva samples via passive drool to measure cotinine
values using a NicAlert® dipstick. The NicAlert® test yields
a semi-quantitative measure of cotinine based on a colori-
metric immunoassay reaction. The test strip displays seven
zones that represent a range of cotinine levels from 0
(0-10 ng/ml) to 6 (>1000 ng/ml). Results are recorded as
values from 0 to 6; a result ≥1 indicated tobacco use [23]. In a further subsample of students from 3 schools (1 in
each intervention group) students provide both objective
outcome measurements described above. The analysis is following a hierarchical testing proced-
ure. In a first step, intervention 2 (student smoking
prevention circuit plus parent intervention) will be
compared to the control group with regard to the pri-
mary outcome (proportion of regular smokers at
24 months). Only if step 1 is significant at the 5% level
(two-sided), will step 2 be tested confirmatively (other-
wise all following analyses will be considered explora-
tive). The
second
step
involves
two
hypotheses:
Intervention 1 (student smoking prevention circuit)
compared to control; and Intervention 2 compared to
Intervention 1, each with regard to the primary out-
come. The sample size determination is based on the
second step (having a smaller assumed intervention ef-
fect, thus requiring a higher sample size than step one)
with the two hypotheses tested equally (significance
level alpha 0.025 each). Control group - student healthy nutrition and School-classes of schools randomized to the control
group participated in the healthy nutrition and exercise
circuit offered by KARUNA e.V. (“Kinderleicht gesund
zu leben” [“Healthy living – as easy as pie”]). The circuit
follows a similar methodological approach to the smok-
ing prevention circuit, but has no smoking related parts. It targets student’s knowledge to make healthy decisions
with a focus on diet and exercise. A trained moderator
leads the small groups in about 2 hours through the five
stations summarized in Table 2 (for more information
on the contents of the nutrition and exercise circuit see
http://www.karuna-prevents.de/index.php). The study questionnaire for adolescents was developed
and pilot tested in a way to allow comparisons with exist-
ing and widely comparable questionnaires investigating
adolescent health behavior and health. It includes ques-
tions related to socio-demographics, smoking and other
health behaviors, such as alcohol consumption, nutrition,
physical activity and sedentary behaviors, as well as height
and weight. In addition to smoking behavior, the question-
naire addresses various other issues related to adolescent
smoking, including smoking behavior of family members
and peers, parental rules and attitudes towards smoking,
as well as peer pressure. The completion of the circuit in small groups is
similar to the smoking prevention circuit. Also, based
on accumulated points a group winner will be identi-
fied. As in the smoking prevention circuit, school clas-
ses will be enrolled in a Berlin wide competition. In
each grade the class with the most points wins the
KARUNA - Champion Award. About two months before the final follow-up of their
children, all parents received a brief self-administered
questionnaire via mail. Primary purpose of this question-
naire was to determine whether parents/caregivers whose
children were allocated to Intervention 2 attended the Page 6 of 10 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Table 2 Description of interactive stations of the student healthy nutrition and exercise circuit
Interactive station
Content
Activity
1. Healthy shopping
• Development of skills for healthy shopping
(balance of carbohydrates, fat and protein)
Students have to choose food out of 90 products which they believe
are healthy. They get points for a well-balanced selection of healthy foods. 2. Flavor bar
• Food flavors
Students taste 7 water solved flavors and have to allocate them to
food products, eg.: Strawberry – strawberry ice-cream; mint – cough drops)
3. Control group - student healthy nutrition and Nutrition pyramid
• Nutritional recommendations
Students get 14 cubes with pictures of food items and have
to build the nutrition pyramid. 4. Exercise
• Exercise and energy expenditure
Students cycle on a bicycle ergometer in order to burn as
many calories as possible. The moderator explains the amount and kind of food that
corresponds to burned calories. 5. Knowledge
• Knowledge on nutrition and exercise
Students complete a quiz about healthy nutrition and health
promoting exercise. Students complete a quiz about healthy nutrition and health
promoting exercise. parent’s night. In addition, the questionnaire addresses
parental smoking behavior, awareness about their child’s
smoking behavior, attitudes and rules towards smoking of
their child or at their home, as well as a brief evaluation of
the parental intervention. Parents whose children were
allocated to Intervention 1 and Control group (parents
who did not receive the parent intervention) received the
same questionnaire except for items related to the evalu-
ation of the parental intervention. data is entered by study personnel into a password pro-
tected database. Data quality is checked by means of
plausibility tests and implausible data are compared
against the original questionnaires. Double data entry of
a random sample of participants is conducted to control
the rate of data entry errors. Access to the data is only
permitted to specific members of the research team. Statistical analysis analysis. Data analysis is conducted with the SAS for
Windows, Version 9.3 or higher (SAS Institute, Cary,
NC, USA) or other software. In general, multi-level regression models (Generalized
Linear Mixed Models (GLMM)) with cluster effects
(school and class within school) will be used for all stat-
istical analyses. For the primary analysis, a logit model
will be used, which will be adjusted for smoking status
at baseline, based on the intention to treat (ITT) popula-
tion. Testing will be hierarchical as described above with
an overall level of significance of 5% (two-sided). All fur-
ther analyses will be considered explorative. Sample size determination
h
l
f ll Taking into account the cluster
design with an assumed intra-cluster correlation coef-
ficient for schools (ICC) of 0.001 and a power of 80%,
15 (14.7 and 13.3 precisely for the two hypotheses)
schools (=clusters) are required in each of the three
arms to detect a 5% difference in the proportion of
regular smokers after two years (assumptions: inter-
vention 1: 30%, intervention 2: 25%, and control group:
35%). Since we assume on average 60 participating stu-
dents per school, this yields a total of 15 × 3 × 60 =
2700 required students. Assuming a drop-out rate of
about 20% this yields a targeted sample size of n = 3375
students to be randomized. Data management at the Institute for Social Medicine,
Epidemiology and Health Economics is conducted ac-
cording to standard operating procedures. Questionnaire Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Page 7 of 10 Recruitment process and distribution of participating
schools and students Schools, school classes and study participants were re-
cruited over a period of two school years. Out of all 214
secondary schools in Berlin that were approached in
2010, a total of 49 secondary schools were enrolled over
two recruitment waves and randomized. Two schools
withdrew despite their initial commitment after being
randomized to control group, leaving a total of 47 par-
ticipating schools. During the school year 2010/11
(wave 1) 32 schools were enrolled. Further 15 schools
were enrolled during the school year 2011/12 (wave 2)). For details of the school and participant recruitment
process see Figure 2. Schools were recruited from all 12
districts of the city state of Berlin (s. Figure 3). With Secondary analysis of the primary endpoint include
models with adjustment for smoking status at baseline and
other baseline variables in case of relevant imbalances be-
tween treatment groups. In addition, missing values will be
imputed as sensitivity analyses. A per-protocol (PP) popula-
tion will be defined and analyzed in a similar manner. Secondary endpoints will be analyzed within similar
frameworks as applicable. All analyses will be specified in detail in a statistical ana-
lysis plan (SAP) which will be finalized prior to data Figure 2 Study-flow up to baseline assessment. Figure 2 Study-flow up to baseline assessment. Page 8 of 10 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Figure 3 Distribution of participating schools across all 12 districts of Berlin. Figure 3 Distribution of participating schools across all 12 districts of Berlin. SD: standard deviation, *Based on the number of students completing the baseline questionnaire. Recruitment process and distribution of participating
schools and students Table 3 Distribution of schools, school classes and students, as well as cluster sizes overall and by intervention groups
Overall
Student intervention
Student-parent intervention
Control group
Total schools
47
17 (36%)
16 (34%)
14 (30%)
Integrated secondary school
32
11 (34%)
11 (34%)
10 (31%)
Gymnasium
15
6 (40%)
5 (33%)
4 (27%)
School classes
161
62 (42%)
58 (31%)
41 (27%)
Students
2801*
1142 (41%)
980 (35%)
679 (24%)
Students per school type
Gymnasium
1173* (41.9%)
525 (46.0%)
394 (40.2%)
254 (37.4%)
Integrated secondary school
1628* (58.1%)
617 (54.0%)
586 (59.8%)
425 (62.6%)
Classes per school (mean ± SD)
3.4 ± 1.8
3.7 ± 1.8
3.6 ± 1.8
3.0 ± 1.9
Students per school (mean ± SC)
59.6 ± 35.3*
67.2 ± 36.2
61.3 ± 38.2
48.5 ± 30.1
Students per class (mean ± SD)
17.4 ± 6.3*
18.4 ± 5.5
17.2 ± 6.5
16.2 ± 7.1
SD: standard deviation, *Based on the number of students completing the baseline questionnaire. f schools, school classes and students, as well as cluster sizes overall and by intervention groups Table 3 Distribution of schools, school classes and students, as well as cluster sizes overall and by Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 Page 9 of 10 regard to the school type, 32 are considered integrated
secondary schools while 15 schools are high schools
(“Gymnasien”). investigate the impact of various individual family charac-
teristics, as well as neighborhood SES on smoking and
other health behaviors. This will make findings highly rele-
vant to populations which are frequently not adequately
reached through prevention efforts. Within these 47 schools, the total number of recruited
school classes is 161, reflecting an average number of
school classes per school of 3.4 (SD: 1.8). The total num-
ber of students meeting all selection criteria and provid-
ing written informant assent and parental consent is n =
3023, reflecting the total study population. For a detailed
description of the allocation of schools, school classes
and students see Table 3. Out of those, 2801 participants
completed the baseline study questionnaire (s. Figure 2). Outcomes of the present school-based RCT are
assessed at baseline, 12 month and 24 month through
self-report, similar to many previous school-based smok-
ing prevention studies [10,11,20]. However, self-report has
its limitations and can result in over- or underreporting of
relevant behaviors. Authors’ contributions FMR, NR, SR, JMN and SNW were conceiving the study. FMR, LK, CB and NSB
conducted the study. FMR drafted the manuscript and all authors were
involved in redrafting of the manuscript. All authors read and approved the
final manuscript. Recruitment process and distribution of participating
schools and students Another strength of the present study
is that in addition to self-report, main study outcomes
will be assessed using objective measures, namely CO
and cotinine measurements. This will provide valuable
additional information to strengthen conclusions based
on self-report outcomes. Competing interests
h
h
d
l
h Competing interests
The authors declare that they have no competing interests. Although our study did not aim to enroll a representa-
tive population of 7th graders in Berlin we were able to re-
cruit schools and students from all 12 districts of the city. This ensures that schools and students are located in dis-
tricts and neighborhoods with diverse characteristics and
SES. While we recruited schools, school classes and stu-
dents from all these districts, the distribution of those does
not fully reflect the general distribution across the city of
Berlin. For instance, certain districts are overrepresented
in terms of the relative number of schools and students en-
rolled, while others underrepresented. In terms of the type
of school, there are 121 integrated secondary schools and
93 “Gymnasien” in Berlin. Within the BEST-Prevention
study we enrolled 32 integrated secondary schools and 15
Gymnasien, reflecting a somewhat different distribution. Our population based sample in a major metropolitan area
in Germany and its diversity will offer the opportunity to Acknowledgments The authors acknowledge support from Ulrike Stasun and Annette Wagner,
data manager and study nurse, as well as from student assistants and
doctoral students. The authors further acknowledge Karuna e.V und KOSS
(Koordinierungsstelle Schulische Suchtvorbeugung Schleswig-Holstein) for
supporting and providing the intervention strategies. The authors also
acknowledge support from the Senatsverwaltung für Bildung, Jugend und
Wissenschaft, Berlin and from participating schools, teachers and students for
supporting the project. Conclusion In summary, the present manuscript provides an overview
of the study design and methodology of a large school-
based smoking prevention cluster randomized controlled
trial in a metropolitan area in Germany. Recruitment of
the BEST-prevention study was successful in enrolling tar-
geted numbers of schools and school classes, although the
number of students was somewhat lower than planned. Moreover, enrolled schools and students represent all dis-
tricts of the city. Findings from our study will soon pro-
vide valuable information with regards to the acceptability
and effectiveness of specific smoking prevention strategies
also in disadvantaged population groups. Furthermore, the
additional effectiveness of strategies targeting parents will
be determined. Despite recruitment challenges due to the implementa-
tion of a school reform in Berlin in 2010, which reduced
the number of potentially eligible schools by about 50%,
the targeted number of 45 schools and 161 school classes
was achieved or even exceeded. However, with 3023 stu-
dents meeting all selection criteria, the estimated sample
size was not fully met. Discussion The BEST-Prevention study is among the largest ran-
domized controlled trials in Germany that investigates
the effectiveness of smoking prevention strategies among
adolescents [10]. It was designed to address important
and unresolved issues in the context of behavioral smok-
ing prevention strategies. Findings will be of importance
for a number of reasons: Firstly, the BEST-Prevention
study targets specific intervention components and ad-
dresses the additional effectiveness of a feasible parental
smoking prevention strategy to reduce smoking rates
among adolescents, which few studies have done so far
[11,20]. Secondly, it targets adolescents at increased risk of
smoking due to their diverse background and their resi-
dence in a large metropolitan area in Germany. Thirdly,
this study assesses the long-term effectiveness of a smok-
ing prevention intervention. Ethics Approval from the Charité-Universitätsmedizin Berlin
institutional review board was obtained. Received: 30 June 2014 Accepted: 14 July 2014
Published: 23 August 2014 y
22. Jarvis MJ, Tunstall-Pedoe H, Feyerabend C, Vesey C, Saloojee Y: Comparison
of tests used to distinguish smokers from nonsmokers. Am J Public Health
1987, 77(11):1435–1438. 22. Jarvis MJ, Tunstall-Pedoe H, Feyerabend C, Vesey C, Saloojee Y: Comparison
of tests used to distinguish smokers from nonsmokers. Am J Public Health
1987, 77(11):1435–1438. References 23. Cooke F, Bullen C, Whittaker R, McRobbie H, Chen MH, Walker N: Diagnostic
accuracy of NicAlert cotinine test strips in saliva for verifying smoking
status. Nicotine Tob Res 2008, 10(4):607–612. 23. Cooke F, Bullen C, Whittaker R, McRobbie H, Chen MH, Walker N: Diagnostic
accuracy of NicAlert cotinine test strips in saliva for verifying smoking
status. Nicotine Tob Res 2008, 10(4):607–612. 1. Ng M, Freeman MK, Fleming TD, Robinson M, Dwyer-Lindgren L, Thomson B,
Wollum A, Sanman E, Wulf S, Lopez AD, Murray CJL, Gakidou E: Smoking
prevalence and cigarette consumption in 187 countries, 1980–2012. JAMA 2014, 311(2):183–192. doi:10.1186/1471-2458-14-871
Cite this article as: Müller-Riemenschneider et al.: Berlin evaluates school
tobacco prevention - BEST prevention: study design and methodology. BMC Public Health 2014 14:871. 2. World Health Organization: WHO Global Report: Mortality Attributable to
Tobacco; 2012. 3. Warren CW, Jones NR, Peruga A, Chauvin J, Baptiste J-P, Costa de Silva V,
el Awa F, Tsouros A, Rahman K, Fishburn B, Bettcher D, Asma S: Global youth
tobacco surveillance, 2000–2007. MMWR Surveill Summ 2008, 57(1):1–28. 3. Warren CW, Jones NR, Peruga A, Chauvin J, Baptiste J-P, Costa de Silva V,
el Awa F, Tsouros A, Rahman K, Fishburn B, Bettcher D, Asma S: Global youth
tobacco surveillance, 2000–2007. MMWR Surveill Summ 2008, 57(1):1–28. 4. BZgA: Der Tabakkonsum Jugendlicher und junger Erwachsener in
Deutschland 2012. Ergebnisse einer aktuellen Repräsentativbefragung
und Trends. In Cologne: Federal Centre for Health Education; 2013. 4. BZgA: Der Tabakkonsum Jugendlicher und junger Erwachsener in
Deutschland 2012. Ergebnisse einer aktuellen Repräsentativbefragung
und Trends. In Cologne: Federal Centre for Health Education; 2013. 5. Richter M, Pfortner TK, Lampert T, Deutschland H-T: Changes in tobacco,
alcohol and cannabis use by adolescents from 2002 to 2010 in Germany. Gesundheitswesen 2012, 74(Suppl):S42–S48. 5. Richter M, Pfortner TK, Lampert T, Deutschland H-T: Changes in tobacco,
alcohol and cannabis use by adolescents from 2002 to 2010 in Germany
Gesundheitswesen 2012, 74(Suppl):S42–S48. 6. Warren CW, Lea V, Lee J, Jones NR, Asma S, McKenna M: Change in
tobacco use among 13–15 year olds between 1999 and 2008: findings
from the Global Youth Tobacco Survey. Glob Health Promot 2009,
16(2 Suppl):38–90. 6. Warren CW, Lea V, Lee J, Jones NR, Asma S, McKenna M: Change in
tobacco use among 13–15 year olds between 1999 and 2008: findings
from the Global Youth Tobacco Survey. Glob Health Promot 2009,
16(2 Suppl):38–90. 7. Funding
h This project was funded by The German Cancer Aid (Deutsche Krebshilfe e.V.). Page 10 of 10 Müller-Riemenschneider et al. BMC Public Health 2014, 14:871
http://www.biomedcentral.com/1471-2458/14/871 References Muller-Riemenschneider F, Nocon M, Willich SN: Prevalence of modifiable
cardiovascular risk factors in German adolescents. Eur J Cardiovasc Prev
Rehabil 2010, 17:204–210. 8. Zatoński W, Przewoźniak K, Sulkowska U, West R, Wojtyła A: Tobacco
smoking in countries of the European Union. Ann Agric Environ Med 2012,
19(2):181–192. 8. Zatoński W, Przewoźniak K, Sulkowska U, West R, Wojtyła A: Tobacco
smoking in countries of the European Union. Ann Agric Environ Med 2012,
19(2):181–192. 9. US Department of Health and Human Services: Preventing Tobacco Use
Among Youth and Young Adults - A report of the Surgeon General. In
US Department of Health and Human Services, Centers for Disease Control and
Prevention, National Center for Chronic Disease Prevention and Health
Promotion, Office on Smoking and Health; 2012. 10. Thomas RE, McLellan J, Perera R: School-based programmes for
preventing smoking. Cochrane Database Syst Rev 2013, 4:CD001293. 11. Muller-Riemenschneider F, Bockelbrink A, Reinhold T, Rasch A, Greiner W,
Willich SN: Long-term effectiveness of behavioural interventions to prevent
smoking among children and youth. Tob Control 2008, 17(5):301–302. 12. Gilman SE, Rende R, Boergers J, Abrams DB, Buka SL, Clark MA, Colby SM,
Hitsman B, Kazura AN, Lipsitt LP, Lloyd-Richardson EE, Rogers ML, Stanton CA,
Stroud LR, Niaura RS: Parental smoking and adolescent smoking initiation:
an intergenerational perspective on tobacco control. Pediatrics 2009, 123(2):e274–e281. 13. Albers AB, Biener L, Siegel M, Cheng DM, Rigotti N: Household smoking
bans and adolescent antismoking attitudes and smoking initiation:
findings from a longitudinal study of a Massachusetts youth cohort. Am J Public Health 2008, 98(10):1886–1893. Author details
1I
i
f
S 18. Curry SJ, Hollis J, Bush T, Polen M, Ludman EJ, Grothaus L, McAfee T: A
randomized trial of a family-based smoking prevention intervention in
managed care. Prev Med 2003, 37(6):617–626. 1Institute for Social Medicine, Epidemiology and Health Economics, Charité
Universitätsmedizin, Luisenstrasse 57, 10117 Berlin, Germany. 2Saw Swee
Hock School of Public Health, National University of Singapore, 16 Medical
Drive, Singapore 117597, Singapore. 3Institute of Nutritional Medicine,
University of Hohenheim, Fruwirthstraße 12, 70599 Stuttgart, Germany. 4Berlin School of Public Health, Charité Universitätsmedizin, Seestraße 73,
13347 Berlin, Germany. 19. Stanton B, Cole M, G J: Randomized trial of a parent intervention: parents
can make a difference in long-term adolescent risk behaviors, perceptions,
and knowledge. JAMA pediatrics 2004, 158(10):947–955. g
p
20. Re T, Pra B, Lorenzetti D: Family-based programmes for preventing smoking
by children and adolescents ( Review ), Volume 4. 2008. 21. Völzke H, Neuhauser H, Moebus S, Baumert J, Berger K, Stang A, Ellert U,
Werner A, Döring A: Regional disparities in smoking among adults in
Germany. Dtsch Ärztbl 2006, 103(42):1–8. 21. Völzke H, Neuhauser H, Moebus S, Baumert J, Berger K, Stang A, Ellert U,
Werner A, Döring A: Regional disparities in smoking among adults in
Germany. Dtsch Ärztbl 2006, 103(42):1–8. Received: 30 June 2014 Accepted: 14 July 2014
Published: 23 August 2014 Received: 30 June 2014 Accepted: 14 July 2014
Published: 23 August 2014 Received: 30 June 2014 Accepted: 14 July 2014
Published: 23 August 2014 References Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: Submit your next manuscript to BioMed Central
and take full advantage of: 14. Sargent JD, Dalton M: Does parental disapproval of smoking prevent
adolescents from becoming established smokers? Pediatrics 2001,
108(6):1256–1262. • Convenient online submission 15. Harakeh Z, Scholte RH, de Vries H, Engels RC: Parental rules and
communication: their association with adolescent smoking. Addiction 2005, 100(6):862–870. 16. Guilamo-Ramos V, Jaccard J, Dittus P, Gonzalez B, Bouris A, Banspach S: The
Linking Lives health education program: a randomized clinical trial of a
parent-based tobacco use prevention program for african american and
latino youths. Am J Public Health 2010, 100(9):1641–1647. 17. Jackson C, Dickinson D: Enabling parents who smoke to prevent their
children from initiating smoking. Arch Pediatr Adolesc Med 2006,
160(1):56–62.
|
https://openalex.org/W4388023518
|
https://cabiagbio.biomedcentral.com/counter/pdf/10.1186/s43170-023-00185-z
|
English
| null |
Does cadmium cause cascading effects on the development and reproduction of the striped stem borer, Chilo suppressalis (Walker)?
|
CABI agriculture and bioscience
| 2,023
|
cc-by
| 7,517
|
© 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. Abstract The heavy metal, cadmium (Cd), causing growth retardation and yield reduction on rice and impacting the fitness
of organisms inhabiting on rice through bottom-up effects, has become a great challenge to rice production. However, the effect of Cd-exposure on the development of an economically important and destructive rice pest,
Chilo suppressalis remains unexplored. By exposing the larvae of C. suppressalis to different Cd-exposed artificial diets
(0, 0.2, 1.0, 2.5, 5.0, and 10.0 mg/kg), we found that Cd exposure did not affect the larval duration or pupation rate
of C. suppressalis, but caused negative effects on pupal weight at high Cd levels (5.0 and 10.0 mg/kg) and on adult
deformity rate from 2.5 and 5.0 mg/kg treatments. Although Cd significantly increased the female pupae ratio, C. suppressalis did not oviposit when Cd treatment was more than 2.5 mg/kg. Meanwhile, Cd transferred to pupae,
females, exuviae of pupa and eggs of C. suppressalis from Cd treated larvae, and exhibited a dose-dependent response
on Cd accumulation. Our results indicated that Cd had a negative effect on rice stem borer and can be transferred
to eggs of C. suppressalis, but more work is needed to further assess the bottom-up effect on third tropic levels
in Cd-polluted fields. Keywords Heavy metal, Cadmium, Rice, Pest, Chilo suppressalis, Bottom-up effect Does cadmium cause cascading effects
on the development and reproduction
of the striped stem borer, Chilo suppressalis
(Walker)? Hexi Huang1,2,3†, Ning Di2,3†, Jie Wang2,3,4, Yuxing Wang2, Zhengyang Zhu2,3, Caige Lu2, Su Wang2,3* and
Liansheng Zang1* Introduction Agricultural soils are under threat of toxic metal
contamination from anthropogenic activities, leading
to excessive accumulation of arsenic (As), cadmium
(Cd), and lead (Pb), and so on, in food crops that poses
significant risks to human health (Liu et al. 2013; Zhao
et al. 2022). Among the toxic metals, Cd ranks seventh in
the list of the top 20 toxic metals due to its high toxicity
and mobility (Wang et al. 2019). China is facing great
challenges in protecting its soil from Cd contamination
caused by rapid industrialization and urbanization
over the last three decades, and 2.786 × 105 hectare
agricultural soils have been reported to be polluted
by Cd (Hussain et al. 2021). Among the soils heavily †Hexi Huang and Ning Di have equally contributed to this work. *Correspondence:
Su Wang
wangsu@ipepbaafs.cn
Liansheng Zang
lsz0415@163.com
1 Key Laboratory of Green Pesticide and Agricultural Bioengineering
of Ministry of Education, Guizhou University, Guiyang 550025, China
2 Institute of Plant Protection, Beijing Academy of Agriculture and Forestry
Sciences, Beijing 100097, China
3 Key Laboratory of Natural Enemies Insects, Ministry of Agriculture
and Rural Affairs, Beijing 100122, China
4 College of Plant Protection, Nanjing Agricultural University,
Nanjing 210095, China 1 Key Laboratory of Green Pesticide and Agricultural Bioengineering
of Ministry of Education, Guizhou University, Guiyang 550025, China
2 Institute of Plant Protection, Beijing Academy of Agriculture and Forestry
Sciences, Beijing 100097, China 3 Key Laboratory of Natural Enemies Insects, Ministry of Agriculture
and Rural Affairs, Beijing 100122, China Huang et al. CABI Agriculture and Bioscience (2023) 4:45
https://doi.org/10.1186/s43170-023-00185-z Huang et al. CABI Agriculture and Bioscience (2023) 4:45
https://doi.org/10.1186/s43170-023-00185-z CABI Agriculture
and Bioscience CABI Agriculture
and Bioscience Huang et al. CABI Agriculture and Bioscience
https://doi.org/10.1186/s43170-023-00185-z Open Access Cd treatment
d According to China Food Safety National Standard for
Maximum Levels of Contaminants in Foods GB2762-
2022 (SAMR and NHCC 2022), and Cd concentrations
in rice storks in paddies of China (Liu 2020), five
different dose levels of Cd2+ in artificial diets (0.2, 1.0,
2.5, 5.0 and 10 mg/kg) was used. Cadmium chloride
(CdCl2, Shanghai Aladdin Bio-Chem Technology Co.,
LTD, China) was dissolved in 10 mL distilled water at
different dose levels, and mixed homogeneously to
1000 g artificial diet to make the Cd-contained artificial
diets. The same volume of distilled water was added
to the control diet. Neonates of SSB were reared on
normal clean diets in plastic boxes (16 × 11 × 8 cm3). Ten days later, the larvae were transferred individually
to 12 well cell culture plates (one larva per well;
125 × 85 × 23 mm3, Nantong Ruique Experimental
Equipment Co., Ltd). Ten g artificial diet cut into cubes
was added to cells. Diets were changed every week until
pupation. Fifty larvae were used in each treatment with
three replications, hundred and fifty larvae were used
in each treatment (N = 150). Pupae in each treatment
were collected and put into 12 well cell culture plates
separately. Adults were paired after eclosion in
cages, and twenty pairs per treatment were used to
evaluate the effect of Cd exposure on the oviposition
of C. suppressalis (N = 20). Rice plants described as
above were provided for oviposition and 10% honey
water solution were supplied as a food source for C. suppressalis adults. f
p
g
y
The bottom-up effects on pests have been considered
key levers for optimizing integrated pest management
(IPM) (Han et al. 2015, 2016; Di et al. 2021; Liu et al. 2022
and see Han et al. 2022 for a thorough review). Increasing
soil pollution stresses the need for a better understanding
of the effects on multi-trophic interactions and their
potential impact on the efficacy of IPM. Rice is a major
component of diet for people worldwide, and can
absorb Cd (Ma et al. 2021). It accumulates more Cd than
other cereal crops such as barley and wheat (Sui et al. 2018). The striped stem borer (SSB), Chilo suppressalis
Walker (Lepidoptera: Pyralidae), is one of the most
economically important and destructive rice pests (Ma
et al. 2020; Zheng et al. 2021). Open Access T (2020) showed that
the life history traits of Mythimna separata (Walker) can
be impacted by Cd, from which the body mass of larvae
was decreased in low Cd treatment, but was increased by
intermediate and high concentrations of Cd treatment. Therefore, it is necessary to assess the dose-dependent
effect of Cd on herbivores in Cd polluted agroecosystems.hf contaminated by Cd, paddy soils show an exceedance
rate of 33.2% of the Chinese national standard (Liu et al. 2016; Wang et al. 2019). Cd exposure in agricultural soils,
not only causes plant growth retardation and decreased
yield (Yu et al. 2022), but also results in bottom-up
effects impacting the fitness of organisms inhabiting
agricultural systems (Butler and Trumble 2008; Han
et al. 2022). Cd can be absorbed and accumulated in
plants, and transferred to herbivores through feeding
(Kaminski et al. 2021; Godinho et al. 2022). Generally,
Cd may cause negative effects on herbivores survival,
development, and behavior, and population dynamics
(Chen et al. 2022; Zhang et al. 2023). For example,
Lin et al. (2020) found that feeding on leaves from
Cd-exposed plants significantly reduced the growth and
survivals of herbivory moths, Botyodes diniasalis Walker
and Spodoptera exigua (Hübner). However, exposure to
mild stress of Cd may also induce hormetic effects in
insects (Cutler et al. 2022). Wei et al. (2020) showed that
the life history traits of Mythimna separata (Walker) can
be impacted by Cd, from which the body mass of larvae
was decreased in low Cd treatment, but was increased by
intermediate and high concentrations of Cd treatment. Therefore, it is necessary to assess the dose-dependent
effect of Cd on herbivores in Cd polluted agroecosystems.hf Materials
Insects rearing Population of the rice stem borer, C. suppressalis,
was initially collected in 2022 from rice paddy fields
in Nanchang County, Jiangxi Province, China and
maintained under laboratory conditions in the Lab
of Applied Entomology (LAE), Institute of Plant
Protection (IPP), Beijing Academy of Agricultural
and Forestry Sciences (BAAFS), China. Larvae were
reared on artificial diets at 28 ± 1 °C and 70 ± 5% relative
humidity with a photoperiod of 16:8 h (L:D). Artificial
diets were prepared according to the protocol from Han
et al. (2012). Clean rice plants at the tillering stage (30
d after sowing, about 35 cm in height) were provided
for oviposition and 10% honey water solution were
supplied as a food source for C. suppressalis adults. Open Access T © 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. Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. CABI Agriculture and Bioscience Page 2 of 8 contaminated food source on pests, and the potential
influence on its natural enemies were discussed. contaminated by Cd, paddy soils show an exceedance
rate of 33.2% of the Chinese national standard (Liu et al. 2016; Wang et al. 2019). Cd exposure in agricultural soils,
not only causes plant growth retardation and decreased
yield (Yu et al. 2022), but also results in bottom-up
effects impacting the fitness of organisms inhabiting
agricultural systems (Butler and Trumble 2008; Han
et al. 2022). Cd can be absorbed and accumulated in
plants, and transferred to herbivores through feeding
(Kaminski et al. 2021; Godinho et al. 2022). Generally,
Cd may cause negative effects on herbivores survival,
development, and behavior, and population dynamics
(Chen et al. 2022; Zhang et al. 2023). For example,
Lin et al. (2020) found that feeding on leaves from
Cd-exposed plants significantly reduced the growth and
survivals of herbivory moths, Botyodes diniasalis Walker
and Spodoptera exigua (Hübner). However, exposure to
mild stress of Cd may also induce hormetic effects in
insects (Cutler et al. 2022). Wei et al. Cd measurements in C. suppressalis Cd measurements were followed by Di et al. (2020)
with adjusts. Insect samples (2 larvae in the late stage
of sixth instar, 2 pupa, 5 female pupal exuviae, 2 female
adults, and 1000 eggs were grounded separately for
each replication, n = 3) were dried at 65 °C for 48 h, and
grounded into a fine powder. Samples were digested by
10 mL of nitric acid and perchloric acid mixed solution
at 10: 1 in microwave dissolver at 170 °C for 30 min. The
digested samples were filtered and diluted to 25 mL with
5% dilute nitric acid. The Cd content in each sample was
analyzed by inductively coupled plasma source mass
spectrometer (ICP-MS) (Thermo Scientific iCAP RQ)
in the standard (STD) mode. Commercially available
standard Cd solutions preserved in nitric acid were used
to prepare the calibration standards. Statistical analysis
All
d All data weretested for normality (Shapiro–Wilk
test, P < 0.05) and homoscedasticity (Levene’s test,
P < 0.05) before analysis of variance. Percentage data for
emergence rate, female pupa ratio, pupa deformity rate,
and adult deformity rate were transformed to arcsine
square root prior to the normality test. A t-test was used
to compare the control with different Cd treatments,
*indicates significant difference between control and
treatment at P < 0.05. One-way ANOVA was used to
analyze the difference of Cd content in different stages
of the pest, following with a Duncan post-hoc pairwise
comparison, and different lowercase letters in the same
stage indicate significant difference at P < 0.05. Statistical
analyses were performed using the SPSS statistical
software package (var. 25.0, IBM, USA). Emergence rate (%) = (number of adult moth/number
of pupae) × 100% Emergence rate (%) = (number of adult moth/number
of pupae) × 100% Emergence rate (%) = (number of adult moth/number
of pupae) × 100% Developmental time of larvae = time (d) from first
instar to prepupae Developmental time of larvae = time (d) from first
instar to prepupae Pupa deformity rate (%) = (number of deformity
pupae/number of pupae) × 100% Adult deformity rate (%) = (number of deformity
adult/number of emergence adult) × 100% Life history traits of C. suppressalis under Cd exposureh The bioconcentration factor (BCF) and translocation
factor (TF) are used to evaluate the ability of C. suppressalis to tolerate and accumulate heavy metals
(Jiang et al. 2020). Life history traits of C. suppressalis under Cd exposureh The alive numbers and died numbers were recorded
every day. Pupae numbers were recorded and were sexed
and weighed on the second day after pupation (N = 30). Then emergence numbers of both females and males
were also recorded. BCF = Metal concentration in the receiving level/
Metal concentration in diet
TF = Metal concentration in receiving level/Metal
concentration in the source level BCF = Metal concentration in the receiving level/
Metal concentration in diet Life history traits were calculated following Zhan et al. (2017): TF = Metal concentration in receiving level/Metal
concentration in the source level Larval mortality (%) = (number of deaths/number of
total larvae) × 100% Larval mortality (%) = (number of deaths/number of
total larvae) × 100% Pupation rate (%) = (number of pupae/number of
sixth instar larvae) × 100% Pupation rate (%) = (number of pupae/number of
sixth instar larvae) × 100% *Indicates significant difference between control and treatment (t-test, P < 0.05) *Indicates significant difference between control and treatment (t-test, P < 0.05) Cd treatment
d There is a great possibility
that the development and population dynamic of SSB
can be affected by Cd cascading from rice plants in
Cd-contained paddy soils, but there are only several
reports on how Cd affect the fitness of SSB (Zhang et al. 2017; Liu 2020).f To investigate the effect of Cd-exposure on SSB,
larvae of C. suppressalis were exposed to different
Cd-contaminated artificial diets (0, 0.2, 1.0, 2.5, 5.0, and
10.0 mg/kg), and (1) the effects of Cd on the development,
and survival of C. suppressalis throughout the entire life
cycle, (2) the accumulation and transfer of Cd through
the main developmental stages of C. suppressalis were
assessed. The bottom-up effects cascading from Cd Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Page 3 of 8 Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. CABI Agriculture and Bioscience Effect of Cd exposure on the performance of C. suppressalis
on artificial diet In our study, Cd exposure in artificial diets showed
negative effects on the performances of C. suppressalis
(Table 1). Although the larval duration and pupation
rate of C. suppressalis did not differ between Cd-exposed
artificial diets and control artificial diets, the emergence
rate and female pupa ratio were significantly affected by
Cd exposure in artificial diet (Table 1). The emergence
rate of C. suppressalis were significantly higher in 1.0 Table 1 Development parameters of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+
Values are given as means +/− SE
*I di
t
i
ifi
t diff
b t
t
l
d t
t
t ( t
t P
0 05)
Cd concentration added in
artificial diet (mg/kg)
Larval duration (d)
Pupation rate (%)
Emergence rate (%)
Female pupa ratio
0 (control)
40.0 ± 1.3
68.00 ± 1.02
74.56 ± 1.89
0.31 ± 0.02
0.2
40.0 ± 1.4
74.67 ± 1.10
74.92 ± 2.37
0.37 ± 0.02
1.0
40.6 ± 0.1
71.33 ± 1.05
80.55 ± 4.46*
0.50 ± 0.01*
2.5
42.9 ± 1.3
68.00 ± 2.26
62.47 ± 2.83*
0.41 ± 0.01*
5.0
42.8 ± 1.3
62.67 ± 1.22
80.63 ± 3.81*
0.56 ± 0.02*
10.0
42.3 ± 1.4
54.67 ± 1.34
75.70 ± 4.46
0.50 ± 0.02* meters of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Page 4 of 8 Fig. 1 Weight of pupa of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ (A, Female pupa; B, Male pupa. Pupae were weighed on the second day after pupation. N = 30, t-test, **P < 0.01, ***P < 0.001) Fig. 1 Weight of pupa of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ (A, Female pupa; B, Male pupa. Pupae were weighed on the second day after pupation. N = 30, t-test, **P < 0.01, ***P < 0.001) Fig. 1 Weight of pupa of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ (A, Fema
Pupae were weighed on the second day after pupation. N = 30, t-test, **P < 0.01, ***P < 0.001) Cd bio-concentrated in larva, pupa, and female in lower
concentration of Cd-exposed artificial diets (≤ 1.0 mg/kg),
consistent with translocation of Cd from diets to larvae
(Table 3). Discussion Cd is one of the most important metal pollutants in
soil, triggering multiple indirect bottom-up effects in
agroecosystems (Dar et al. 2019; Liu et al. 2023; Yan et al. 2023). It can be absorbed by plants, and transported to g
Cd exposure in artificial diets didn’t affect the
pupation, but resulted in significantly negative effects
on the emergence of C. suppressalis (Table 2). The adult
deformity rate of C. suppressalis in 2.5 and 5.0 mg/kg
Cd-exposed artificial diets were significantly higher than
that in control diets (2.5 mg/kg: t = − 3.487, P = 0.025;
5.0 mg/kg: t = − 2.919, P = 0.043, Table 2). Table 2 Pupa and adult deformity rate of Chilo suppressalis fed
with the artificial diets containing different concentrations of
Cd2+ Table 2 Pupa and adult deformity rate of Chilo suppressalis fed
with the artificial diets containing different concentrations of
Cd2+
Values are given as means +/− SE
*Means the significant difference between control and treatment (t-test,
Cd concentration added in
artificial diet (mg/kg)
Pupa deformity
rate (%)
Adult
deformity
rate (%)
0 (control)
1.04 ± 0.85
5.34 ± 1.22
0.2
3.59 ± 0.75
5.90 ± 0.87
1.0
0.81 ± 0.66
7.03 ± 0.34
2.5
3.94 ± 0.80
16.17 ± 2.53*
5.0
5.42 ± 1.02
13.64 ± 2.22*
10.0
2.59 ± 1.10
4.94 ± 0.41 V l
i
/
SE
Cd concentration added in
artificial diet (mg/kg)
Pupa deformity
rate (%)
Adult
deformity
rate (%)
0 (control)
1.04 ± 0.85
5.34 ± 1.22
0.2
3.59 ± 0.75
5.90 ± 0.87
1.0
0.81 ± 0.66
7.03 ± 0.34
2.5
3.94 ± 0.80
16.17 ± 2.53*
5.0
5.42 ± 1.02
13.64 ± 2.22*
10.0
2.59 ± 1.10
4.94 ± 0.41 Effect of Cd exposure on the performance of C. suppressalis
on artificial diet However, when Cd treatment concentration
was ≥ 2.5 mg/kg, Cd did not bio-concentrated in larvae,
pupae, or female adults, except for females from 5.0 mg/
kg treatment groups (1.08, Table 3). When the larvae
were fed with the artificial diets containing Cd, Cd can be
transferred to the eggs of C. suppressalis but can’t be bio-
concentrated in eggs (Table 3). and 5.0 mg/kg Cd-exposed artificial diets than that in
control diets (1.0 mg/kg: t = − 3.514, P = 0.024; 5.0 mg/
kg: t = − 3.334, P = 0.029, Table 1). Moreover, the female
pupa ratio of C. suppressalis was significantly higher
in Cd-exposed artificial diets (expected for 0.2 mg/
kg) than that in control diets (Table 1). Cd exposure in
artificial diets showed negative effects on pupal weight
of C. suppressalis in high concentrations (i.e., 5.0 mg/
kg, female: t = 3.668, P = 0.005, Fig. 1). Compared to the
control, the pupal weight of C. suppressalis in 10.0 mg/
kg Cd-exposed artificial diets was significantly lower
no matter with female and male (10.0 mg/kg, female:
t = 3.668, P < 0.001; male: t = 4.154, P < 0.001, Fig. 1). Means the significant difference between control and treatment (t test,
P < 0.05). – means that female can’t oviposit under such Cd stress Values are given as means +/− SE Contamination and transfer of Cd in C. suppressalis
on artificial dieti When the larvae were fed with the artificial diets
containing different concentrations of Cd, Cd can be
transferred to the larvae, pupa, female, and eggs of C. suppressalis (Fig. 2). And C. suppressalis exhibited a
dose-dependent response to Cd accumulation. We found
that the exuviae of C. suppressalis can also accumulate
significant amount of Cd (F = 628.22, df = 5, 17, P < 0.001,
Fig. 2). Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. CABI Agriculture and Bioscience Page 5 of 8 Fig. 2 Cd content of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ (N = 3, one way ANOVA, different
lowercase letters in the same stage indicate significant difference at P < 0.05) Fig. 2 Cd content of Chilo suppressalis fed with the artificial diets containing different concentrations of Cd2+ (N = 3, one way ANOVA, different
lowercase letters in the same stage indicate significant difference at P < 0.05) Table 3 Bioconcentration (BCF) and translocation factors (TF) of Cd different developmental stages of Chilo suppressalis fed with the
artificial diets containing different concentrations of Cd2+
Cd concentration added in
artificial diet (mg/kg)
BCF
TF
Larva
Pupa
Female
Egg
Diet-larva
Larva-pupa
Pupa-female
Female-egg
0.2
1.64
1.51
1.35
0.63
1.64
0.92
0.90
0.47
1.0
1.10
1.18
1.51
0.58
1.10
1.08
1.28
0.38
2.5
0.94
0.50
0.45
–
0.94
0.53
0.90
–
5.0
0.97
0.55
1.08
–
0.97
0.57
1.96
–
10.0
0.99
0.60
0.92
–
0.99
0.61
1.53
– Table 3 Bioconcentration (BCF) and translocation factors (TF) of Cd different developmental stages of Chilo suppressalis fed with the
artificial diets containing different concentrations of Cd2+ phytophagous insects through feeding (Lin et al. 2020;
Chen et al. 2022). Many studies have confirmed that Cd
causes negative effects on the survival and development
of lepidopterous insects (Lin et al. 2020; Luo et al. 2020). In our study, Cd exposure did not affect the
larval duration of C. suppressalis, which is consistent
with the results of Zhang et al. (2017), from which the
larval duration was prolonged once C. suppressalis was
exposed to 15 mg/kg Cd diet. Cd exposure in artificial
diets negatively affected the female pupal weight of
C. suppressalis in high Cd concentrations. This was
consistent to the performance of Spodoptera frugiperda
feeding on Cd-exposed artificial diets (Wang et al. 2023). Contamination and transfer of Cd in C. suppressalis
on artificial dieti CABI Agriculture and Bioscience Page 6 of 8 Page 6 of 8 Furthermore, studies have examined the molecular
mechanisms underlying the influence of heavy mental
exposure on insect reproduction (Chen et al. 2022;
Zhang et al. 2023). RNA-Seq was used to investigate
changes in ovary gene expression in newly emerged
female of beet army worms (Su et al. 2020). Pb stress
causes inhibition on insect life-history traits at the
transcriptome and proteome levels of S. litura (Chen
et al. 2021). Furthermore, other abiotic factors could
impact pests, e.g., low lethal and sublethal concentrations
of insecticides can modulate mRNA transcript level of
Vg gene in Aphis gossypii (Ullah et al. 2019) and silicon
accumulation in maize could affect S. exigua (Leroy et al. 2022). Therefore, further research needs to be done in
mating behavioral and molecular levels, accounting for
the reasons of C. suppressalis can not oviposit under Cd
exposure.f Fe2+ and Mg2+, inhibits the metabolism of N, cuts down
the transportation of water and minerals, and destroys
the homeostasis (Hussain et al. 2021). (
)
It is widely accepted that heavy metals can accumulate
in insects through food chains (Green et al. 2010; Di et al. 2016; Tibbett et al. 2021). In our study, we found that
Cd concentrations in different developmental stages of
C. suppressalis exhibited a significant dose-dependent
relationship. Although Cd could be excreted outside by
female exuviae, Cd still significantly bio-accumulated in
larvae, pupae and females, especially from the lowest two
treatment levels. High concentration of metal treatment
may cause antifeedant effects (Di et al. 2016), leading to
relatively lower bioconcentration factors. Pupation is a
critical remodeling period in metamorphosed insects. All treatment levels show the lowest translocation
factor in the larvae to pupae period, indicating that Cd
could be metabolized through this period. However, it is
significantly transferred from larva to pupa from 1 mg/
kg treatment groups. Due to the toxic effects of Cd, only
females from the lowest two treatments laid enough eggs
for detecting the metal levels. The next generation may
also be affected since eggs could also accumulate Cd. This
should be further studied to the colony levels for more
generations. Heavy metals transfer along the food chain can affect
the growth and physiological activities of predators and
parasitoids (Liu et al. 2023; Tan et al. 2023; Wang et al. 2023). Contamination and transfer of Cd in C. suppressalis
on artificial dieti Cd has been documented to be directly toxic to many
herbivores, including Lepidoptera (Luo et al. 2020; Wei
et al. 2020) and Orthoptera (Zhang et al. 2014). This
effect is suggested as “element defense” (Boyd 2007). We
found Cd exposure in artificial diets did not affect the pupation, but resulted in significantly negative effects
on the emergence of C. suppressalis. In contrast to our
study, Wei et al. (2020) found Cd exposure increased
the pupation rate but did not affect the emergence rate
of M. separata. The differences may be caused by Cd
treatment levels, where herbivores treated under a low
Cd concentration (0.15–0.60 mg/kg, Wei et al. 2020)
than our study (0.2–10.0 mg/kg). Moreover, studies
confirmed that Cd exposure increased the pupation
deformed rate of M. separata and C. suppressalis (Zhang
et al. 2017; Wei et al. 2020). However, there were no
significant differences in the pupation deformed rates
under our Cd-exposed concentrations, and Cd exposure
resulted in a significantly higher adult deformed rate
of C. suppressalis in 2.5 and 5.0 mg/kg Cd-exposed
artificial diets. C. suppressalis from these two treatments
suffered from significantly heavier Cd burden, since Cd
accumulation affects the absorption of basic ions such as phytophagous insects through feeding (Lin et al. 2020;
Chen et al. 2022). Many studies have confirmed that Cd
causes negative effects on the survival and development
of lepidopterous insects (Lin et al. 2020; Luo et al. 2020). In our study, Cd exposure did not affect the
larval duration of C. suppressalis, which is consistent
with the results of Zhang et al. (2017), from which the
larval duration was prolonged once C. suppressalis was
exposed to 15 mg/kg Cd diet. Cd exposure in artificial
diets negatively affected the female pupal weight of
C. suppressalis in high Cd concentrations. This was
consistent to the performance of Spodoptera frugiperda
feeding on Cd-exposed artificial diets (Wang et al. 2023). Cd h
b
d
d
b d
l i
Cd has been documented to be directly toxic to many
herbivores, including Lepidoptera (Luo et al. 2020; Wei
et al. 2020) and Orthoptera (Zhang et al. 2014). This
effect is suggested as “element defense” (Boyd 2007). We
found Cd exposure in artificial diets did not affect the Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. Contamination and transfer of Cd in C. suppressalis
on artificial dieti From our study, Cd accumulation was detected in
the eggs of C. suppressalis. Thus, there is a risk that the
higher trophic level, such as egg parasitoids and predators
will also suffer from Cd burdens. While egg parasitoids
in the genus Trichogramma have been used successfully
in biological control of C. suppressalis (Wang et al. 2021;
Zang et al. 2021), more works are needed to achieve
fully effective IPM (e.g., see Wang et al. 2022). It is an
advantage that Cd adversely affect the fitness of pests,
but there is also a great possibility that the bottom-up
effects mediated by Cd may disrupt the efficiency of pest
biological control in Cd-polluted soils. Therefore, more
efforts are required to access the potential impacts of Cd
exposure in agroecosystems to ensure pest control for
crops and ultimately food security. g
The fecundity of herbivores is an important indicator
for pest management programs. However, the effect of
Cd on the fecundity of herbivores are poorly documented
(Jiang and Yan 2017; Zhan et al. 2017; Liu et al. 2023). Our results showed that the number of eggs per C. suppressalis females under Cd stress was significantly
lower than that in the control diets, indicating there is an
inhibition of insect fecundity by Cd stress. Furthermore,
we found that C. suppressalis didn’t oviposit when the Cd
concentration in artificial diets was more than 2.5 mg/kg. Spodoptera exigua (Hübner) and Helicoverpa armigera
(Hübner) can oviposit under Cd exposure in artificial
diets with 51.2 mg/kg and 50.0 mg/kg, respectively (Zhan
et al. 2017; Su et al. 2021). These results demonstrate that
there is interspecific variability in herbivore response to
elemental defenses.hf References Liu Y, Wen C, Liu X. China’s food security soiled by contamination. Science. 2013;339:1382–3. Boyd RS. The defense hypothesis of elemental hyperaccumulation: status,
challenges and new directions. Plant Soil. 2007;293:153–76. Liu X, Tian G, Jiang D, Zhang C, Kong LQ. Cadmium (Cd) distribution and
contamination in Chinese paddy soils on national scale. Environ Sci Pollut
Res. 2016;23(18):17941–52. Butler CD, Trumble JT. Effects of pollutants on bottom-up and top-down
processes in insect–plant interactions. Environ Pollut. 2008;156:1–10. Butler CD, Trumble JT. Effects of pollutants on bottom-up and top-down
processes in insect–plant interactions. Environ Pollut. 2008;156:1–10. Chen J, Guo Y, Huang S, Zhan H, Zhang M, Wang J, Shu Y. Integration of
transcriptome and proteome reveals molecular mechanisms underlying
stress responses of the cutworm, Spodoptera litura, exposed to different
levels of lead (Pb). Chemosphere. 2021;283:131205. Liu H, Su X, Sun Z, Wang C, Shi J, Foba CN, Jin H, Wang M. Nitrogen and plant
pathogens alter rice plant volatiles mediating host location behavior of
Nilaparvata lugens and its parasitoid Anagrus nilaparvatae. Entomol Gen. 2022;42(2):549–57. Chen Y, Huang J, Wei J, Liu X, Lu J, Baldwin IT, Lou Y, Li R. Low-level cadmium
exposure influences rice resistance to herbivores by priming jasmonate
signaling. Environ Exp Bot. 2022;194:104741. Chen Y, Huang J, Wei J, Liu X, Lu J, Baldwin IT, Lou Y, Li R. Low-level cadmium
exposure influences rice resistance to herbivores by priming jasmonate
signaling. Environ Exp Bot. 2022;194:104741. Chen Y, Huang J, Wei J, Liu X, Lu J, Baldwin IT, Lou Y, Li R. Low-level cadmium
exposure influences rice resistance to herbivores by priming jasmonate
signaling. Environ Exp Bot. 2022;194:104741. Liu J, Di N, Zhang K, Trumble JT, Zhu Z, Wang S, Zang L. Cadmium
contamination triggers negative bottom-up effects on the growth
and reproduction of Frankliniella occidentalis (Thysanoptera:
Thripidae) without disrupting the foraging behavior of its predator,
Orius sauteri (Heteroptera: Anthocoridae). Environ Sci Pollut Res. 2023;30(15):43126–36. Cutler GC, Amichot M, Benelli G, Guedes RNC, Qu Y, Rix RR, Ullah F, Desneux
N. Hormesis and insects: Effects and interactions in agroecosystems. Sci
Total Environ. 2022;825:153899. Dar MI, Green ID, Khan FA. Trace metal contamination: transfer and fate in food
chains of terrestrial invertebrates. Food Webs. 2019;20:e00116. Dar MI, Green ID, Khan FA. Trace metal contamination: transfer and fate in food
chains of terrestrial invertebrates. Food Webs. 2019;20:e00116. Luo M, Cao H, Fan Y, Zhou X, Chen J, Chung H, Wei H. Conclusionh This study showed that the heavy metal, cadmium can
cascade from artificial diets to rice pest, C. suppressalis. Cd exposure did not affect the larval duration or pupation
rate of C. suppressalis, but caused negative effects
on pupal weight at high Cd levels (5.0 and 10.0 mg/
kg) and on adult deformity rate from 2.5 and 5.0 mg/
kg treatments. Although Cd significantly increased the
female pupae ratio, C. suppressalis did not oviposit when
Cd treatment was more than 2.5 mg/kg. Meanwhile, Cd
transferred to pupae, females, exuviae of pupa and eggs
of C. suppressalis from Cd treated larvae, and exhibited
a dose-dependent response on Cd accumulation. Our results indicated that Cd had a negative effect on
rice stem borer and can be transferred to eggs of C. suppressalis, which may influence the parasitism of their
parasitoids, but more work is needed to further assess the The mechanisms underlying the effects of heavy mental
on the reproduction of insects have been explored in
behavioral and molecular levels (Luo et al. 2020; Chen
et al. 2021). Courtship behavior is vital for copulation. Many abiotic and biotic factors can affect courtship
behavior (Owens et al. 2020; Su et al. 2021). Luo et al. (2020) showed that Cd exposure during an early life
stage result in adverse effects on mating behavior of O. furnacalis male, and Su et al. (2021) indicated that Cd
exposure can disrupt the courtship rhythm for females
and has negative influences on copulation behavior and
high cadmium stress can reduce fecundity of S. exigua. Page 7 of 8 Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Huang et al. CABI Agriculture and Bioscience bottom-up effect on third tropic levels in Cd-polluted
fields. Di N, Wang S, Ridsdill-Smith J, Chen Y, Harwood JD, Zhang K, Liu T. Nitrogen
and plant growth regulator affect plant detoxification metabolism and
tritrophic interactions among Triticum aestivum, Sitobion avenae and
Aphelinus asychis. Entomol Gen. 2021;41(4):369–84.i Consent for publication
Not applicable. Kaminski P, Barczak T, Bennewicz J, Jerzak L, Kavanagh BP, Tkachenko
H, Stuczynski T, Baszynski J, Szady-Grad M, Wozniak A. The role of
aphids in the transfer of chemical elements in disturbed Polish saline
environments. Sci Total Environ. 2021;776:145980. Funding This work was supported by Beijing Innovation Consortium of Agriculture
Research System (BAIC08-2023), and Technology Innovation Program of
Beijing Academy of Agriculture and Forestry Sciences (KJCX20210401,
KJCX20230115, KJCX20230417). Han P, Lavoir AV, Rodriguez-Saona C, Desneux N. Bottom-up forces in
agroecosystems and their potential impact on arthropod pest
management. Annu Rev Entomol. 2022;67(1):239–59. Received: 31 August 2023 Accepted: 16 October 2023 Received: 31 August 2023 Accepted: 16 October 2023 Received: 31 August 2023 Accepted: 16 October 2023 Liu Z. Transfer of heavy metal Cadmium in rice paddy field and its effects on
the growth and reproductive behaviors of Chilo suppressalis (Walker). Nanchang: Jiangxi Agricultural University; 2020. Availability of data and materials Hussain B, Ashraf MN, Shafeeq-Ur-R, Abbas A, Li J, Farooq M. Cadmium stress
in paddy fields: effects of soil conditions and remediation strategies. Sci
Total Environ. 2021;754:142188. The data set used/analyzed during the current study is available from the
corresponding author on reasonable request. Jiang D, Yan S. Effects of Cd, Zn or Pb stress in Populus alba berolinensis on
the development and reproduction of Lymantria dispar. Ecotoxicology. 2017;26(10):1305–13. Acknowledgements Godinho DP, Branquinho C, Magalhães S. Intraspecific variability in herbivore
response to elemental defences is caused by the metal itself. J Pest Sci. 2022;96:797–806. We thank Dr. Yuyong Liang (Institute of Plant Protection, Jiangxi Academy
of Agricultural Sciences) and Prof. Lanzhi Han (Institute of Plant Protection,
Chinese Academy of Agricultural Sciences) for assistance in the insect
collecting and rearing. Green ID, Diaz A, Tibbett M. Factors affecting the concentration in seven-
spotted ladybirds (Coccinella septempunctata L.) of Cd and Zn transferred
through the food chain. Environ Pollut. 2010;158:135–41. Declarations Jiang Y, Jiang S, Yan X, Qin Z, Jia C, Li Z, Zhang J, Huang R. The mobility of
cadmium and lead in the soil–mulberry–silkworm system. Chemosphere
2020;242:125179. Author contributions Han L, Li S, Liu P, Peng Y, Hou M. New artificial diet for continuous rearing
of Chilo suppressalis (Lepidoptera: Crambidae). Ann Entomol Soc Am. 2012;105(2):253–8. HH, JW, and YW performed the experimental trial. HH, ND and JW conceived
of the study, analyzed data, and interpreted results. HH, ND, and JW wrote
the paper. CL, SW and LZ reviewed the manuscript prior to submission and
provided valuable comments on the interpretation and presentation of
results. All authors read and approved the final manuscript. Han P, Dong Y, Lavoir AV, Adamowicz S, Bearez P, Wajnberg E, Desneux N. Effect
of plant nitrogen and water status on the foraging behavior and fitness
of an omnivorous arthropod. Ecol Evol. 2015;5(23):5468–77. Han P, Desneux N, Michel T, Le Bot J, Wajnberg E, Amiens-Desneux E, Seassau
A, Lavoir AV. Does plant cultivar difference modify the bottom-up effects
of resource limitation on plant-insect herbivore interactions? J Chem
Ecol. 2016;42:1293–303. Competing interests Leroy N, Hanciaux N, Cornélis JT, Verheggen F. Silicon accumulation in maize
negatively impacts the feeding and life history traits of Spodoptera exigua
(Hübner). Entomol Gen. 2022;42(3):413–20. The authors declared that they have no competing interest in connection
with the evaluated manuscript. Lin T, Chen J, Zhou S, Yu W, Chen G, Chen L, Wang X, Shi H, Han S, Zhang F. Testing the elemental defense hypothesis with a woody plant species:
Cadmium accumulation protects Populus yunnanensis from leaf herbivory
and pathogen infection. Chemosphere. 2020;247:125851. Publisher’s Note Su H, Yang Y, Zou J, Cheng Y, Yang Y, Wu J, Pollak P, Yang Y. Transcriptome
analysis of the ovary of beet armyworm Spodoptera exigua under
different exposures of cadmium stress. Chemosphere. 2020;251:126372. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Su H, Wu J, Zhang Z, Ye Z, Chen Y, Yang Y. Effects of cadmium stress at different
concentrations on the reproductive behaviors of beet armyworm
Spodoptera exigua (Hubner). Ecotoxicology. 2021;30(3):402–10. Sui F, Chang J, Tang Z, Liu W, Huang X, Zhao F. Nramp5 expression and
functionality likely explain higher cadmium uptake in rice than in wheat
and maize. Plant Soil. 2018;433(1):377–89. Tan M, Wu H, Li Y, Zhang A, Xu J, Chai R, Meng Z, Yan S, Jiang D. Cadmium
exposure through the food chain reduces the parasitic fitness of Chouioia
cunea to Hyphantria cunea pupae: an ecotoxicological risk to pest control. Sci Total Environ. 2023;887:164106. Tibbett M, Green I, Rate A, De Oliveira V, Whitaker J. The transfer of trace metals
in the soil-plant-arthropod system. Sci Total Environ. 2021;779:146260. Ullah F, Gul H, Desneux N, Qu Y, Xiao X, Khattak AM, Gao X, Song
D.Acetamiprid-induced hormetic effects and vitellogenin gene
(Vg) expression in the melon aphid, Aphis gossypii. Entomol Gen. 2019;39:259–70. Wang P, Chen H, Kopittke P, Zhao F. Cadmium contamination in agricultural
soils of China and the impact on food safety. Environ Pollut. 2019;249:1038–48. Wang P, Li M, Bai Q, Ali A, Desneux N, Dai H, Zang L. Performance of
Trichogramma japonicum as a vector of Beauveria bassiana for parasitizing
eggs of rice striped stem borer. Chilo suppressalis Entomol Gen. 2021;41(2):147–55. Wang X, Tian L, Ricupero M, Harwood JD, Liang Y, Zang L, Wang S. Hormesis
effects of chlorantraniliprole on a key egg parasitoid used for
management of rice lepidopterans. Entomol Gen. 2022;42(6):941–8. Wang J, Liu S, Gan T, Di N, Wang S, Li Y. Effects of cadmium stress on the
development of Spodoptera frugiperda (Lepidoptera: Noctuidae)
and parasitism by Trichogramma dendrolimi (Hymenoptera:
Trichogrammatidae). Acta Entomol Sin. 2023;66(2):209–18. Wei Z, Wang X, Li P, Tan X, Yang X. Diet-mediated effects of cadmium on
fitness-related traits and detoxification and antioxidative enzymes in the
oriental armyworm. Mythimna separata Entomol Gen. 2020;40(4):407–19. Yan S, Tan M, Zheng L, Wu H, Wang K, Chai R, Jiang D. References Bioaccumulation of
cadmium affects development, mating behavior, and fecundity in the
Asian Corn Borer. Ostrinia Furnacalis. Insects. 2020;11:7. Di N, Hladun KR, Zhang K, Liu T, Trumble JT. Laboratory bioassays on the
impact of cadmium, copper and lead on the development and survival
of honeybee (Apis mellifera L.) larvae and foragers. Chemosphere. 2016;152:530–8. Ma W, Zhao X, Yin C, Jiang F, Du X, Chen T, Zhang Q, Qiu L, Xu H, Joe Hull J, Li
G, Sung WK, Li F, Lin Y. A chromosome-level genome assembly reveals
the genetic basis of cold tolerance in a notorious rice insect pest. Chilo
Suppressalis Mol Ecol Resour. 2020;20(1):268–82. Di N, Zhang K, Hladun KR, Rust M, Chen Y, Zhu Z, Liu T, Trumble JT. Joint effects
of cadmium and copper on Apis mellifera forgers and larvae. Comp
Biochem Physiol C Toxicol Pharmacol. 2020;237:108839. Ma J, Shen R, Shao J. Transport of cadmium from soil to grain in cereal crops: a
review. Pedosphere. 2021;31(1):3–10. Huang et al. CABI Agriculture and Bioscience (2023) 4:45 Page 8 of 8 Zheng Y, Zhang X, Liu X, Qin N, Xu K, Zeng R, Liu J, Song Y. Nitrogen supply
alters rice defense against the striped stem borer Chilo suppressalis. Front
Plant Sci. 2021;12:691292. Zheng Y, Zhang X, Liu X, Qin N, Xu K, Zeng R, Liu J, Song Y. Nitrogen supply
alters rice defense against the striped stem borer Chilo suppressalis. Front
Plant Sci. 2021;12:691292. Zheng Y, Zhang X, Liu X, Qin N, Xu K, Zeng R, Liu J, Song Y. Nitrogen supply
alters rice defense against the striped stem borer Chilo suppressalis. Front
Plant Sci. 2021;12:691292. Owens ACS, Cochard P, Durrant J, Farnworth B, Perkin EK, Seymoure B. Light
pollution is a driver of insect declines. Biol Conserv. 2020;241:108259. SAMR (State Administration for Market Regulation), NHCC (National Health
Commission of China). GB 2762-2022 National food safety standard
maximum levels of contaminants in foods; 2022. Publisher’s Note Defense response
of Fraxinus mandshurica seedlings to Hyphantria cunea larvae under
Cd stress: a contradiction between attraction and resistance. Sci Total
Environ. 2023;859:160390. Yu E, Wang W, Yamaji N, Fukuoka S, Che J, Ueno D, Ando T, Deng F, Hori K,
Yano M, Shen R, Ma J. Duplication of a manganese/cadmium transporter
gene reduces cadmium accumulation in rice grain. Nature Food. 2022;3:597–607. Zang L, Wang S, Zhang F, Desneux N. Biological control with Trichogramma
in China: history, present status, and perspectives. Annu Rev Entomol. 2021;66:463–84. Zhan H, Zhang J, Chen Z, Huang Y, Ruuhola T, Yang S. Effects of Cd2+
exposure on key life history traits and activities of four metabolic
enzymes in Helicoverpa armigera (Lepidopteran: Noctuidae). Chem Eco. 2017;33:325–38. Zhang Y, Song D, Wu H, Yang H, Zhang J, Li L, Ma E, Guo Y. Effect of
dietary cadmium on the activity of glutathione S-transferase and
carboxylesterase in different developmental stages of the Oxya chinensis
(Orthoptera: Acridoidea). Environ Entomol. 2014;43(1):171–7. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: Zhang Y, Hu X, Zheng L, Wei H. Effect of Cd2+ on development and male
orientational behaviors to the conspecific female pheromones in
the striped stem borer, Chilo suppressalis (Walker). J Environ Entomol. 2017;39(2):423–30. Zhang J, Guo X, Xu F, Chen L, Wei H. The molecular characteristics of two
metallothioneins in Ostrinia furnacalis and expression under conditions of
heavy metal stress. J Asia Pac Entomol. 2023;26:102006. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: Publisher’s Note Zhao F, Tang Z, Song JJ, Huang X, Wang P. Toxic metals and metalloids: Uptake,
transport, detoxification, phytoremediation, and crop improvement for
safer food. Mol Plant. 2022;15:27–44.
|
https://openalex.org/W4289781640
|
https://hal.science/hal-01193811/document
|
English
| null |
Digestive efficiency, gut microbiota and genetics – Are they interrelated?
|
HAL (Le Centre pour la Communication Scientifique Directe)
| 2,014
|
cc-by
| 19,799
|
To cite this version: Irène Gabriel, Fanny Calenge, Agnès Narcy, Sandrine Mignon-Grasteau. Digestive efficiency, gut
microbiota and genetics – Are they interrelated?. 5. International Broiler Nutritionists’ Conference,
Apr 2014, Queenstown, New Zealand. hal-01193811 Distributed under a Creative Commons Attribution 4.0 International License Digestive efficiency, Gut Microbiota and Genetics – Are they interrelated? Irène Gabriel1, Fanny Calenge2, Agnès Narcy1, Sandrine Mignon-Grasteau1 1 INRA, UR83, Recherches Avicoles, Centre Val de Loire, 37380 Nouzilly France
2 INRA, UMR1313 GABI, Génétique Animale et Biologie Intégrative, Domaine de Vilvert,
78352 Jouy en Josas cedex, France 1 INRA, UR83, Recherches Avicoles, Centre Val de Loire, 37380 Nouzilly France
2 INRA, UMR1313 GABI, Génétique Animale et Biologie Intégrative, Domaine de Vilvert,
78352 Jouy en Josas cedex, France HAL Id: hal-01193811
https://hal.science/hal-01193811v1
Submitted on 18 Jun 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Introduction Digestive efficiency (DE) is a part of feed efficiency, and its improvement is a major goal in
poultry production for economic, environmental and sociological objectives. It allows
reducing production costs, using feedstuffs of low or variable quality or alternative feedstuffs
and decreases the animal manure. Indeed, feed costs are the main part of production cost and
shows high fluctuations (ITAVI, 2014). For meat-type chickens, it is ranging from 55 to 65 %
depending on production type (Riffard et al., 2011). Regarding social demands, an increased
in digestibility would lead to an improved animal health and welfare, through a decreased
nutrient content in the intestine and a lower microbiota development or a better equilibrium
between favorable and unfavorable microorganisms (Crévieu-Gabriel and Naciri, 2001; Klis
and Lensing, 2007; Timbermont et al., 2011). Moreover, the undigested dietary compounds
increase the quantity of fermentation substrates in the litter and the frequency of
pododermatitis (Shepherd and Fairchild, 2010). With increase of human population, an increase in need for poultry meat and for the crops
required for both animal and human consumptions will follow. Moreover, feedstuffs are used
by the industry for biofuel. To decrease the competition between these different uses of
feedstuffs, for animal nutrition conventional crop vegetal resources are replaced by
unconventional feedstuffs as by-products. Moreover in several countries, the use of local
feedstuffs is needed to decrease the dependence toward soya as protein source, as it mainly
comes from importation, and other cereal than corn is introduce in diet. However most of
these alternatives feedstuffs have variable or low nutritional values as wheat or oilseed a
rapeseed meal used in many countries throughout the world, thus leading to reduced
digestibility and increased animal wastages. It has been shown from several years that nutrient digestibility depends on animal
development, diet composition and feed technology. Moreover, to improve digestibility of
low quality feedstuffs, additives as enzyme have been proposed to increase hydrolysis of
macromolecules of the diet, and also additives to control digestive microbiota that is involved
in this physiological function of the host. However, this microbiota has been shown to present
a high individual variability, whatever the animal species. Introduction Thus, in human, although main
bacteria are shared between individuals (the core microbiome), a very high variability of
abundance has been observed for the most common species (Li et al., 2013; Qin et al., 2010;
Salonen et al., 2012; Tap et al., 2009; Turnbaugh and Gordon, 2009). In animal such as
chicken, a considerable variation is also observed between individual microbiota although
under carefully controlled environmental conditions, as feed and rearing conditions (Gabriel
et al., 2007; Stanley et al., 2013b ; Torok et al., 2008; Zhu et al., 2002). Understanding the
origin of this individual variability is required to allow manipulating digestive microbial
communities in order to improve DE. This high individual variability is probably due to the
fact that after their first contact, the host and its digestive commensal microbiota co-evolve. These latest years, several studies have search to evaluate the relative role of the potential
contributing factors to this variation, environment origin and also genetics. In this review, after reminding how can be defined and measured DE in chicken and what is at
the present day known in its digestive microbiota, we will present relationships observed
between these two entities. In a second part, the present knowledge on genetic control of these
two entities and the availability of new approaches to study it will be displayed. In this
review, a tentative has been made to gather present knowledge on this topic, particularly in
chicken, however it can not be considered as exhaustive due to the huge number of studies,
and new proposed concept by research teams involved in this topic. In the last part, we will
present which further studies are needed to increase our knowledge to improve DE in chicken. 1.1. Digestive efficiency and digestive microbiota : some reminders Digestive efficiency for animal : difference between the measure at the ileal and 1.1.1. Digestive efficiency for animal : difference between the measure at the ileal and
faecal level faecal level Faecal digestibility has been the most used method to determine digestibility in chicken as
observed by published studies on Web of Knowledge. This measure of digestibility may be
considered as the global use of nutrients by the digestive tract (Fuller et al., 2012). It can be
considered that animal digestive capacity correspond to its capacity to extract compounds
from the diet for it. It is performed by hydrolyzing macromolecules to absorbable molecules
and absorbing them, with its enzymatic / absorbent system, but also with the help or
competition of its digestive microbiota. Moreover faecal digestibility allow maintaining
animal alive for further measurements, and to measure digestibility on a period of one or
several days. However due to hind gut fermentation (mainly in caeca, but also potentially in the rectum), as
well as use of undigested compounds by digestive microbiota in post ileal segments and
production of bacterial biomass (mainly protein and lipid), estimated to represent 20% of DM
in feces, ileal digestibility is a more accurate measurement of nutrient availability from the
point of view of animal. Indeed, it allows separating two different phenomena: digestion until
end of small intestine due mainly to direct host factors, and post-ileal digestion mainly due to
digestive microbiota. In chicken, although this hindgut digestion is lower developed than in
mammal as pig, ileal digestibility shows different value than faecal digestibility (Dublecz et
al., 2006; Ravindran et al., 1999). However one need to take into account that digestive microbiota is implied in ileal digestion
as we will see later, and that digestive microbiota in the caeca produced nutrients that can be
absorbed by the host, as carbohydrates and amino acids (Moreto et Planas 1989) although the
quantitative importance of these phenomena is not known, and supposed to be of very lower
amplitude than post-ileal modifications. 1.1.2. Digestive microbiota in chicken The microbiota of the gastrointestinal tract is a complex community of many different species
of microorganisms, mainly bacteria. Thus, in the following text, when we used the term
microbiota, it refers to bacteria. Until recently, one of the main problems with studying microbiota, was the availability of
appropriate methods as a large majority of species cannot be easily cultivated (70-90%). Thanks to the development of new independent approaches to culture, molecular methods
based on 16 S ribosomal DNA, new tools are available. Among these methods, qualitative methods have been developed such as fingerprint techniques (DGGE, TGGE, TTGE, t-RFLP,
ARISA …). Quantitative methods as fluorescent in situ hybridization (FISH), quantitative
PCR and low throughput clone analysis have also been used. These methods allow obtaining a
more precise and complete image of the microbial diversity than cultures. More recently, high
throughput sequencing of variable regions of 16S rDNA (pyrosequencing belonging to Next
Generation Sequencing, NGS) has been developed, and begin to be used for poultry digestive
microbiota. It allows again more deep insight in digestive microbiota. In the following part,
we will see what is known in chickens. It is generally admitted that digestive microbiota of animals depends on its development after
the first contact between early-colonizing bacteria and digestive tract, and later, depends on
the conditions meet by the bacteria in the digestive tract along the bird live. These conditions
are within bacteria cross talk and the interactions of bacteria with the digestive tract
environment. This latest is composed of host-microbial interaction and digestive biotope
composition. This biotope may depend on animal environmental factor as diet (feedstuff,
technological treatment, feed additives) and rearing environment of which stress, and host
factors, as host genetics and animal physiological state (age, health status). All these
conditions lead to a strong selective pressure allowing only microbial populations that are able
of establishing a mutualistic relation with the host to be maintained in this gut ecosystem, and
to cooperate with other bacteria in microbial food networks, where fermentation end products
from one organism can act as a substrate for another, a phenomenon known as “cross-
feeding” (El Aidy et al., 2013). As the digestive microbiota and the host co-evolved after their
first contact, they are considered as a supraorganism with numerous cross-talk between
microbial and host cells (Lederberg, 2000). They seem to be in a mutualistic relationship
when the equilibrium is reached. 1.1.2. Digestive microbiota in chicken Moreover stochastic phenomena are implied as in whatever
microbial ecosystem. It is generally thought that constant community structure is likely to
provide continuity of microbial metabolic activities for the host and give rise to stable
microbe-dependent phenotypic traits. On the contrary, an unsteady digestive microbiota is
thought to have negative consequence on animals. In chicken, as reviewed recently, implantation and localization of digestive microbiota is
specific to these birds and their rearing conditions (Apajalahti et al., 2004; Dibner et al, 2008;
Gabriel et al., 2006, 2012; Yeoman et al, 2012). Before hatch, digestive tract may not be
sterile, but the content in bacteria seems to be very low. After hatch, further development of
digestive microbiota of birds depends on the environment of the eggs. In modern commercial chicken hatcheries, eggs are fumigated by formol to remove bacterial contamination, thus
limiting the bacterial source to incubator and hatcher environment, which are also disinfected,
and maintained in surpressure with filtered air. Next chickens are manipulated for sexing and
vaccination. In the boxes for transportation, bacteria from outside and from other chickens can
be sampled by individuals thanks to retrograde movement of the cloaca and rectum (Bar-Shira
and Friedman 2005; Clench, 1999). When chicks arrive in the farm, new bacterial sources are
people handling, first feed and water, and litter. Thus, in one day old chickens, bacterial load
has been measured to be 108 and 1010 bacteria per g of content in ileal and caecal content
respectively, and in three days old chickens, a stable quantity is reached, 109 and 1011
respectively (Apajalahti et al., 2004). In the chicken, the major sites of bacterial localization are the crop and the caeca, and to a
lesser extent the small intestine. For example Guardia et al., (2011) determined that the total
bacterial load was 5.5 × 1011 , 5.3 × 1010 and 7.4 × 1012 copies of 16S rDNA/g of fresh
samples in the crop, the terminal ileum and the caeca respectively. In the chicken, the major sites of bacterial localization are the crop and the caeca, and to a
lesser extent the small intestine. For example Guardia et al., (2011) determined that the total
bacterial load was 5.5 × 1011 , 5.3 × 1010 and 7.4 × 1012 copies of 16S rDNA/g of fresh
samples in the crop, the terminal ileum and the caeca respectively. 1.1.2. Digestive microbiota in chicken The crop is considered as the inoculum of the following digestive tract, with the dominant
group being Lactobacillus, although not the unique genera (Gong et al., 2007; Guardia et al.,
2011; Peinado et al., 2013). After a fall in the total amount in the proventriculus and the gizzard, due to their low pH,
biotope conditions at the beginning of the small intestine are not in favor of developing
microbiota with high oxygen pressure, high concentration in antimicrobial compounds such as
digestive enzymes and bile salts. In the following small intestine, the environment becomes
more favorable to bacterial growth thanks to lower oxygen pressure, and lower digestive
enzyme and bile acids concentrations. Due to these changes along the small intestine,
microbiota evolved between the upper part (duodenum-jejunum) to the lower part of the small
intestine, with Lactobacillus being always the dominant bacteria. However, it can be noted that due to reverse peristaltic contractions in chicken from the
cloaca to the gizzard, the digestive biotope is subjected to fast modification that may be not in
favor of bacteria development (Sacranie et al., 2012). In the lower digestive tract, the caeca, the biotope is the most favorable for bacterial growth. Indeed, oxygen pressure is lower and this biotope is relatively stable, due to the low renewal
of the digestive content (Gabriel et al., 2012). Thus, it is the major site of bacterial
fermentation in chicken, and it has been evaluated that 50% of the biomass would be of
bacterial origin (Clench, 1999). It may contribute to energy extracted from the feed by the host-microbiota association, thanks to reabsorption of bacterial metabolites. Contrarily to the
crop and the small intestine, the bacterial composition of the caeca is more diverse, and the
major phylum are Firmicutes and Bacteroidetes, mainly genera Clostridium (Guardia et al.,
2011; Lu et al., 2003; Stanley et al., 2013a). Apajalahti et al., (2004) found 640 different
species, and more recent studies using 16S rRNA pyrosequencing showed as much as 783
operational taxonomic units or OTU (Danzeisen et al., 2011; Moore et al., 2011; Nordentoft
et al., 2011). It was also observed that chicken caecal microbiota was markedly different from
faecal microbiota, and display more diversity (Lei et al., 2012) This digestive microbiota can be located in the lumen, in the mucus layer(s) or at the mucosal
surface. 1.1.2. Digestive microbiota in chicken Moreover, interactions between the microbiota and the
host are being considered pivotal in the early programming of gut function that may be
mediated by epigenetic mechanisms with consequence throughout life (Lalles, 2012). It may be assumed that both quantitatively rich microbiota, as caecal content, and poorer, as
small intestinal content or mucosal microbiota, may have effects, that may be due to their high
metabolic activity or more targeted action respectively. Moreover, due to the distance away
effect of microbiota, via neurohormonal effect or via their metabolites or constituents, or even
themselves, that pass through the digestive epithelium, and carried out in blood, it is not
necessarily in proximity of the highest load of bacteria in the lower part of the digestive tract
that the highest effects are reached. Thus, Larsson et al.(2012) observed by comparing germ-
free and conventionally raised mice, a higher effect on the gene expression of the small
intestine than in the colon. chain fatty acids (SCFA) that have regularly functions via neural and humoral pathways (Al-
Lahham et al., 2010; Mroz et al., 2006; Reilly et al., 1995; Tappenden et McBurney, 1998) or
steroid compounds (Groh et al., 1993). Moreover, interactions between the microbiota and the
host are being considered pivotal in the early programming of gut function that may be
mediated by epigenetic mechanisms with consequence throughout life (Lalles, 2012). It may be assumed that both quantitatively rich microbiota, as caecal content, and poorer, as
small intestinal content or mucosal microbiota, may have effects, that may be due to their high
metabolic activity or more targeted action respectively. Moreover, due to the distance away
effect of microbiota, via neurohormonal effect or via their metabolites or constituents, or even
themselves, that pass through the digestive epithelium, and carried out in blood, it is not
necessarily in proximity of the highest load of bacteria in the lower part of the digestive tract
that the highest effects are reached. Thus, Larsson et al.(2012) observed by comparing germ-
free and conventionally raised mice, a higher effect on the gene expression of the small
intestine than in the colon. The question can be raised if modifiable microbiota as in the upper digestive tract of chicken,
due to reverse peristaltic contractions, can have a stable effect on the host, contrarily to high
stable microbiote in caecal contents. 1.1.2. Digestive microbiota in chicken The luminal microbiota depends on available nutrients, transit rate and the presence
of antibacterial compounds. It is the main studied microbiota. The mucosal microbiota in the
crop, has been described as adhesive to the mucosa developing several cell layers. Mucosal
microbiota, in the small intestine of chicken, would be adherent to the epithelial cells, or
localized in the single mucus layer. In the caeca, digestive microbiota as been described to
form a 200 cells deep layer, or may be localized in the upper layer of mucus near the intestinal
content, and not in the lower layer of mucus. This mucosal microbiota depends on available
substrates coming from the mucosa and molecules coming from the digestive content
diffusing into the protein matrix of mucus. It also depends on bacteria adhesins, specific
adhesive sites on the mucus or mucosa, on mucus or cell renewal rates, on antibacterial
substances contributing to the innate immune system of the digestive tract. As these bacteria
are in narrow contact with the host, it may be supposed that they have a more direct effect on
the host. In the chicken, this mucosal microbiota, is different from the luminal microbiota, but
has been relatively little studied compared to the studies in digestive contents. Moreover, in
the digestive tract, distribution of microbiota is not homogenous due to different biotopes
present in the digestive tract, and there are niches with specific microbiota (Pédron et al.,
2012). In recent years evidence has accumulated to support the idea that the community structure of
the gut microbiota is a major contributor to the phenotype of the animal host, due to its
narrow contact with the host. Digestive microbiota can be considered as an organ in the
digestive tract that uses nutrients and products metabolites, recognizes and synthesizes
neuroendocrine hormones, may interface with the nervous system that innervates the
gastrointestinal tract or via neuropeptides (Holzer et al., 2012; Lyte, 2010). Digestive
microbiota may act on several host functions as digestive physiology via its products as short chain fatty acids (SCFA) that have regularly functions via neural and humoral pathways (Al-
Lahham et al., 2010; Mroz et al., 2006; Reilly et al., 1995; Tappenden et McBurney, 1998) or
steroid compounds (Groh et al., 1993). 1.2. Digestive efficiency and gut microbiota composition relationship In mammals, with a high bacterial load in the colon, it is generally admitted that digestive
microbiota is implicated in digestive physiology and digestion (March, 1979). We will see in
the following part, that in birds as chickens, several data are accumulated to show link
between digestive microbiota and digestion. 1.1.2. Digestive microbiota in chicken Among the various effects of microbiota on the host physiology, digestive microbiota has
effect on the digestive area. It contributes to the development, morphology and functionality
of the digestive tract. These effects may have consequences on animal digestion of nutrient as
we will detail later. The commensal microbiota is also implicated in digestive health. It
contributes to the protection against harmful microorganisms (barrier effect) and stimulates
immune system, leading the host digestive tract in a physiological inflammatory states
corresponding to homeostasie. Digestive microbiota contributes to detoxification of some
compounds, but also to production of toxic substances. The digestive microbiota can also influence extra-digestive physiology of the host. Indeed, in
mammals, effects have been observed on the animal metabolism as fattening, or on the central
nervous system with effects on behavior. In chicken, the presence of microbiota has been
shown to increase total protein synthesis of 6-8%, and energy requirement (Furuse and
Okumura, 1994; Muramatsu et al., 1987). Digestive microbiota may also contribute to
mineral and vitamins nutrition. Moreover the bacterial activity has consequences on health,
and thus animal welfare, such as conjunctivitis and respiratory problems due to irritant
compounds products released by bacterial fermentation in the litter material. These fermentations also have consequences on contact dermatitis or pathogen development in the
litter. All these effects have consequences on animal production, as well as on growth
performance and product quality. 1.2.1.1. Parallel change in digestive efficiency and digestive microbiota Several studies have been performed between 1970 and 1985 by using germ-free chickens
compared to conventional animals to study the effect on DE. Thus, for lipids, in young
chicken of 3 weeks, microbiota led to a decrease of 2 points of apparent faecal digestibility for
vegetal oil and 10 points for animal fat (Boyd and Edwards, 1967, Kussaibati et al., 1982b). Digestibility of saturated fatty acids as palmitic and stearic acids are highly decreased whereas
this of unsaturated fatty acids as oleic and linoleic acids is not modified by microbiota (Boyd
and Edwards, 1967). However the change in faecal digestibility may be due in part to
endogenous lipids and bacterial biomass. For protein digestibility, effect of microbiota may
depend on sensitivity to hydrolysis of proteins, bacteria being able to hydrolyze some resistant
proteins for enzyme host (Kussaibati et al., 1982a; Salter 1973; Salter and Fulford, 1974). Due to the effect of digestive microbiota on nutrient digestibility, it can have an effect on
metabolisable energy, positive or negative, (Furuse and Okumura, 1994; Kussaibati et al.,
1982b). In a study comparing conventional chickens to chickens with limited microflora obtained by
rearing birds in sterilized conditions, Maisonnier et al., (2003) also observed a significant
decrease of faecal lipid digestibility. This negative effect of digestive microbiota on lipid
digestibility and the apparent metabolizable energy value of diet, corrected to zero nitrogen
retention (AMEn) was also observed in a study by using high dose of antibiotics (Garcia et
al., 2007). Following the ban of the use of antibiotic growth promoters (AGP) in feed, several studies
have been performed to explore alternative feed management strategies as feed additives or
cereal form, to improve digestive health of chickens, particularly to control digestive
microbiota (Alloui et al., 2013; Gabriel et al., 2013). Moreover, consequences of other factors
as type of cereals or rearing conditions have been studied. Among these works, some of them
also studied if the DE is also modified. Indeed some of these additives can be used as first
objective to modify digestive microbiota, but they may also have on DE. In these studies following both digestive microbiota and DE, some showed that the two are
modified, but some of them observed that only one of these two markers is modified, with no
consequence on the other marker. 1.2.1.1. Parallel change in digestive efficiency and digestive microbiota The type of cereal grains has been shown to lead to change in ileal digestibility as well as
change in digestive microbiota in ileal and caecal content, but also in gizzard content and
mucosa of the small intestine (Baurhoo et al., 2011a; Rodriguez et al., 2012; Shakouri et al.,
2009; Yang et al., 2008a). Technological treatments of cereals have been shown to improve
ileal digestibility and change microbial digestive contents (Amerah et al., 2011; Bhuiyan et
al., 2010). Rearing conditions have also been shown to lead to change in ileal digestibility and digestive
microbiota, as delay feeding after hatching and immunological stress (Sarica and Corduk
2013; Yang et al., 2011). These observations lead to the common idea of beneficial effect of some bacteria as
Bacteroides and Lactobacillus Bifidobacteria and negative effect of Clostridium and E. coli. However, this is more complex, because all species of a bacteria genera and all strains of a
bacteria species have not necessarily the same properties, and their biological effects depend
on the digestive biotope (Roy et al., 2008; Yuan et al., 2008). Thus whereas Lactobacillus is
most of the time associated with beneficial bacteria genera, it was also associated with
negative effect on digestibility (Knarreborg, 2002; Ramasamy et al., 2010). Moreover the
effect of digestive microbiota on DE depends on localization of this microbiota (digestive
segment, localization in content or mucus). Recently, in our laboratory, Poultry Research Unit of INRA, studies have been performed to
search for difference in digestive microbiota between birds having different digestibility
estimated by AMEn. These chickens were from a divergent line using AMEn as the criteria
for selection (Mignon-Grateau et al., 2004). As explained previously faecal digestibility is a
complex phenotypic traits, including animal digestion and microbiota digestion, but this
choice was performed in order to maintain animal alive for selection purpose, and in order to
study digestion in its globality for research on animal excreta for environment objective. As
this selection has been performed with vegetal feedstuffs, it can be expected that difference
between ileal and faecal digestibility is small (Ravindran et al., 1999). However, works are in
progress in our laboratory to compare these two measurements of digestibility in these
animals. Briefly, the selection of these chickens was performed with a diet composed of
compounds able to lead to low digestibility, in order to increase difference between
individuals. 1.2.1.1. Parallel change in digestive efficiency and digestive microbiota Thus, the majority of studies reporting effects of AGP or
their alternatives, studying ileal digestibility and digestive microbiota, observed positive
effect on digestibility. However, one must keep in mind that all of the studies performed on
alternatives to AGP are not necessarily publicly available, thus this representation may be a
biased image of the reality. Indeed, although to our knowledge, no study reports negative
effect of alternatives to AFC on ileal digestibility, this may happen. Indeed, the additives may
have a nutrient cost for the host at the digestive level if they lead to increase bacterial load in
the digestive tract that have nutrient requirement for their growth, as the objective of these
alternatives is not necessarily to reduce the digestive bacterial population but to enhance the
gut health function via the increase of beneficial microorganism, competitive exclusion of
pathogens and stimulation of the immune system. Thus, use of virginiamycin was observed by Zulkifli et al., (2012) to lead to increase ileal
protein digestibility and increase of Lactobacillus and E. coli counts in ileal content. Introduction of enzymes in diets has been shown in several studies to lead to increase ileal
digestibility and change in digestive microbiota count or their products of fermentation
mainly in caeca, but also in ileum (Baurhoo et al., 2011b; Bhuiyan et al., 2010 ; Choct et al.,
1999; Cowieson et Masey O’Neill 2013; Nian et al., 2011b). By the use of herbal product or
essential oils, increase ileal digestibility has been reported with modifications of ileal
microbiota (Amerah et al., 2011; Zulkifli et al., 2012). With insoluble fiber, Amerah et al.,
(2009) observed an increase in ileal starch digestibility and a difference in ileal microbiota
observed by fingerprint. Use of mannanoligosaccharide in a wheat diet was observed to
increase ileal digestibility and led to change in microbiota of the small intestinal mucosa and
caecal content (Yang et al., 2008a). The type of cereal grains has been shown to lead to change in ileal digestibility as well as
change in digestive microbiota in ileal and caecal content, but also in gizzard content and
mucosa of the small intestine (Baurhoo et al., 2011a; Rodriguez et al., 2012; Shakouri et al.,
2009; Yang et al., 2008a). Technological treatments of cereals have been shown to improve
ileal digestibility and change microbial digestive contents (Amerah et al., 2011; Bhuiyan et
al., 2010). 1.2.1.1. Parallel change in digestive efficiency and digestive microbiota The diet contains a high level of wheat (525 g/kg) of high viscosity due to its
richness in soluble non-starch polysaccharides (NSP), arabinoxylan, that may be a substrate for digestive microbiota, and of medium hard value (Rialto cultivar). Moreover, the fat
concentration was high (60-80 g/kg of added vegetal oil) which represent a difficulty for
digestion for young chickens. The diet was pelleted. In order to maintain performances at a
common level between lines, body weight was constrained among both lines. The lines were
selected at 3 weeks of age, as it represents a key period in gastrointestinal tract development. The two lines were named D+ (high digester) and D- (low digester) and have been selected on
AMEn during 10 generations. This model has been developed to study the physiological
limiting factors of digestion. To study links between DE and digestive microbiota, we used a F2 cross between the D+ and
D- lines with 144 animals. Studied microbiota was those of caecal contents. Links between
AMEn and microbiota were observed. High AMEn was associated with low amounts of E. coli expressed in absolute values, and also in relative values compared to all other bacterial
groups (Lactobacillus, L. salivarius, L. crispatus, C. coccoides, C. leptum). On the contrary, a
low AMEn was associated with high amounts of E. coli expressed in absolute, and high
amounts of E. coli relative to Clostridium. These low AMEn are also associated with high
amounts of L. salivarius expressed in absolute, and a higher proportion of L. salivarius
compared to Lactobacillus groups and Clostridium groups (C. coccoides and C. leptum). Moreover, this F2 cross of D+ and D- lines allows showing that a significant amount of
variability of the AMEn can be explained with some components of caecal microbiota. Thus,
L. salivarius amounts can explain significantly 9% of this variability, with a negative effect. And the bacterial ratio of Log L. salivarius to Log C. leptum explains a greater part of the
variability (13%), with a negative effect. 1.2.1.2. Hypotheses on the mechanisms implied in the relationships between digestive
efficiency and gut microbiota Changes in digestibility in parallel with change in digestive microbiota can affect different
nutrients as protein, lipid, starch and minerals. The mechanisms responsible for the
relationship between digestibility and digestive microbiota are not accurately described at the
present time. It is needed to determine how animal digestion leads to change in the digestive
microbiota and how digestive microbiota leads to change in DE. However hypothesis can be
proposed. DE may impact small intestinal microbiota because digestion leads to undigested particles
that can be used as substrates by microorganisms as bacteria. Caecal microbiota may also be modified by small intestinal digestion as caecal microbiota substrates depend on undigested
compounds at the end of the small intestine that can enter in the caeca, with the filter at the
caeca entrance. Digestive microbiota, from the small intestine and caeca, may act on DE, directly or
indirectly. In chicken, crop microbiota can be considered as an help for starch hydrolysis in
the crop, with amylase coming mainly from Lactobacillus species (Champs et al., 1981;
Szylit et al., 1980). Microbiota along the following digestive tract may also provide
hydrolytic enzymes that may contribute to hydrolysis of polysaccharides as raw starch, or
resistant proteins (Kau et al., 2011). Hydrolysis of fiber compounds may also allow release of
entrapped molecules and thus enhance nutrient availability. It can also not be ruled out that if
chickens have access to a litter it may allow them to practice coprophagy that may allow them
to benefit from the bacterial cell composition as proteins or vitamins, although the
quantitative importance of this phenomenon is not known, and deserves to be studied. However, in the small intestine, it is generally assumed that digestive microbiota acts mainly
as competitor of the host, due to their high metabolic potential with high hydrolysis activity
due to their high area links to their very small size. Moreover, microbiota has negative effect
of lipid digestion due to deconjugation of bile salts by some bacterial species as Lactobacillus
as indicated previously (Engberg et al., 2004; Kim and Lee, 2005). Microbiota is also
considered to be involved in the negative effect observed in digestion of diet rich in soluble
NSP, leading to increase viscosity of digestive content (Bedford and Cowieson, 2012),
although according to Maisonnier et al., (2003) it is not the main factor. 1.2.1.3. No parallel change in digestive efficiency and digestive microbiota Some studies shown change in ileal digestibility, but not in digestive microbiota, and on the
reverse, no change in ileal digestibility, and change in digestive microbiota. Thus, in some studies, although AGP as avilamycin, or alternatives to AGP, as enzyme and
products of essential oils, were observed to improve ileal digestibility, not effect was observed
on digestive microbiota (Cao et al., 2010; Mountzouris et al., 2010; Sarica and Corduk 2013). On the contrary, by using germ-free chicken compared to conventional animal Kussaibati et
al., (1982a) observed no change for starch digestibility with maize starch. Moreover, in
several studies observing change in microbiota with inclusion of additives (enzyme, vegetal
products, prebiotic, probiotic) or modification of cereal structure and type, no change in ileal
digestibility was observed (Amerah et al., 2009; Baurhoo et al., 2011a; Bhuiyan et al., 2013;
Cao et al., 2010; Nian et al., 2011a; Ouhida et al., 2002; Rodriguez et al., 2012; Yang et al.,
2008b). 1.2.1.2. Hypotheses on the mechanisms implied in the relationships between digestive
efficiency and gut microbiota However as major site of digestion is jejunum, and small intestinal microbiota mainly
concentrated in the ileum, the role of microbiota in digestion may be low. However, in the
case of diet difficult to digest, and with more importance of the terminal steps of small
intestinal digestion in the ileum, effect of microbiota may be important. Moreover due to
retroperistaltis from caeca to ileum and further anterior (Sacranie et al., 2007, 2012), caecal
microbiota and its products can influence directly small intestinal microbiota and thus
digestive efficiency, although only caecal bacteria able to growth in small intestine can
develop in this digestive segment. Moreover microbiota can modify digestive tract structure and digestive physiology as
enzymatic activities or transit as well as positively than negatively. These effects of
microbiota may be direct or indirect by mean of bacterial products that can have a systemic
effect. These modifications of digestive physiology may have consequence on DE. For
example, as proposed by Cowieson and Massey’O’Neill (2013), caecal digestive microbiota may have an effect on ileal digestion via ileal brake mechanism, via release of peptide YY
secreted by the neuroendocrine cells in the distal tract in response to the presence of SCFA. Thus DE may be modulated by change of digestive microbiota. However the latter can be
modified by the former and is affected by several other factors, as microbiota is as an organ in
links with other animal physiological functions. All this equilibria must be taken into account
to proposed control of DE by digestive microbiota. 1.2.2. Conclusion on these observed relationships between digestive efficiency and gut
microbiota and further works With the differents studies presented above, we can see that various results have been
observed. These discrepancies may be due to different experimental conditions concerning
both animal experiment and sample analyses. Indeed concerning animal studies, experimental
conditions can be very different, from animal history before arrival for experiment to rearing
conditions and diet compositions. Mode of sampling and sample treatments may differ from
one study to another. For digestibility assessment, all the nutrients are not necessarily studied,
according to objectives of the works, and thus some nutrient having a change in its
digestibility may have been not highlighted. Another difference can come from microbiota analysis. Targeted approaches as bacterial counts of main bacteria can miss difference
contrary to new untargeted approaches. However parallel modifications of digestive microbiota and DE have been observed. This not
necessarily involves a links between these parameters. An additive may act on these two
parameters via different mechanisms as vegetal products, which, on one hand, may act on
digestive physiology and on the other hand may have an antibacterial activity (Gabriel et al.,
2013). However DE may be controlled via digestive microbiota via several mechanisms that
need to be studied. For example, as proposed by Cowieson et Massey’O’Neill (2013) by
decreasing transit rate with higher products of fermentation (SCFA) as modern broilers may
have a too rapid feed passage rate particularly due to lower gizzard activity (Croom et al.,
1999). The modification of microbiota can be performed by environmental factor as diet composition
or rearing conditions, and also by genetics as we will see in the following part. 2. Genetic control of digestive efficiency and digestive microbiota Beside technological treatment of diet and feed additives to improve DE via or independently
from digestive microbiota, genetic selection may be an interesting tool. Until now, genetic
selection of animals has been focused on the improvement of the performances of animals as
growth and feed efficiency. These phenotypes are complex traits. Although DE is one of the
components of feed efficiency, the selection on feed efficiency did not improve DE. Indeed,
in most studies of genetics, a highly digestible diet was used to enable animals to express their
genetic potential for feed efficiency and growth. With such diets, DE of birds is not
sollicitated, which explains that this component was not found to vary in most studies. At the
opposite, a strong genetic link was observed between feed efficiency and DE when using a
challenging diet, more difficult to digest (Carré et al., 2008). It also appears that to improve
DE by genetics, it must be directly targeted. However, even if this trait is less complex than
feed efficiency as it does not include components as heat production, protein and fat
deposition or energy expenditure for activity, it is still a complex trait. Digestive microbiota, which is also a complex trait, may also be under genetic control. In the following part, the concept of quantitative genetics will be used. This part of the
genetics deals with the way that continuous traits (such as body weight, feed efficiency, DE or
bacterial species content) are inherited. This approach includes the estimation of the heritability (h²) of these traits, i.e. the proportion of phenotypic variation in a population that
is attributable to genetic variation among individuals. The level of heritability indicates
whether selecting on the trait will be easy (high heritability) or difficult (low heritability). In
addition, quantitative genetics aims at identifying QTLs, which are genomic regions which
variation are associated with the quantitative variation in a phenotypic trait. Identifying QTLs
regions is necessary to do a selection assisted by markers (SAM) which takes into account the
genotypic information for selection. It is also the first step to identify genes on which the
variability of the trait relies on. QTL can be found at the chromosome level, or at the genome
level, the latest one using more strict statistical test than at the chromosome level. 2.1.1. General observation in monogastric farm animals This selection, performed within a breed of chicken fed with a diet rich in wheat,
concerned faecal digestive efficiency, and has proven the existence of individual variability of
digestive efficiency for lipid, protein, starch and energy (h² between 0.33 and 0.47). In poultry, two attempts to select directly for different digestive efficiencies in broiler
chickens have been performed. Zhang et al., (2003, 2005) attempted to select for phytate
phosphorous (P) bioavailability in a randomly bred chicken population. Selection for this trait
for three generations improved P bioavailability. Although the heritability of the trait was
very low (h²=0.05), it was significantly different from zero. The other selection on digestive
efficiency was performed in our laboratory as indicated previously (Mignon-Grasteau et al.,
2004). This selection, performed within a breed of chicken fed with a diet rich in wheat,
concerned faecal digestive efficiency, and has proven the existence of individual variability of
digestive efficiency for lipid, protein, starch and energy (h² between 0.33 and 0.47). 2.1.1. General observation in monogastric farm animals In poultry, contrarily to diets based on maize and soybean meal, a diet containing a high level
of wheat (336 g/kg) induced a rather high difference in metabolizable energy between lines of
chicken (Pym et al., 1984). Indeed, wheat diets were often observed to result in low
digestibilities when compared with maize diets and to lead to high variability between birds
(Carré et al., 2002; Hughes and Choct, 1997) suggesting a genetic factor in digestibility. These problems of wheat digestion largely come from high viscosity and hardness, which
decreases digestibility and then the value of wheat. These factors vary largely between wheat
varieties. In pig, the digestive loss of nutrients and energy ranges from 15% to 25% of the total intake in
usual rearing systems (Le Goff and Noblet, 2001). In addition to variations in diet
composition, feed technology and animal development (body weight), animal breed can
impact digestibility coefficients of nutrients and energy (Le Gall et al., 2009). Differences in
energy digestibility have been observed between lines selected for differences in adiposity
(Sundstöl et al., 1979). Comparing Asian breeds with commercial European lines has
generally shown that the latter have a lower digestive efficiency than the former, especially
when given fibrous feeds (Février et al., 1992; Kemp et al., 1991; Len et al., 2009; Morel et
al., 2006). Recently, in a study on 20 pigs, originating from four boars and three to four sows
per boar, and fed a diet with high dietary fibre content, Noblet et al., (2013) observed that the
apparent faecal digestibility was affected by boar origin, with a difference of 2.2 and 2.6 points between the extremes for energy and nitrogen, respectively. These preliminary results
suggest the possibility of selecting growing pigs for an increased digestive efficiency when
fed high DF diets. However, these preliminary data deserve a confirmation on a much greater
number of pigs to achieve estimations of genetic parameters for digestibility. In poultry, two attempts to select directly for different digestive efficiencies in broiler
chickens have been performed. Zhang et al., (2003, 2005) attempted to select for phytate
phosphorous (P) bioavailability in a randomly bred chicken population. Selection for this trait
for three generations improved P bioavailability. Although the heritability of the trait was
very low (h²=0.05), it was significantly different from zero. The other selection on digestive
efficiency was performed in our laboratory as indicated previously (Mignon-Grasteau et al.,
2004). 2.1.2. Current knowledge in chicken As indicated previously, the divergent lines available in our laboratory at the Poultry Research
Unit of INRA, have been divergently selected using AMEn as the criteria for digestive
efficiency (Mignon-Grasteau et al., 2004). The two lines were named D+ (high digester) and
D- (low digester) and have been selected on AMEn during 10 generations. By using wheat diet, the AMEn at 3 weeks of age showed a higher value of +13.2%
between D+ and D- birds at the 2nd generation (Mignon-Grasteau et al., 2004), and +33.5% at
the 8th generation (de Verdal et al., 2011), showing an increased divergence in this selected
trait. At 8 weeks of age, this difference between the lines disappeared at the 2nd generation
(Carré et al., 2005), whereas it persisted at the 9th generation (de Verdal et al., 2010b). Thus
the results observed at 3 weeks of age would still hold for the whole production cycle. As D+ birds fed with wheat diets were characterized at the 5th generation by a higher
AMEn than D- birds (+36.5%), the D+ birds were characterized by higher faecal
digestibilities of lipids, starch, and proteins, with a highest difference observed for lipid
(+58.0%), intermediate for starch (+39.3%), and lowest although significant for protein
(+13.3%) (Carré et al., 2007). Heritability of AMEn has been estimated in different experiments on the first 2th and 8th
generations and is between 0.30 and 0.38 when birds were fed with wheat (de Verdal et al.,
2011; Mignon-Grasteau et al., 2004, 2010). Heritabilities of faecal digestibility of lipids,
starch and proteins for the 8th first generations were 0.25 to 0.29 (Mignon-Grasteau et al.,
2010). Feed by genotype interactions were observed. Thus, the results are highly diet-dependent. When birds were fed a corn diet easier to digest, differences between lines were still
significant but much lower for AMEn and coefficients of digestive utilization, values in D+
line being 1 to 8 % higher than in D- line (Rougière et al., 2009). Whilst D+ chickens showed
a small variation in AMEn between maize and wheat (2.9%), D- chickens displayed a high
AMEn variation (10.3%) (Carré et al., 2008). 2.1.2. Current knowledge in chicken With a maize diet, at the 6th generation,
differences between lines were lower as well as for AMEn (+6.4%), and for digestibility, the
higher difference was observed for protein digestibility (+9.1%), followed by lipid
digestibility (+5.6%), and the lowest difference although significant was for starch
digestibility (+1.3%) (Rougière et al., 2009). Thus the limiting factors for the D- birds
digestibility were dependent on the cereal source. Heritability of AMEn is also diet-
dependent. It has been estimated to be only 0.15 when they were fed with maize (Mignon-
Grasteau et al., 2010). As for AMEn, heritability of digestibility was much lower when birds
were fed with maize (0.04 to 0.09), except for starch that presented equivalent levels of
heritability with both diets. These differences in digestive efficiency were associated with anatomical and
physiological differences between these two lines in all the parts of the digestive system as
previously reviewed in Gabriel et al., (2012). It can be noted that the lower feed intake of D+
birds compared to D- birds (-21.5% at the 8th generation), cannot be the only cause of
difference between the lines (de Verdal et al., 2011). Briefly, the most striking differences between D+ and D- birds are observed for the
proventriculus-gizzard complex. The relative weights of the proventriculus and gizzard are
higher in D+ birds than D- birds, +21.9% and +34.0% respectively, at 3 weeks of age at the
8th generation and pH of gizzard content was lower in D+ birds than in D- birds (de Verdal et
al., 2011, 2013). Pepsin activity in the proventriculus tissue was observed to be higher in D+
birds when expressed as per animal body weight (Péron et al., 2007). The isthmus area
between the proventriculus and the gizzard showed a 4 times larger lumen and a 1.4 larger total area of this region for D+ than for D- birds (Rideau et al., In press). This is the region
where are located the interstitial cells of Cajals, the pacemaker of gizzard contraction
(Reynhout and Duke, 1999). In D- birds, the isthmus mucosa has a more oval shape and more
twisted, and its muscular part is more developed than in D+ birds. A higher mean retention
time was observed in the stomach of D+ than in D- chickens at 9 and 29 d with a maize diet
(Rougière and Carré, 2010). 2.1.2. Current knowledge in chicken This may improve nutrient accessibility in D+ birds by
increasing time for grinding and enzymatic activity. This physiological parameter may be the
major factor associated with genotype differences between the D+ and D- genetic lines
(Rougière and Carré, 2010). A higher relative intestinal weight was observed in D- birds at 3 weeks of age. It concerns
each of the three segments, although less pronounced in duodenum (+15%) than in jejunum
(+37%) and ileum (+40%) (de Verdal et al., 2011), mainly due to increased density (weight to
length ratio), +12%, +30% and + 31%, for duodenum, jejunum and ileum respectively. This
increased density may be explained in part by an increased development of epithelium area
and thicker tunica muscularis. Moreover more goblet cells per villus were observed in D-
birds in jejunum and ileum (de Verdal et al., 2010a). Digestive contents of D+ and D- lines
showed some difference in their composition in terms of pH and bile salts. The pH of the
intestinal content is higher for D+ birds in duodenum and ileum at 21 d and in ileum at 53 d
(de Verdal et al., 2013, de Verdal, pers.comm.). Intestinal contents of D+ birds show more
conjugated bile acids and total bile acids (Garcia et al., 2007). In the caeca, heavier digestive contents were observed in D+ birds at the 8th generation at 3
weeks of age (+80%; H. de Verdal, comm pers.) and a higher relative weight tissue was
observed with a maize/soybean diet at 4 weeks of age (+29%; Rougière and Carré, 2010) . Moreover, the transit time was twice as long in D+ birds (Rougière and Carré, 2010). Thus at
3 weeks of age, caecal functions appeared more developed in D+ than in D- birds. Detection of QTLs controlling DE was undertaken on 820 chickens of the F2 cross between
the D- and D+ lines fed the wheat diet used for the selection and leads to the identification of
several genomic regions involved in this trait (Tran et al., In press). Nine QTLs were detected. They were mainly found for components of AMEn (mainly digestibility of starch) and only
one for AMEn itself. This result is consistent with the results of previous QTL studies devoted
to FCR, that found few QTLs for this trait, but rather QTLs on components of FCR, such as
feed intake, growth and body composition. 2.2.1. Observation in animal From about seven years according to Web of Science database, an increasing number of
studies are now evaluating the contribution of host genetics to the diversity of the microbial
community in the digestive tract, and the analysis of host genetics is just beginning to be
incorporated into studies to estimate its contribution to the diversity of the gut bacteria. 2.1.2. Current knowledge in chicken Moreover the fact that most QTL were found for
digestibility of starch is probably due to the fact that starch has the highest proportion in dietary content. For DE, two QTL significant at the genome level were present at the same
position for digestibility of starch and dry matter, two traits highly genetically correlated. Moreover, chromosome wide QTLs were detected for AMEn, digestibility of starch, dry
matter and protein at the same region on other chromosomes. Other chromosome wide QTLs
were identified for digestibility of starch and dry matter on three other chromosomes. Moreover, 11 QTL controlling digestive anatomy-related traits were observed. On two
chromosomes, co-localization between QTL controlling digestive efficiency and the ratio of
intestine length to body weight were observed. However, it is to note that most of these QTLs
are only significant at the chromosome level. This is probably linked to the fact that these
QTLs are not fixed in the F0 population. Moreover, digestive efficiency traits are probably
polygenic in their determinism, as it includes a large number of physiological processes such
as digestive secretions, enzymatic hydrolyses, absorption, motility and neurohormonal
regulations. Most of the QTL have a low effect in favor of a polygenic control, with the
exception of QTLs controlling digestibility of starch on one chromosome. Now, further
studies are needed to refine the position of these QTLs, in order to be able to identify the
candidate genes underlying these effects. An ongoing study is performed in our laboratory for
this (ADIGEN Project AGENAVI 2014-2015). In a further phase, it will be needed to validate
these results in commercial populations and breeding environments. 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals There is increasing evidence that genetics of the host influences and interacts with gut
microbiota in various mammals. Results of different types of studies on the role of the
genetics in shaping the composition of the gut microbiota, have recently been gathered in a
review by Spor et al., (2011) and more recent studies presented below. Comparing the faecal microbiota in human twins or between human differing by varying
degrees of genetic relatedness, has led to some conflicting results. Whereas some of these
studies using fingerprint technique suggest a strong influence of host genotype (Dicksved et
al., 2008; Van de Merwe et al., 1983; Stewart et al., 2005; Zoetendal et al., 2001), more
recent studies using sequencing did not (Turnbaugh et al., 2010) although these approaches
yield support for a role of host genetics. The effects are likely to be small, and detecting them
in a healthy population will require a large number of subjects, because of confounding
factors due to genetic diversity of human populations and strong environmental effects,
primarily diet, but also gene–environment interactions that may overshadow the effect of
genetics. In animals, results were more conclusive. With laboratory mice, comparison of caecal
microbiota showed more similar fingerprint between more related animals than with the
others (Hufeldt et al., 2010). The analysis of faecal samples from twin calves revealed higher
similarity in fingerprint profiles for twins compared to their coresident indicating that the
individual microflora might be genetically or epigenetically influenced (Mayer et al., 2012). In a study comparing the faecal microbiota of humans and 59 other mammalian species by
16S rRNA gene sequencing, Ley et al., (2008) observed that host diet and phylogeny both
influence bacterial diversity. In a study of human and four species of great apes, the host
phylogeny explained 25% of the variation in the faecal microbial community analyzed by
pyrosequencing with a high sampling depth (10 000 sequences per individual) to accurately
assess the diversity present in these complex microbial communities (Ochman et al., 2010). In animals, results were more conclusive. With laboratory mice, comparison of caecal
microbiota showed more similar fingerprint between more related animals than with the
others (Hufeldt et al., 2010). The analysis of faecal samples from twin calves revealed higher
similarity in fingerprint profiles for twins compared to their coresident indicating that the
individual microflora might be genetically or epigenetically influenced (Mayer et al., 2012). 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals Linnenbrink et al., (2013) studying the caecal microbiota by D-loop sequencing of house mice
from eight locations across Western Europe (France, Germany) found a small influence of
genetics, assessed by the genetic distance between populations. Thus, for these mice
populations, genetics has a weaker influence than environment to explain the diversity in the
digestive microbiota. Moreover, the authors observed that the influence of host genetics was
limited to the mucosa communities, this environment being more intimately dependent from
the host. Thus in caecal mucosa, geography and genetics explained 16% and 6% of the
microbiota variation respectively, and in caecal content, geography explained 11% of the
microbiota variation, whereas genetics was not significantly implied. However, the small
scale of genetic divergence among the populations included in this study may be limited with
regard to the potential influence on microbial communities. Genes of the host affecting digestive microbiota have also been observed in animal or human. For example, in pig, Meijerink et al., (2000) found that the adhesion of F18 fimbriated E. coli
to intestinal mucosa and subsequent susceptibility to swine edema disease was controlled by
fucosyl-transferase 1 gene, implied in H antigen production on red blood cells. In human, a
mutation of the gene encoding for the protein pyrin, a regulator of innate immunity, was
shown to be linked to significant changes in bacterial community structure of feces studied by
sequencing of 16S rDNA clones libraries and FISH (Khachatryan et al., 2008). In the
pathogenesis of inflammatory bowel diseases, two genes implied in mucosal immunity were
significantly associated with shifts in microbial compositions obtained by sequencing clones
libraries from intestinal tissues (Frank et al., 2011). Candidate gene approaches, in which one gene is deleted or added to a model host organism,
show that a single host gene can have a tremendous effect on the diversity and population
structure of the gut microbiota (Spor et al., 2011). Most of the genes shown to have an impact
on the composition of the gut microbiome are components of the immune system, and a few
others have roles in metabolism. Genes implied in immunity affecting digestive microbiota
are for example genes coding for defensing, cytokine, IgA, HLA of the major
histocompatibility complex, receptor or signaling molecule implied in innate immune system. 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals Thus, several studies in monogenic models (knockout mice) have demonstrated the role of
innate immune response in altering the composition of mouse gut microbiota and disease
susceptibility. For example, deficiency in Toll-like receptor 5 (TLR5) alters the abundance of
microbiota at species level leading to features characteristic of metabolic syndrome (Vijay-
Kumar et al., 2010). Mice deficient in a regulatory factor implied in immune functions had
greater instability in the composition of the faecal microbiota, both daily and over 5-day
intervals, than control mice (Thompson et al., 2010). Regarding genes implied in metabolism, a few host genes have been studied for their impact
on the gut microbiota. One is the gene coding apolipoprotein AI, another example is the
leptin-encoding gene, OB (also known as LEP) (Spor et al., 2011). More recently, Buhnik-
Rosenblau et al., (2012) observed that the deletion of iron metabolism genes in the mouse
host affects the composition of its faecal bacteria observed with fingerprint by t-RFLP and
sequencing. It may be due to change in luminal iron content of the gut which is one of the
important elements essential for bacterial growth. Gene involved in the regulation of
developmental processes, have also been observed to have consequences on digestive microbiota as deficiency in T-bet (Tbx2), that promotes a colitogenic microbial population
and ulcerative colitis (Garrett et al., 2007). However, some of these studies have shown a much weaker effect of genetics than a change
in diet on the microbial composition (Hildebrandt et al., 2009; Zhang et al., 2010). However, some of these studies have shown a much weaker effect of genetics than a change
in diet on the microbial composition (Hildebrandt et al., 2009; Zhang et al., 2010). Studies have been performed with genetically inbred mouse lines that provide a resource for
high resolution analysis of complex traits. The interest in using such genetic lines, instead of
approaches such as polymorphism genotyping or knockout animals, lies in the fact that a
hierarchy in genetic determinants can be proposed. With eight inbred mouse lines, Kovacs et al., (2011) concluded that the faecal microbiota was
substantially different in different genetic backgrounds by using DNA fingerprinting methods
based on length variability regions within the bacterial ribosomal RNA operon. 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals Nevertheless,
the small sample size, and the pooling of samples, which is known to result in
underrepresentation of rare taxa, may have hindered the assessment of the full magnitude of
the genotype effect (Manter et al., 2010). By studying the intestinal microbiota by pyrosequencing of 10 genetically distinct inbred
mouse strains, Campbell et al., (2012) studied the effects of host genetics and environmental
on caecal microbiota. They found significant correlations between the mouse strains and their
gut microbiota, reflected by distinct bacterial communities. Common environment had a
reduced, although detectable effect, and the microbiota response to this factor varied by strain. They identified discriminative and strain-specific bacterial phylotypes. Cohabitation of
different strains of mice revealed an interaction between host genetics and environmental
factors in shaping gut bacterial consortia, as bacterial communities became more similar
under common environments but remained strain specific. However, in these studies with inbred mouse lines, the maternal effect was confounded with
genotype effects. To disentangle between maternal and genotype effects, different methods
have been used as cross-fostering (swapping offspring between two mothers after birth),
uterine transplants of embryos of one genotype into a dam of another genotype, and
inoculation of one microbiota into a set of germ-free mice. Thus, in a study involving
reciprocal transplantations of digestive microbiota between germ-free zebra fish and mice, it
has been observed that after transplantation, the bacterial lineages found in each recipient
animal resembled the donor microbiota, but the relative abundance of each taxonomic group
was altered to be more similar to the typical microbiota of the recipient (Rawls et al., 2006). It shows that selection pressure in each host acts to influence community structure during and
after colonization. Now, quantitative genetics (QTL mapping) is emerging as a highly promising approach that
can be used to better understand the overall architecture of host genetics influence on the
microbiota, and to uncover potential candidate gene controlling microbial diversity in the gut
(Spor et al., 2011). These studies are performed on large number of animals, from divergently
selected lines, that can be intercrossed between 2 generations (producing a F2 cross) or during
more than 6 generations (production of an advanced intercross lines, AIL). The F2 crosses
have been widely used to detect the presence of QTL. As continuous intercrosses used to
produce AIL result in animals with many recombinations in the genome, AIL allow a better
mapping resolution (Darvasi et Soller, 1995). 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals A recent study using QTL mapping method detected genome-wide linkages with the relative
abundance of several taxa (Benson et al., 2010). The faecal microbiota of AIL population of
mice selected for wheel-running behavior was analyzed with pyrosequencing on 645 mice. The authors identified 13 significant QTLs and 5 suggestive QTLs for which host genetic
variation is significantly linked with relative abundances of 26 of the 64 conserved taxonomic
groups (defined as core microbiota) that varied quantitatively across most animals in the
population. In several instances, one QTL was associated with more than one taxon,
indicating that host genetic composition can influence population structure. These QTL
regions contain genes implied in immunity. More recently, McKnite et al., (2012) used this genetic mapping approach with recombinant
inbred strain of mice in combination with gene expression within the gastrointestinal tract. This population resulted from the combination of 2 mice strains and displays important
differences in susceptibility to obesity and other morphologic, immunologic, behavioral and
metabolic traits. The faecal microbiota studied by next-generation sequencing revealed
important quantitative differences in microbial composition among these strains. These
differences in gut microbial composition are influenced by host-genetics, which is complex
and involves many loci. Linkage analysis defined QTLs restricted to a particular taxon,
branch or that influenced the variation of taxa across phyla. Study of gene expression within
the gastrointestinal tract and analysis of the sequence of the parental genomes in the QTL
regions uncovered candidate genes with potential to alter gut immunological profiles and
impact the balance between gut microbial communities. With the exponential increase in the number of SNP (eg. 6K 5 years ago, 600K today in
chicken), the interval mapping approach used for QTL detection studies evolved toward
association studies. This approach is called Genome-wide association studies (GWAS). The
aim is to relate genome-wide variation in genetic markers to variation in digestive microbiota. GWAS studies identify SNPs and other variants in DNA which are associated with
components of digestive microbiota. Such studies in large populations will enable the
detection of genes for which variation is related to complex phenotypes that can be triggered
by differences in the digestive microbiota. However these studies cannot conclude on the
causal effect of a given gene. This systemic genetics approach was used by Parks et al., (2013) with more than 100 inbred
strains of mice to assess gene-by-diet interactions common to obesity, allowing an improved
mapping resolution. 2.2.1.1. Effect of genetics on digestive microbiota in mammals and model animals They observed a strong relationship between genotype and caecal
microbiota composition analysed by pyrosequencing. Indeed a strong effect of genetics was
observed on the composition and plasticity of the gut microbiota in response to altered dietary
composition, after feeding a high-fat and high sucrose diet instead of a control diet. A genome
region which contains three amylase genes may contribute to gut microbiota composition. In pigs, the SUS FLORA project (2011-2014 Genomic ANR; C. Rogel-Gaillard) being
developed at INRA has created Large White cohorts allowing to uncover the contribution of
genetics of the host to the microbiota composition via GWAS (Estellé et al., personal
communication). In fact, the intestinal microbiota has been associated to porcine immune and
health traits (Lepage et al., 2012) that are themselves under genetic control (Flori et al.,
2011). 2.2.1.2. Effect of genetics on digestive microbiota in birds Advantage to work on chicken is that, as in mice used as human model (Gootenberg and
Turnbaugh, 2011), all the environmental conditions can be controlled. Moreover, there is no
influence of maternal environment and the maternal effect is limited to the eggs formation,
and it allows working directly on the target animal species, avoiding the difference of
microbiota between the model and the target species as between mice and human (Ley et al.,
2005). In chicken, first studies with AGP showed that their effects on growth was dependent on the
animal strain suggesting that genetics may have an effect on digestive microbiota (Nordskog
et Johnson, 1953). By comparing the molecular fingerprint of digestive microbiota of ileal content of 3 genetic
lines of chicken feeding the same diet, Lumpkins et al., (2010) observed difference between
two modern genetic lines and an historic strain with a much slower rate of development than
the 2 other lines. In duck, by comparing the digestive microbiota analyzed by pyrosequencing
of two genotypes of duks (Pekin and Muscovy), fed ad libitum or overfed, Vasai et al., (2014)
observed that digestive microbiota of caecal content is more affected by genetics than by
overfeeding, whereas digestive microbiota of ileal content is affected by overfeeding. As for laboratory mice, used as human model, several well characterized divergent genetic
lines of chickens have been developed as research tools for agronomic research. It allows
using a QTL detection approach that allows measuring the heritability of the digestive
microbiota as explained for mice previously. As it can be expected that a genetic selection on DE, that has a direct effect on the biotope of
digestive microbiota has an impact on the microbiota, in our laboratory, we used our
divergent lines D+ and D- to study effect of genetics on digestive microbiota in chicken. Digestive microbiota of the two divergent chicken lines was studied in birds from the 10th
generation, in the terminal ileum and the caeca. For these studies, birds were reared on litter
the first 10 days of life to be in contact with the environmental microbiota from other birds,
and then reared in individual cages for AMEn determination. The analyses showed clear
differences between microbiota of the two lines in both the digestive contents and in the
mucosa (Gabriel et al., 2011, 2012; Konsak et al., 2011, 2012). 2.2.1.2. Effect of genetics on digestive microbiota in birds In the ileum contents, comparison of bacterial fingerprint of the two bird lines showed
significant difference between them. Identification of specific bacteria showed a higher
amount of a strain of a long segmented filamentous organism in D+ birds, belonging to
cluster I of Clostridium, and a strain of L. crispatus in D- birds. Moreover, quantitative
analysis by qPCR of the main bacterial groups found in the digestive tract of chickens showed
in D+ chickens a higher amount of C. coccoides and a higher amount of E. coli in D-
chickens. In the ileal mucosa, more L. salivarius per segment were observed in D- birds. In the caeca contents, in D+ birds, total bacterial biomass is higher than those of their small
intestine, contrarily to D- birds that may have a similar or slightly higher bacterial load in the
small intestine than in their caeca. Moreover, a high difference between the fingerprints of the
two bird lines was observed, and a higher relative amount of an E. coli strain was found in D-
birds. Moreover, in D+ birds, higher amounts of C. leptum group were observed, and in D-
birds more Lactobacillus, and particularly L. salivarius, a dominant lactic acid bacteria in
broiler digestive tract and more E. coli. In the caecal mucosa, as in the caecal content, a high
difference between fingerprints of the two bird lines was observed, and a higher relative
amount of an E. coli strain in D+ birds, and a L. salivarius strain in D- birds. In this mucosa,
in D- birds, more Lactobacillus per segment, as well as L. salivarius and L. crispatus were
observed. Thus contrarily to the observation of Linnenbrink et al., (2013) in mice, digestive
microbiota was modified both in content and in mucosa of caeca. Using a high number of F2 birds (144 animals) of these divergently selected line, D+ and D-
with high range of AMEn (from 7.6 to 16.1 MJ/kg), and on one of the more discriminant
biotope observed previously, caecal content, genetic parameters were determined. Significant
heritability estimates were observed for bacterial numbers, or higher for bacterial ratios
(Gabriel et al., 2012). Thus heritability ranged between 0.11 and 0.14 for the genus
Lactobacillus, and more precisely with L. salivarius. Higher heritability estimates were
obtained (h2 close to 0.20) for the ratios of L. salivarius to C. leptum and of C. leptum to C. coccoides. 2.2.1.2. Effect of genetics on digestive microbiota in birds The chickens were reared in individual cages to prevent exchange between
birds and fed a corn-soybean diet. Birds were sampled at 8 months. The digestive microbiota
that was targeted was faecal microbiota, and the method used to study it was next generation
sequencing (NGS), with high sequencing depth (about 20 000 sequences reads per sample,
between 7 600 and 32 900). They showed that quantitative genetic background of the host
influences gut microbiomes in chickens, as they observed difference in microbiota at the
genera and species level: 29 species were affected by genotype. However they did not obtain
significant heritability, although some show high value maybe because of low number of
birds used in this study (56 animals, with 12-15 animals per group, 2 sexes, 2 strains). 2.2.1.2. Effect of genetics on digestive microbiota in birds The highest heritability was estimated for the ratio of C. coccoides to Lactobacillus
(h2=0.34). These estimates imply that the development of microbiota is partly controlled by
the genetics of the host. Moreover, 15 QTLs regions controlling variations of caecal microbiota composition have
been identified in our F2 cross. These regions are under analysis to detect candidate genes. Moreover, gene expression within the gastrointestinal tract, in parallel to the determination of
major bacteria groups, will allow refining these candidate genes (ADIGEN Project,
AGENAVI 2014-2015). It may contribute to explain the observed link between host genetics
and its digestive microbiota. A new study is also in progress on AIL population of D+D- lines, generation 7, with 216 birds
(ongoing project: INRA Project GISA Galmide 2014-2015). It will allow refining the
relationship between microbiota composition, innate immune system parameters and digestive
efficiency (Calenge et al., 2013). In this study, digestive microbiota will be studied by
pyrosequencing allowing a more precise description of digestive microbiota. Recently, Zhao et al., (2013) used two divergent chicken lines to study the genetic effect on
digestive microbiota. These two lines of chickens were selected during 54 generations for
high (HW) or low (LW) 56-day body weight, and maintained at the same location and reared
on the same diets. The chickens were reared in individual cages to prevent exchange between
birds and fed a corn-soybean diet. Birds were sampled at 8 months. The digestive microbiota
that was targeted was faecal microbiota, and the method used to study it was next generation
sequencing (NGS), with high sequencing depth (about 20 000 sequences reads per sample,
between 7 600 and 32 900). They showed that quantitative genetic background of the host
influences gut microbiomes in chickens, as they observed difference in microbiota at the
genera and species level: 29 species were affected by genotype. However they did not obtain
significant heritability, although some show high value maybe because of low number of
birds used in this study (56 animals, with 12-15 animals per group, 2 sexes, 2 strains). Recently, Zhao et al., (2013) used two divergent chicken lines to study the genetic effect on
digestive microbiota. These two lines of chickens were selected during 54 generations for
high (HW) or low (LW) 56-day body weight, and maintained at the same location and reared
on the same diets. 2.2.2. Assessment of present knowledge and hypotheses on this relationship Published studies in mammals, fish and birds, show an effect of host genetics on digestive
microbiota that may explain a part of individual variability in the case of animal reared in
very similar environment, as poultry. Host genotype may exert an effect on the composition
of the microbiota by a selective pressure imposed within the gut habitat through secretions as
bile acids and other antimicrobial compounds as defensins, control of digestion, gut motility
or modification of epithelial cell surfaces. This selective pressure is not the same according to
each digestive compartment as different physiochemical properties exist, and depend on host
genotype and gene expression in these compartments. At the present day, studies in mammals have highlighted the implication of genes implied
mainly in immunity and metabolism to control digestive microbiota. However, there is bias in
the genes that could be found by these studies, which mostly relied on animal models used in
these studies, focused on human health diseases as metabolic, immunity or neuropsychiatric
disease. However, it is interesting to note that in our QTL detection study, several QTLs for
digestive efficiency have been identified on chromosome 16, which role in immunity of
chicken is well-known. Other sets of genes, than those of immunity and metabolism, may be
implied in the control of digestive microbiota, as the genes implied in the characteristic of the
digestive biotopes, as suggested by results of Parks et al., (2013) who found a genome region
which contains three amylase genes that may contribute to gut microbiota composition. Genes implied in digestive physiology will influence the composition of the undigested compounds
of the diet in the digestive tract, and then in turn, substrates than can be used by microbiota. Thus genes implied in the digestion steps as hydrolysis and absorption, or digestive transit via
neurohormonal regulation, may control microbiota. In poultry, researches ongoing in our
laboratory are precisely designed to determine the relationship between microbiota and
digestibility, which increase the chance to find such genes. This effect of host genetics on digestive microbiota is probably the results of adaptation
during host speciation and the evolution of pluricellular eukaryotes to their surrounding
prokaryotes to evolve to a mutualistic relationship between the host and the colonizers as
proposed by Benson et al., (2010). This evolution may imply complex sets of loci, that may
have been implied during stepwise change of host and its microbiota. 2.2.2. Assessment of present knowledge and hypotheses on this relationship This could explain the
evolution of highly specialized digestive organs (e.g. crop and ceca in poultry) that harness
microbes for fermentation. By exerting this selective pressure, host genetics control would
control microbiota within the gut ecosystem to promote beneficial microbes. This hypothesis
is compatible with the suggestion that the adaptive immune system has specifically evolved in
vertebrates to regulate and maintain beneficial microbial communities (McFall-Ngai, 2007). 3. Conclusion and further knowledge needed Digestive efficiency is a major goal in poultry production for several reasons, economic,
environmental and sociological. As shown by the works performed in our laboratory, DE is
under genetic control. Moreover, DE depends on digestive microbiota composition. This later
one and its stability appear also to depend on genetics, via or not via DE, whose results lead to
microbial substrates. Thus DE, host genetics and digestive microbiota are linked together. Further studies are needed to understand these genetic controls and these interactions. It is
made easier with the development of high throughput method to study digestive microbiota. Work in progress in our laboratory will contribute to improve knowledge concerning genetic
control of DE, with finer delimitation of QTLs. Moreover, if identified gene candidates are
confirmed in our further work, after being validated in commercial populations and breeding
environment, they can be included in selection schemes, in selection assisted by markers. Concerning genetic control of digestive microbiota, analysis of identified QTLs region will
allow detecting candidate genes. Moreover parallel study of gene expression within the gastrointestinal tract will allow refining these candidate genes, and may contribute to explain
the observed link between host genetics and its digestive microbiota. Assessing the causal role
of host genetic variation in gut microbiota composition and dynamics will enable an
understanding of the mechanisms of colonization, and the relation to DE. Correlations of QTL
and host gene expression to bacterial diversity will likely shed more light on potential
physiological roles of digestive bacteria. Understanding the mechanisms of community
selection and genetic influences on community structure will have many implications for
attempts to alter this community structures in order to increase DE. Moreover, future isolation
and physiological studies of bacterial taxa that were discriminatory among the divergent lines
selected for DE may improve our understanding of the role of these bacteria. However, one question to answer when studying digestive microbiota, genetics and host
phenotype as DE, is which digestive microbiota has the highest influence on the host : the one
with highest concentration or whose with highest contact with the host ? The one of content or
mucosa ? The one of small intestine or caeca, and also crop as inoculum of the following
digestive tract ? 3. Conclusion and further knowledge needed And can feces (intestinal or caecal) be used to represent these microbiota in
order to collect samples without killing birds to follow microbiota evolution on the same
animal or to work on farm ? Moreover, several parameters need to be taken into account from sampling conditions, choice
of analysis of individual or pool samples, and number of samples per treatment that need to be
high due to high animal variability to have a faithful representation of the reality. The
different steps of sample processing need also to be adequately chosen to obtain the image the
more representative of the reality. These different steps include sample storage conditions,
bacterial DNA extraction adapted to the specificity of digestive samples (types of potential
bacteria and digestive matrix), PCR conditions, pyrosequencing parameters as choice of
sequencing depth, bioinformatics analysis of generated data, and statistical analysis of these
data, as all of these steps have impact on the final results. These different steps need
standardization and compromises are needed to be practical. Although genetics appear to have an important part in the control of digestive microbiota,
numerous environmental factors are also implied. Thus, environment of first days of life may
be crucial in the development of digestive microbiota as proposed by Stanley et al., (2013a). First bacteria in contact with the bird are at hatcher, and further in post-hatch environment
during vaccination, boxes for transportation. During this fasting and other stress that birds undergo (temperature changes, vibrations), digestive microbiota depends on animal
endogenous products as mucus, intestinal cells and yolk sac content. The consequences of
these post-hatching conditions on digestive microbiota will be studied in our laboratory
(ongoing project: INRA Project GISA Whelp 2014-2016; L. Guilloteau and C. Leterrier). At
arrival on the farm, bacteria come from the diet, water, litter, and other birds. On farm,
digestive microbiota can be impacted by rearing environment as animal density (Guardia et
al., 2011) and diet composition as explained previously. The sum of the small differences in
each step of microbial colonization may be responsible in part to the variability between
individual birds. Maternal effect, although potential not so important than in mammals, may
be implied as proposed by Torok et al., (2011). Indeed, maternal nutrition has been shown to
alter chick gut development (Rebel et al., 2006), which has been linked indirectly to
microbiota development and biological variation between birds (Lumpkins et al., 2010). 3. Conclusion and further knowledge needed Moreover, in microbial ecology, it is know that a same initial biocenose can evolve to
different states. The objective will be to perform a hierarchy in all these factors, genetic and
environmental, in order to manipulate microbiota to improve DE. Another essential point is that not only the composition but also the functional capacity of the
digestive microbiota is highly important regarding the host physiology as DE and
performances. Indeed, different bacterial strains of the same species can have profound
differences in the interaction with their host. However current phylogenetic characterization
of digestive microbiota by pyrosequencing does not allow to the determination of bacteria
strain because of information in databases that depend on current knowledge, yet a high
proportion of bacteria in complex system as in the digestive tract are not known, as the
exploration of these systems are in progress. That’s why recently was developed metagenomic
approach in order to highlighted all the potential functions of digestive microbiota in human
(Qin et al., 2010; Turnbaugh et al., 2009) and also in chicken (Yeoman et al., 2012). However
the detection of genes in a metagenomic library does not necessarily means that they are
functionally important, as microbiota effect on the host depends on bacterial metabolism that
depends to their environment (Roy et al., 2008). As bacteria fulfil their effect on the host by
the intermediate of excreted products as protein or metabolites and/or their direct action, to
gain better insight into the activity and functionality of the intestinal microbiota, its products
can be studied as RNA, proteins and metabolites (Blottière et al., 2013). Metatranscriptomics
remains a challenging step due to the difficulty of obtaining intact RNA. Bacterial proteins
and metabolites can be studied by targeted approach. Thus specific bacterial enzymes have been studied. Particularly, the metabolites which represent the actual end products of
metabolism are of high significance for the links with host physiology. Large amounts and
various compounds are produced by microbiota. Specific metabolites can be studied as those
from carbohydrates as it is the main substrate for microbial fermentation. As the main
products of carbohydrate metabolism are SCFA, they have been the most studied. As bacteria
fermentations switch to protein fermentation when carbohydrate source are depleted, products
of protein fermentation have also been studied. Fermentation of bile acids has also been
studied due to the various modifications by microbial fermentation and consequences on the
host. 3. Conclusion and further knowledge needed However, these latest years, metaproteomics and metabolomics are rapidly developing
and applied to digestive samples. They allow following all the proteins and metabolites
respectively. By following these approaches, novel proteins and metabolites, and mechanisms
involved in host physiology as DE, can be identified. Metaproteomic has been used to study
digestive microbiota in human (Kolmeder et al., 2012) and in chicken (Tang et al., 2014). Metabolomic has also been applied in human (Nicholson et al., 2012) and in chicken (Bailey,
2010; Gabriel et al., unpublished), and we will study these metabolites in our ongoing project
on AIL population of D+D- lines (INRA Project GISA Galmide 2014-2015). To move
forward in this area, knowledge about the exact microbiota-derived metabolites that can
induce host responses need to be increased. Digestive microbiota has a complex position in the relation to the host. It can be view as a
phenotype trait as whatever other phenotypes, being the results of genetic and environmental
factors, but it can also be viewed as an environmental factor that contribute with genetic
components to multifactorial phenotype traits of animals as DE. To study these interactions, it
appears that an holistic approach is needed which will be possible thanks to the development
of the different OMIC methods, and the use of divergent line of animals (Blottière et al.,
2013). The main challenge is the integration of complex data in order to identify meaningful
relationships. This knowledge of host-gut microbe interrelationships will rapidly increase and
allow orientating digestive microbiota to the targeted phenotype. However, it must be noted that this search in optimal digestive microbiota to improve DE,
must take into accounts other effects on animal phenotype, and that there are no trade-off with
other important phenotypic traits as immunity, as we will studied in our ongoing project
(INRA Project GISA Galmide 2014-2015). To conclude, knowledge of microbiota composition is important to understand in which
digestive biotopes these populations lead to which products having an effect on the host
physiology. Deciphering these microbial signatures and their metabolites that govern short
and long-term equilibrium, as well as imbalances in host-microbial relationships, may provide
novel physiological markers of the targeted phenotype traits, as DE. Thus, increase in knowledge on the complex dynamic of poultry digestive tract ecosystem is
needed in the future, in order to improve phenotypic traits as DE. References Al-Lahham, S.H., Peppelenbosch, M.P., Roelofsen, H., Vonk, R.J., Venema, K., 2010. Biological effects of propionic acid in humans; metabolism, potential applications and
underlying mechanisms. Biochim. Biophys. Acta 1801, 1175-1183. Alloui, M.N., Szczurek, W., Swiatkiewicz, S., 2013. The usefulness of prebiotics and
probiotics in modern poultry nutrition : a review. Ann. Anim. Sci. 13, 17-32. Amerah, A.M., Peron, A., Zaefarian, F., Ravindran, V., 2011. Influence of whole wheat
inclusion and a blend of essential oils on the performance, nutrient utilisation, digestive
tract development and ileal microbiota profile of broiler chickens. Br. Poult. Sci. 52, 124-
132. Amerah, A.M., Ravindran, V., Lentle, R.G., 2009. Influence of insoluble fibre and whole
wheat inclusion on the performance, digestive tract development and ileal microbiota
profile of broiler chickens. Br. Poult. Sci. 50, 366-375. Apajalahti, J., Kettunen, A., Graham, H., 2004. Characteristics of the gastrointestinal
microbial communities, with special reference to the chicken. Worlds Poult. Sci. J. 60,
223-232. Bailey, R.A., 2010. Intestinal microbiota and the pathogenesis of dysbacteriosis in broiler
chickens. Thesis, Institute of Food Research, Norwich Research Park, Colney Lane,
Norwich, Norfolk, NR4 7UA, United Kingdom, 198 pages. Bar-Shira, E., Friedman, A., 2005. Ontogeny of gut associated immune competence in the
chick. Refu. Vet. 60, 42-50. Baurhoo, N., Baurhoo, B., Mustafa, A.F., Zhao, X., 2011a. Comparison of corn-based and
Canadian pearl millet-based diets on performance, digestibility, villus morphology, and
digestive microbial populations in broiler chickens. Poult. Sci. 90, 579-586. Baurhoo, N., Baurhoo, B., Zhao, X., 2011b. Effects of exogenous enzymes in corn-based and
Canadian pearl millet-based diets with reduced soybean meal on growth performance,
intestinal nutrient digestibility, villus development, and selected microbial populations in
broiler chickens. J. Anim. Sci. 89, 4100-4108. Bedford, M.R., Cowieson, A.J., 2012. Exogenous enzymes and their effects on intestinal
microbiology. Anim. Feed Sci. Technol. 173, 76-85. Benson, A.K., Kelly, S.A., Legge, R., Ma, F., Low, S.J., Kim, J., Zhang, M., Oh, P.L.,
Nehrenberg, D., Hua, K., Kachman, S.D., Moriyama, E.N., Walter, J., Peterson, D.A.,
Pomp, D., 2010. Individuality in gut microbiota composition is a complex polygenic trait
shaped by multiple environmental and host genetic factors. Proc. Natl. Acad. Sci. U.S.A. 107, 18933-18938. Bhuiyan, M.M., Islam, A.F., Iji, P.A., 2010. Response of broiler chickens to diets containing
artificially dried high-moisture maize supplemented with microbial enzymes. S. Afr. J. Anim. Sci. 40, 348-362. Bhuiyan, M.M., Islam, A.F., Iji, P.A., 2013. References High levels of maize in broiler diets with or
without microbial enzyme supplementation. S. Afr. J. Anim. Sci. 43, 44-55. Blottiere, H.M., de Vos, W.M., Ehrlich, S.D., Dore, J., 2013. Human intestinal metagenomics:
state of the art and future. Curr. Opin. Microbiol. 16, 232-239. Boyd, F.M., Edwards, H.M., 1967. Fat absorption by germ-free chicks. Poult. Sci. 46, 1481-
1483. Buhnik-Rosenblau, K., Moshe-Belizowski, S., Danin-Poleg, Y., Meyron-Holtz, E.G., 2012. Genetic modification of iron metabolism in mice affects the gut microbiota. Biometals 25,
883-892. Calenge, F., Quéré, P., Trapp, S., Grasteau, S., Le-Bihan-Duval, E., Gabriel, I., Bed’Hom, B.,
2013. Improving immune competence: screening for new parameters describing the innate
immune status in chicken. In : 8th European Symposium on Poultry Genetics (Venice,
Italy). Campbell, J.H., Foster, C.M., Vishnivetskaya, T., Campbell, A.G., Yang, Z.K., Wymore, A.,
Palumbo, A.V., Chesler, E.J., Podar, M., 2012. Host genetic and environmental effects on
mouse intestinal microbiota. ISME J. 6, 2033-2044. Cao, P.H., Li, F.D., Li, Y.F., Ru, Y.J., Peron, A., Schulze, H., Bento, H., 2010. Effect of
essential oils and feed enzymes on performance and nutrient utilization in broilers fed a
corn/soy-based diet. Int. J. Poult. Sci. 9, 749-755. Carré, B., Idi, A., Maisonnier, S., Melcion, J.P., Oury, F.X., Gomez, J., Pluchard, P., 2002. Relationships between digestibilities of food components and characteristics of wheats
(Triticum aestivum) introduced as the only cereal source in a broiler chicken diet. Br. Poult. Sci. 43, 404-415. Carré, B., Mignon-Grasteau, S., Svihus, B., Péron, A., Bastianelli, D., Gomez, J., Besnard, J.,
Sellier, N., 2005. Nutritional effects of feed form, and wheat compared to maize, in the D+
and D- chicken lines selected for divergent digestion capacity. In : 15th European
Symposium on Poultry Nutrition (Budapest, Hungary), pp. 42-44. Carre, B., Mignon-Grasteau, S., Peron, A., Juin, H., Bastianelli, D., 2007. Wheat value:
improvements by feed technology, plant breeding and animal genetics. Worlds Poult. Sci. J. 63, 585-596. Carré, B., Mignon-Grasteau, S., Juin, H., 2008. Breeding for feed efficiency and adaptation to
feed in poultry. Worlds Poult. Sci. J. 64, 377-390. Champ, M., Szylit, O., Gallant, D.J., 1981. The influence of microflora on the breakdown of
maize starch granules in the digestive tract of chicken. Poult. Sci. 60, 179-187. Choct, M., Hughes, R.J., Bedford, M.R., 1999. Effects of a xylanase on individual bird
variation, starch digestion throughout the intestine, and ileal and caecal volatile fatty acid
production in chickens fed wheat. Br. Poult. Sci. References 40, 419-422. Clench, M.H., 1999. The avian cecum: update and motility review. J. Exp. Zool. 283, 441-
447. Clench, M.H., 1999. The avian cecum: update and motility review. J. Exp. Zool. 283, 441-
447. Cowieson, A.J., Massey O'Neill, H.V., 2013. Effects of exogenous xylanase on performance,
nutrient digestibility and caecal thermal profiles of broilers given wheat-based diets. Br. Poult. Sci. 54, 346-354. Crévieu-Gabriel, I., Naciri, M., 2001. Dietary effect on chicken coccidiosis. Prod. Anim. 14,
231-246. Croom, W.J., Brake, J., Coles, B.A., Havenstein, G.B., Christensen, V.L., McBride, B.W.,
Peebles, E.D., Taylor, I.L., 1999. Is intestinal absorption capacity rate-limiting for
performance in poultry. J. Appl. Poult. Res. 8, 242-252. Danzeisen, J.L., Kim, H.B., Isaacson, R.E., Tu, Z.J., Johnson, T.J., 2011. Modulations of the
chicken cecal microbiome and metagenome in response to anticoccidial and growth
promoter treatment. PLoS One 6, e27949. Darvasi, A., Soller, M., 1995. Advanced intercross lines, an experimental population for fine
genetic mapping. Genetics 141, 1199-1207. De Verdal, H., Mignon-Grasteau, S., Jeulin, C., Le Bihan-Duval, E., Leconte, M., Mallet, S.,
Martin, C., Narcy, A., 2010a. Digestive tract measurements and histological adaptation in
broiler lines divergently selected for digestive efficiency. Poult. Sci. 89, 1955-1961. De Verdal, H., Narcy, A., Le Bihan-Duval, E., Mignon-Grasteau, S., 2010b. Excretion and
gastro-intestinal tract development in chickens divergently selected on their capacity of
digestion. In : 13th European Poultry Conference (Tours, France), 6 pages. De Verdal, H., Narcy, A., Bastianelli, D., Chapuis, H., Meme, N., Urvoix, S., Le Bihan-
Duval, E., Mignon-Grasteau, S., 2011. Improving the efficiency of feed utilization in
poultry by selection. 1. Genetic parameters of anatomy of the gastro-intestinal tract and
digestive efficiency. BMC Genet. 12, 59. De Verdal, H., Mignon-Grasteau, S., Bastianelli, D., Même, N., Le Bihan-Duval, E., Narcy,
A., 2013. Reducing the environmental impact of poultry breeding by genetic selection. J. Anim. Sci. 91, 613-622. Dibner, J. J., Richards, J. D., Knight, C. D., 2008. Microbial imprinting in gut development
and health. J. Appl. Poult. Res. 17, 174-188. Dicksved, J., Halfvarson, J., Rosenquist, M., Jarnerot, G., Tysk, C., Apajalahti, J., Engstrand,
L., Jansson, J.K., 2008. Molecular analysis of the gut microbiota of identical twins with
Crohn's disease. ISME J. 2, 716-727. Dublecz, K., Pál, L., Wágner, L., Bartos, Á., Bányai, A., Tóth, S., 2006. Differences between
the faecal and ileal amino acid digestibility values of soybean meal, determined with
broiler chicks at different ages. References In : 12th European Poultry Conference (Verona, Italy), pp. 317. El Aidy, S., Van den Abbeele, P., Van de Wiele, T., Louis, P., Kleerebezem, M., 2013. Intestinal colonization: how key microbial players become established in this dynamic
process: microbial metabolic activities and the interplay between the host and microbes. Bioessays 35, 913-923. Engberg, R.M., Hedemann, M.S., Steenfeldt, S., Jensen, B.B., 2004. Influence of whole wheat
and xylanase on broiler performance and microbial composition and activity in the
digestive tract. Poult. Sci. 83, 925-938. Février, C., Bourdon, D., Aumaitre, A., 1992. Effects of level of dietary fiber from wheat bran
on digestibility of nutrients, digestive enzymes and performance in the European Large
White and Chinese Mei Shan pig. J. Anim. Physiol. Anim. Nutr. 68, 60-72. Flori, L., Gao, Y., Laloë, D., Lemonnier, G., Leplat, J.J., Teillaud, A., Cossalter, A.M.,
Laffitte, J., Pinton, P., de Vaureix, C., Bouffaud, M., Mercat, M.J., Lefèvre, F., Oswald,
I.P., Bidanel, J.P., Rogel-Gaillard, C., 2011. Immunity traits in pigs: substantial genetic
variation and limited covariation. PLoS One 6, e22717. Frank, D.N., Robertson, C.E., Hamm, C.M., Kpadeh, Z., Zhang, T., Chen, H., Zhu, W.,
Sartor, R.B., Boedeker, E.C., Harpaz, N., Pace, N.R., Li, E., 2011. Disease phenotype and
genotype are associated with shifts in intestinal-associated microbiota in inflammatory
bowel diseases. Inflamm. Bowel Dis. 17, 179-184. Fuller, M., 2012. Determination of protein and amino acid digestibility in foods including
implications of gut microbial amino acid synthesis. Br. J. Nutr. 108, S238-S246. Furuse, M., Okumura, J., 1994. Nutritional and physiological characteristics in germ-free
chickens. Comp. Biochem. Physiol. 109A, 547-556. Gabriel, I., Lessire, M., Mallet, S., Guillot, J.F., 2006. Microflora of the digestive tract:
critical factors and consequences for poultry. Worlds Poult. Sci. J. 62, 499-511. Gabriel, I., Leconte, M., Guillon, J., Rideaud, P., Moreau-Vauzelle, C., Dupont, C., 2007. Individual variability in the digestive flora of the broiler chicken analysed by molecular
fingerprint. In : 16th European Symposium on Poultry Nutrition (Strasbourg, France), pp. 305-308. Gabriel, I., Guardia, S., Konsak, B., Leconte, M., Rideaud, P., Moreau-Vauzelle, C., Dupont,
C., Mignon-Grasteau, S., 2011. Comparison of chicken digestive microbiota selected on
their metabolizable energy. In : 9th Poultry Research Days (Tours, France), pp. 760-764. Gabriel, I., Konsak, B., Mignon-Grasteau, S., 2012. Genetic selection of poultry based on
digestive capacity – Impact on gut microbiota. In : Wiseman, J., Garnsworthy, P.C. (Eds.),
Recent Advances in Animal Nutrition. Nottingham (UK), Nottingham University Press,
pp. 197-238. Gootenberg, D.B., Turnbaugh, P.J., 2011. Companion animals symposium: humanized animal
models of the microbiome. J. Anim. Sci. 89, 1531-1537. References Gabriel, I., Alleman, F., Dufourcq, V., Perrin, F., Gabarrou, J.F., 2013. Essential oils in
poultry feeding. 2. Hypotheses of modes of action implicated. Prod. Anim. 26, 13-24. Garcia, V., Gomez, J., Mignon-Grasteau, S., Sellier, N., Carré, B., 2007. Effect of xylanase
and antibiotic supplementations on the nutritional utilisation of a wheat diet in growing
chicks from genetic D+ and D- lines selected for divergent digestion efficiency. Animal 1,
1435-1442. Garrett, W.S., Lord, G.M., Punit, S., Lugo-Villarino, G., Mazmanian, S.K., Ito, S., Glickman,
J.N., Glimcher, L.H., 2007. Communicable ulcerative colitis induced by T-bet deficiency
in the innate immune system. Cell 131, 33-45. Gong, J., Si, W., Forster, R.J., Huang, R., Yu, H., Yin, Y., Yang, C., Han, Y., 2007. 16S
rRNA gene-based analysis of mucosa-associated bacterial community and phylogeny in
the chicken gastrointestinal tracts: from crops to ceca. FEMS Microbiol. Ecol. 59, 147-157. Gootenberg, D.B., Turnbaugh, P.J., 2011. Companion animals symposium: humanized animal
models of the microbiome. J. Anim. Sci. 89, 1531-1537. Groh, H., Schade, K., Hörhold-Schubert, C., 1993. Steroid metabolism with intestinal
microorganisms. J. Basic. Microbiol. 33, 59-72. Groh, H., Schade, K., Hörhold-Schubert, C., 1993. Steroid metabolism with intestinal
microorganisms. J. Basic. Microbiol. 33, 59-72. Guardia, S., Konsak, B., Juin, H., Lessire, M., Rideaud, P., Moreau-Vauzelle, C., Dupont, C.,
Guillot, J.F., Gabriel, I., 2011. Effects of stocking density on growth performance and
digestive microbiota of broiler chickens. Poult. Sci. 90, 1878-1889. Hildebrandt, M.A., Hoffman, C., Sherrill-Mix, S.A., Keilbaugh, S.A., Hamady, M., Chen,
Y.Y., Knight, R., Ahima, R.S., Bushman, F., Wu, G.D., 2009. High Fat Diet Determines
the Composition of the Murine Gut Microbiome Independently of Obesity. Gastroenterology 137, 1716-1724. e1-2. Holzer, P., Reichmann, F., Farzi, A., 2012. Neuropeptide Y, peptide YY and pancreatic
polypeptide in the gut-brain axis. Neuropeptides 46, 261-274. Hufeldt, M.R., Nielsen, D.S., Vogensen, F.K., Midtvedt, T., Hansen, A.K., 2010. Family
relationship of female breeders reduce the systematic inter-individual variation in the gut
microbiota of inbred laboratory mice. Lab. Anim. 44, 283-289. Hughes, R., Choct, M., 1997. Low-ME wheat or low-ME chickens?-Highly variable
responses by birds on the same low-ME wheat diet. In : Aust. Poult. Sci. Symp., pp. 138–
141. ITAVI,
2014. Index
number
of
feedstuff
cost
in
poultry
diet
(http://www.itavi.asso.fr/economie/conjoncture/volailles.php; consulted 25/01/14). Kau, A.L., Ahern, P.P., Griffin, N.W., Goodman, A.L., Gordon, J.I., 2011. Human nutrition,
the gut microbiome and the immune system. Nature 474, 327-336. Kemp, B., den Hartog, L.A., Klog, J.J., Zandstra, T., 1991. References The digestibility of nutrients,
energy and nitrogen in the Meishan and Dutch landrace pig. J. Anim. Physiol. Anim. Nutr. 65, 263–266. Khachatryan, Z.A., Ktsoyan, Z.A., Manukyan, G.P., Kelly, D., Ghazaryan, K.A., Aminov,
R.I., 2008. Predominant role of host genetics in controlling the composition of gut
microbiota. PLoS One 3, e3064. Kim, G.B., Lee, B.H., 2005. Biochemical and molecular insights into bile salt hydrolase in the
gastrointestinal microflora - A review. Asian Australas. J. Anim. Sci. 18, 1505-1512. Klis, J.D.v.d., Lensing, M., 2007. Wet litter problems relate to host-microbiota interactions. World Poult. 23, 20-22. Knarreborg, A., 2002. The impact of microbial deconjugation of bile salts on fat digestion in
broiler chickens. Thesis. Department of animal nutrition and physology (Danish institute of
agricultural sciences research centre foulum DK 8830 tjele), 121 pages. Kolmeder, C.A., de Been, M., Nikkila, J., Ritamo, I., Matto, J., Valmu, L., Salojarvi, J., Palva,
A., Salonen, A., de Vos, W.M., 2012. Comparative metaproteomics and diversity analysis
of human intestinal microbiota testifies for its temporal stability and expression of core
functions. PLoS One 7, e29913. Konsak, B., Guardia, S., Leconte, M., Moreau-Vauzelle, C., Dupont, C., Mignon-Grasteau, S.,
Gabriel, I., 2011. Comparison of the digestive microbiota between two divergent lines of
chickens selected based on digestive capacity. In : 7th International Symposium of
Anaerobic Microbiology, ISAM 2011 (Smolenice, Slovakia), pp. 17. Konsak, B., Guardia, S., Leconte, M., Moreau-Vauzelle, C., Dupont, C., Doré, J., Levenez, F.,
Mignon-Grasteau, S., Gabriel, I., 2012. Comparison of the digestive microbiota between
two divergent lines of chickens selected based on apparent metabolisable energy. In : 8th
INRA-Rowett Symposium on Gut Microbiology (Clermont-Ferrand, France), pp. 86. Kovacs, A., Ben-Jacob, N., Tayem, H., Halperin, E., Iraqi, F.A., Gophna, U., 2011. Genotype
is a stronger determinant than sex of the mouse gut microbiota. Microb. Ecol. 61, 423-428. Kussaibati, R., Guillaume, J., Leclercq, B., 1982a. The effect of gut microflora on the
digestibility of starch and proteins in young chicks. Annal. Zoot. 31, 483-488. Kussaibati, R., Guillaume, J., Leclercq, B., Lafont, J.P., 1982b. Effect of the intestinal
microflora and added bile salts on the metabolisable energy and digestibility of saturated
fats in the chicken. Arch. Geflugelkd. 46, 42-46. Lalles, J.P., 2012. Long term effects of pre- and early postnatal nutrition and environment on
the gut. J. Anim. Sci. 90, 421-429. Larsson, E., Tremaroli, V., Lee, Y.S., Koren, O., Nookaew, I., Fricker, A., Nielsen, J., Ley,
R.E., Backhed, F., 2012. References Analysis of gut microbial regulation of host gene expression along the length of the gut and regulation of gut microbial ecology through MyD88. Gut
61, 1124-1131. along the length of the gut and regulation of gut microbial ecology through MyD88. Gut
61, 1124-1131. Lederberg, J., 2000. Infectious history. Science 288, 287–293. Le Gall, M., Warpechowski, M., Jaguelin-Peyraud, Y., Noblet, J., 2009. Influence of dietary
fibre level and pelleting on digestibility of energy and nutrients in growing pigs and adult
sows. Animal 3, 352–359. Le Goff, G., Noblet, J., 2001. Comparative digestibility of dietary energy and nutrients in
growing pigs and adult sows. J. Anim. Sci. 79, 2418–2427. Lei, F., Yin, Y., Wang, Y., Deng, B., Yu, H.D., Li, L., Xiang, C., Wang, S., Zhu, B., Wang,
X., 2012. Higher-level production of volatile fatty acids in vitro by chicken gut microbiotas
than by human gut microbiotas as determined by functional analyses. Appl. Environ. Microbiol. 78, 5763-5772. Len, N.T., Hong, T.T.T., Ogle, B., Lindberg, J.E., 2009. Comparison of total tract
digestibility, development of visceral organs and digestive tract of Mong Cai and
Yorkshire × Landrace piglets fed diets with different fibre sources. J. Anim. Physiol. Anim. Nutr. 93, 181–191. Lepage, P., Mach, N., Levenez, F., Lemonnier, G., Denis, C., Bailly, J., Leplat, J.J., Doré, J.,
Estelle, J., Rogel-Gaillard, C., Consortium TS., 2012. Gut microbiota and immunity traits
in a swine cohort. In 8th INRA-Rowett Symposium on Gut Microbiology : Gut microbiota
: friend or foe ? (Clermond-Ferrand, France), pp. 18. Ley, R.E., Backhed, F., Turnbaugh, P., Lozupone, C.A., Knight, R.D., Gordon, J.I., 2005. Obesity alters gut microbial ecology. Proc. Natl. Acad. Sci. U.S.A. 102, 11070-11075. Ley, R.E., Hamady, M., Lozupone, C., Turnbaugh, P.J., Ramey, R.R., Bircher, J.S., Schlegel,
M.L., Tucker, T.A., Schrenzel, M.D., Knight, R., Gordon, J.I. 2008. Evolution of mammals
and their gut microbes. Science 320, 1647-1651. Li, K., Bihan, M., Methe, B.A., 2013. Analyses of the stability and core taxonomic
memberships of the human microbiome. PLoS One 8, e63139. Linnenbrink, M., Wang, J., Hardouin, E.A., Kunzel, S., Metzler, D., Baines, J.F., 2013. The
role of biogeography in shaping diversity of the intestinal microbiota in house mice. Mol. Ecol. 22, 1904-1916. Lu, J., Idris, U., Harmon, B., Hofacre, C., Maurer, J., Lee, M.D., 2003. Diversity and
succession of the intestinal bacterial community of the maturing broiler chicken. Appl. Environ. Microbiol. 69, 6816-6824. Lumpkins, B.S., Batal, A.B., Lee, M.D., 2010. References Evaluation of the bacterial community and
intestinal development of different genetic lines of chickens. Poult. Sci. 89, 1614-1621. Lyte, M., 2010. The microbial organ in the gut as a driver of homeostasis and disease. Med. Hypotheses 74, 634-638. Maisonnier, S., Gomez, J., Bree, A., Berri, C., Baeza, E., Carré, B., 2003. Effects of
microflora status, dietary bile salts and guar gum on lipid digestibility, intestinal bile salts
and histo-morphology, in broiler chickens. Poult. Sci. 82, 805-814. Manter, D.K., Weir, T.L., Vivanco, J.M., 2010. Negative effects of sample pooling on PCR-
based estimates of soil microbial richness and community structure. Appl. Environ. Microbiol. 76, 2086–2090. March, B.E., 1979. The host and its microflora : an ecological unit. J. Anim. Sci. 49, 857-867. Mayer, M., Abenthum, A., Matthes, J.M., Kleeberger, D., Ege, M.J., Holzel, C., Bauer, J.,
Schwaiger, K., 2012. Development and genetic influence of the rectal bacterial flora of
newborn calves. Vet. Microbiol. 161, 179-185. McFall-Ngai, M., 2007. Adaptive immunity - Care for the community. Nature 445, 153-153. McKnite, A.M., Perez-Munoz, M.E., Lu, L., Williams, E.G., Brewer, S., Andreux, P.A.,
Bastiaansen, J.W., Wang, X., Kachman, S.D., Auwerx, J., Williams, R.W., Benson, A.K.,
Peterson, D.A., Ciobanu, D.C., 2012. Murine gut microbiota is defined by host genetics
and modulates variation of metabolic traits. PLoS One 7, e39191. Meijerink, E., Neuenschwander, S., Fries, R., Dinter, A., Bertschinger, H.U., Stranzinger, G.,
Vögeli, P., 2000. A DNA polymorphism influencing alpha(1,2)fucosyltransferase activity
of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to
Escherichia coli F18 adhesion. Immunogenetics 52, 129–136. Mignon-Grasteau, S., Juin, H., Sellier, N., Bastianelli, D., Gomez, J., Carré, B., 2010. Genetic
Parameters of Wheat- or Corn-based Diets in Chickens. In : 9th World Congres of Applied
Livestock Production (Leipzig, Allemagne), 4 pages. Mignon-Grasteau, S., Juin, H., Sellier, N., Bastianelli, D., Gomez, J., Carré, B., 2010. Genetic
Parameters of Wheat- or Corn-based Diets in Chickens. In : 9th World Congres of Applied
Livestock Production (Leipzig, Allemagne), 4 pages. Mignon-Grasteau, S., Muley, N., Bastianelli, D., Gomez, J., Peron, A., Sellier, N., Millet, N.,
Besnard, J., Hallouis, J.M., Carre, B., 2004. Heritability of digestibilities and divergent
selection for digestion ability in growing chicks fed a wheat diet. Poult. Sci. 83, 860-867. Moore, R.J., Stanley, D., Konsak, B.M., Haring, V.R., Hughes, R.J., Geier, M.S., Crowley,
T.M., 2011. Correlations between variable broiler performance and gene expression and
microflora in the gut. References In : Proceedings of the 22nd Annual Australian Poultry Science
Symposium, Sydney, New South Wales, 14-16th February 2011, pp. 9-16. Morel, P.C.H., Lee, T.S., Moughan, P.J., 2006. Effect of feeding level, live weight and
genotype on the apparent faecal digestibility of energy and organic matter in the growing
pig. Anim. Feed Sci. Technol. 126, 63–74. Moreto, M., Planas, J.M., 1989. Sugar and amino acid transport properties of the chicken
caeca. J. Exp. Zool. sup 3, 111-116. Mountzouris, K.C., Tsitrsikos, P., Palamidi, I., Arvaniti, A., Mohnl, M., Schatzmayr, G.,
Fegeros, K., 2010. Effects of probiotic inclusion levels in broiler nutrition on growth
performance, nutrient digestibility, plasma immunoglobulins, and cecal microflora
composition. Poult. Sci. 89, 58-67. Mroz, Z., Koopmans, S.J., Bannink, A., Partanen, K., Krasucki, W., Overland, M., Radcliffe,
S., 2006. Carboxylic acids as bioregulators and gut growth promoters in nonruminants. In :
Mosenthin, R. Zentek, J. Zebrowska, T. (Eds.), Biology of Nutrition in Growing Animals. Edinburgh (UK), Elsevier, pp. 81-133. Muramatsu, T., Takasu, O., Furuse, M., Tasaki, I., Okumura, J., 1987. Influence of the gut
microflora on protein synthesis in tissues and in the whole body of chicks. Biochem. J. 246, 475-479. Nian, F., Guo, Y.M., Ru, Y.J., Li, F.D., Peron, A., 2011a. Effect of exogenous xylanase
supplementation on the performance, net energy and gut microflora of broiler chickens fed
wheat-based diets. Asian Australas. J. Anim. Sci. 24, 400-406. Nian, F., Guo, Y.M., Ru, Y.J., Peron, A., Li, F.D., 2011b. Effect of xylanase supplementation
on the net energy for production, performance and gut microflora of broilers fed corn/soy-
based diet. Asian Australas. J. Anim. Sci. 24, 1282-1287. Nicholson, J.K., Holmes, E., Kinross, J., Burcelin, R., Gibson, G., Jia, W., Pettersson, S.,
2012. Host-gut microbiota metabolic interactions. Science 336, 1262-1267. Noblet, J., Gilbert, H., Jaguelin-Peyraud, Y., Lebrun, T., 2013. Evidence of genetic variability
for digestive efficiency in the growing pig fed a fibrous diet. Animal 7, 1259-1264. Nordentoft, S., Molbak, L., Bjerrum, L., De Vylder, J., Van Immerseel, F., Pedersen, K.,
2011. The influence of the cage system and colonisation of Salmonella Enteritidis on the
microbial gut flora of laying hens studied by T-RFLP and 454 pyrosequencing. BMC
Microbiol. 11, 187. Nordskog, A.W., Johnson, E.L., 1953. Breed differences in response to feeding antibiotics. Poult. Sci. 32, 1046-1051. Ochman, H., Worobey, M., Kuo, C.H., Ndjango, J.B., Peeters, M., Hahn, B.H., Hugenholtz,
P., 2010. Evolutionary relationships of wild hominids recapitulated by gut microbial
communities. References PLoS Biol. 8, e1000546. Ouhida, I., Perez, J.F., Anguita, M., Gasa, J., 2002. Influence of beta-mannase on broiler
performance, digestibility, and intestinal fermentation. J. Appl. Poult. Res. 11, 244-249. Parks, B.W., Nam, E., Org, E., Kostem, E., Norheim, F., Hui, S.T., Pan, C., Civelek, M., Rau,
C.D., Bennett, B.J., Mehrabian, M., Ursell, L.K, He, A., Castellani, L.W., Zinker, B.,
Kirby, M., Drake, T.A., Drevon, C.A., Knight, R., Gargalovic, P., Kirchgessner, T., Eskin,
E., Lusis, A.J., 2013. Genetic control of obesity and gut microbiota composition in
response to high-fat, high-sucrose diet in mice. Cell. Metab. 17, 141-152. Pédron, T., Mulet, C., Dauga, C., Frangeul, L., Chervaux, C., Grompone, G., Sansonetti, P.J.,
2012. A crypt-specific core microbiota resides in the mouse colon. MBio 3, e00116-12. Peinado, M.J., Ruiz, R., Echavarri, A., Aranda-Olmedo, I., Rubio, L.A., 2013. Garlic
derivative PTS-O modulates intestinal microbiota composition and improves digestibility
in growing broiler chickens. Anim. Feed Sci. Technol. 181, 87-92. Péron, A., Svihus, B., Gabriel, I., Bérot, S., Tanguy, D., Bouchet, B., Gomez, J., Carré, B.,
2007. Effects of two wheat cultivars on physico-chemical properties of wheat flours and
digesta from two broiler chicken lines (D+ and D-) differing in digestion capacity. Br. Poult. Sci. 48, 370-380. Péron, A., Svihus, B., Gabriel, I., Bérot, S., Tanguy, D., Bouchet, B., Gomez, J., Carré, B.,
2007. Effects of two wheat cultivars on physico-chemical properties of wheat flours and
digesta from two broiler chicken lines (D+ and D-) differing in digestion capacity. Br. Poult. Sci. 48, 370-380. Pym, R.A.E., Nicholls, P.J., Thomson, E., Choice, A., Farrell, D.J., 1984. Energy and
nitrogen-metabolism of broilers selected over 10 generations for increased growth-rate,
food-consumption and conversion of food to gain. Br. Poult. Sci. 25, 529-539. Qin, J., Li, Y., Cai, Z., Li, S., Zhu, J., Zhang, F., Liang, S., Zhang, W., Guan, Y., Shen, D.,
Peng, Y., Zhang, D., Jie, Z., Wu, W., Qin, Y., Xue, W., Li, J., Han, L., Lu, D., Wu, P., Dai,
Y., Sun, X., Li, Z., Tang, A., Zhong, S., Li, X., Chen, W., Xu, R., Wang, M., Feng, Q.,
Gong, M., Yu, J., Zhang, Y., Zhang, M., Hansen, T., Sanchez, G., Raes, J., Falony, G.,
Okuda, S., Almeida, M., LeChatelier, E., Renault, P., Pons, N., Batto, J.M., Zhang, Z.,
Chen, H., Yang, R., Zheng, W., Li, S., Yang, H., Wang, J., Ehrlich, S.D., Nielsen, R.,
Pedersen, O., Kristiansen, K., Wang, J., 2012. References A metagenome-wide association study of
gut microbiota in type 2 diabetes. Nature 490, 55-60. Ramasamy, K., Abdullah, N., Wong, M., Karuthan, C., Ho, Y.W., 2010. Bile salt
deconjugation and cholesterol removal from media by Lactobacillus strains used as
probiotics in chickens. J. Sci. Food Agric. 90, 65-69. Ravindran, V., Hew, L.I., Ravindran, G., Bryden, W.L., 1999. A comparison of ileal digesta
and excreta analysis for the determination of amino acid digestibility in food ingredients
for poultry. Br. Poult. Sci. 40, 266-274. Rawls, J.F., Mahowald, M.A., Ley, R.E., Gordon, J.I., 2006. Reciprocal gut microbiota
transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127, 423-433. Rebel, J.M., Van Hemert, S., Hoekman, A.J., Balk, F.R., Stockhofe-Zurwieden, N., Bakker,
D., Smits, M.A., 2006. Maternal diet influences gene expression in intestine of offspring in
chicken (Gallus gallus). Comp. Biochem. Physiol. A Mol. Integr. Physiol. 145, 502-508. Reilly, K.J., Frankel, W.L., Bain, A.M., Rombeau, J.L., 1995. Colonic short chain fatty acids
mediate jejunal growth by increasing gastrin. Gut 37, 81-86. Reynhout, J.K., Duke, G.E., 1999. Identification of interstitial cells of Cajal in the digestive
tract of turkeys (Meleagris gallopavo). J. Exp. Zool. 283, 426-440. Rideau, N., Godet, E., Combémorel, C., Chaudeau, M., Carré, B., Mignon-Grasteau, S., 2014. The gastric isthmus from D+ and D- broiler lines divergently selected for digestion
efficiency shows histological and morphological differences. Poult. Sci., in press. Riffard, C., Gallot, S., Magdelaine, P., 2011. Technical performances and production costs for
chicken and hen. Results 2010. In : ITAVI, Paris, pp. 1-57. Rodriguez, M.L., Rebole, A., Velasco, S., Ortiz, L.T., Trevino, J., Alzueta, C., 2012. Wheat-
and barley-based diets with or without additives influence broiler chicken performance,
nutrient digestibility and intestinal microflora. J. Sci. Food Agric. 92, 184-190. Rougière, N., Carré, B., 2010. Comparison of gastrointestinal transit times between chickens
from D+ and D- genetic lines selected for divergent digestion efficiency. Animal 4, 1861-
1872. Rougière, N., Gomez, J., Mignon-Grasteau, S., Carré, B., 2009. Effects of diet particle size on
digestive parameters in D(+) and D(-) genetic chicken lines selected for divergent digestion
efficiency. Poult. Sci. 88, 1206-1215. Roy, K., Meyrand, M., Corthier, G., Monnet, V., Mistou, M.Y., 2008. Proteomic investigation
of the adaptation of Lactococcus lactis to the mouse digestive tract. Proteomics 8, 1661-
1676. Sacranie, A., Iji, P.A., Mikkelsen, L.L., Choct, M., 2007. Occurrence of reverse peristalsis in
broiler chickens. References In : Proceedings of the 19th Australian Poultry Science Symposium
(Sydney, New South Wales, Australia), pp. 161-164. Sacranie, A., Svihus, B., Denstadli, V., Moen, B., Iji, P.A., Choct, M., 2012. The effect of
insoluble fiber and intermittent feeding on gizzard development, gut motility, and
performance of broiler chickens. Poult. Sci. 91, 693-700. Salonen, A., Salojarvi, J., Lahti, L., de Vos, W.M., 2012. The adult intestinal core microbiota
is determined by analysis depth and health status. Clin. Microbiol. Infect. 18, 16-20. Salter, D.N. (1973). The influence of gut micro-organisms on utilization of dietary ptotein. Proc. Nutr. Soc. 32, 65-71. Salter, D.N., Fulford, R.J., 1974. The influence of the gut microflora on the digestion of
dietary and endogenous proteins: studies of the amino acid composition of the excreta of
germ-free and conventional chicks. Br. J. Nutr. 32, 625-637. Sarica, S., Corduk, M., 2013. Effects of oregano essential oil supplementation to diets for
broiler chicks with delayed feeding after hatching. 1. Performances and digestibility of
nutrients. Arch. Geflugelkd. 77, 81-89. Shakouri, M.D., Iji, P.A., Mikkelsen, L.L., Cowieson, A.J., 2009. Intestinal function and gut
microflora of broiler chickens as influenced by cereal grains and microbial enzyme
supplementation. J. Anim. Physiol. Anim. Nutr. 93, 647-658. Shepherd, E.M., Fairchild, B.D., 2010. Footpad dermatitis in poultry. Poult. Sci. 89, 2043-
2051. Spor, A., Koren, O., Ley, R., 2011. Unravelling the effects of the environment and host
genotype on the gut microbiome. Nat. Rev. Microbiol. 9, 279-290. Stanley, D., Geier, M.S., Denman, S.E., Haring, V.R., Crowley, T.M., Hughes, R.J., Moore,
R.J., 2013a. Identification of chicken intestinal microbiota correlated with the efficiency of
energy extraction from feed. Vet. Microbiol. 164, 85-92. Stanley, D., Geier, M.S., Hughes, R.J., Denman, S.E., Moore, R.J., 2013b. Highly variable
microbiota development in the chicken gastrointestinal tract. Plos One 8, e84290. Stewart, J.A., Chadwick, V.S., Murray, A., 2005. Investigations into the influence of host
genetics on the predominant eubacteria in the faecal microflora of children. J. Med. Microbiol. 54, 1239-1242. Sundstöl, F., Standal, N., Vangen, O., 1979. Energy metabolism in lines of pigs selected for
thickness of backfat and rate of gain. Acta Agric. Scand. 29, 337–345. Szylit, O., Champ, M., Aitabdelkader, N., Raibaud, P., 1980. Role of 5 Lactobacillus Strains
on Carbohydrate Degradation in Monoxenic Chickens. Reprod. Nutr. Develop. 20, 1701-
1706. Tang, Y., Underwood, A., Gielbert, A., Woodward, M.J., Petrovska, L., 2014. Metaproteomics analysis reveals the adaptation process for the chicken gut microbiota. References Appl. Environ. Microbiol. 80, 478-485. Tap, J., Mondot, S., Levenez, F., Pelletier, E., Caron, C., Furet, J.P., Ugarte, E., Munoz-
Tamayo, R., Paslier, D.L., Nalin, R., Doré, J., Leclerc, M., 2009. Towards the human
intestinal microbiota phylogenetic core. Environ. Microbiol. 11, 2574-2584. Tappenden, K.A., McBurney, M.L., 1998. Systemic short-chain fatty acids rapidly alter
gastrointestinal structure, function, and expression of early response genes. Dig. Dis. Sci. 43, 1526-1536. Thompson, C.L., Hofer, M.J., Campbell, I.L., Holmes, A.J., 2010. Community dynamics in
the mouse gut microbiota: a possible role for IRF9-regulated genes in community
homeostasis. PLoS One 5, e10335. Timbermont, L., Haesebrouck, F., Ducatelle, R., Van Immerseel, F., 2011. Necrotic enteritis
in broilers: an updated review on the pathogenesis. Avian Pathol. 40, 341-347. Torok, V.A., Ophel-Keller, K., Loo, M., Hughes, R.J., 2008. Application of methods for
identifying broiler chicken gut bacterial species linked with increased energy metabolism. Appl. Environ. Microbiol. 74, 783-791. Torok, V.A., Hughes, R.J., Mikkelsen, L.L., Perez-Maldonado, R., Balding, K., MacAlpine,
R., Percy, N.J., Ophel-Keller, K., 2011. Identification and Characterization of Potential
Performance-Related Gut Microbiotas in Broiler Chickens across Various Feeding Trials. Appl. Environ. Microbiol. 77, 5868-5878. Tran, T.S., Narcy, A., Carré, B., Gabriel, I., Rideau, N., Gilbert, H., Demeure, O., Bed’Hom,
B., Chantry-Darmon, C., Boscher, M.Y., Bastianelli, D., Sellier, N., Chabault, M.,
Calenge, F., Le Bihan-Duval, E., Beaumont, C., Mignon-Grasteau, S. Detection of QTL
controlling digestive efficiency and anatomy of the digestive tract in chicken fed a wheat-
based diet. Genet. Sel. Evol. In press. Turnbaugh, P.J., Gordon, J.I., 2009. The core gut microbiome, energy balance and obesity. J. Physiol.-London 587, 4153-4158. Turnbaugh, P.J., Hamady, M., Yatsunenko, T., Cantarel, B.L., Duncan, A., Ley, R.E., Sogin,
M.L., Jones, W.J., Roe, B.A., Affourtit, J.P., Egholm, M., Henrissat, B., Heath, A.C.,
Knight, R., Gordon, J.I., 2009. A core gut microbiome in obese and lean twins. Nature 457,
480-484. Turnbaugh, P.J., Quince, C., Faith, J.J., McHardy, A.C., Yatsunenko, T., Niazi, F., Affourtit,
J., Egholm, M., Henrissat, B., Knight, R., Gordon, J.I., 2010. Organismal, genetic, and
transcriptional variation in the deeply sequenced gut microbiomes of identical twins. Proc. Natl. Acad. Sci. U.S.A. 107, 7503-7508. Van de Merwe, J.P., Stegeman, J.H., Hazenberg, M.P., 1983. The resident faecal flora is
determined by genetic characteristics of the host. Implications for Crohn's disease? Antonie
Van Leeuwenhoek 49, 119-124. Vasai, F., Brugirard Ricaud, K., Bernadet, M.D., Cauquil, L., Bouchez, O., Combes, S.,
Davail, S., 2014. References Overfeeding and genetics affect the composition of intestinal microbiota
in Anas platyrhynchos (Pekin) and Cairina moschata (Muscovy) ducks. FEMS Microbiol. Ecol. 87, 204-216. Vijay-Kumar, M., Aitken, J.D., Carvalho, F.A., Cullender, T.C., Mwangi, S., Srinivasan, S.,
Sitaraman, S.V., Knight, R., Ley, R.E., Gewirtz, A.T., 2010. Metabolic syndrome and
altered gut microbiota in mice lacking Toll-like receptor 5. Science 328, 228–231. Yang, X.J., Li, W.L., Feng, Y., Yao, J.H., 2011. Effects of immune stress on growth
performance, immunity, and cecal microflora in chickens. Poult. Sci. 90, 2740-2746. Yang, Y., Iji, P.A., Kocher, A., Mikkelsen, L.L., Choct, M., 2008. Effects of dietary
mannanoligosaccharide on growth performance, nutrient digestibility and gut development
of broilers given different cereal-based diets. J. Anim. Physiol. Anim. Nutr. 92, 650-659. Yang, Y., Iji, P.A., Kocher, A., Mikkelsen, L.L., Choct, M., 2008. Effects of xylanase on
growth and gut development of broiler chickens given a wheat-based diet. Asian Australas. J. Anim. Sci. 21, 1659-1664. Yeoman, C.J., Chia, N., Jeraldo, P., Sipos, M., Goldenfeld, N.D., White, B.A., 2012. The
microbiome of the chicken gastrointestinal tract. Anim. Health Res. Rev. 13, 89-99. Yuan, J., Wang, B., Sun, Z., Bo, X., Yuan, X., He, X., Zhao, H., Du, X., Wang, F., Jiang, Z.,
Zhang, L., Jia, L., Wang, Y., Wei, K., Wang, J., Zhang, X., Sun, Y., Huang, L., Zeng, M.,
2008. Analysis of host-inducing proteome changes in bifidobacterium longum NCC2705
grown in Vivo. J. Proteome Res. 7, 375-385. Zhang, C.H., Zhang, M.H., Wang, S.Y., Han, R.J., Cao, Y.F., Hua, W.Y., Mao, Y.J., Zhang,
X.J., Pang, X.Y., Wei, C.C., Zhao, G.P., Chen, Y., Zhao, L.P., 2010. Interactions between
gut microbiota, host genetics and diet relevant to development of metabolic syndromes in
mice. ISME J. 4, 232-241. Zhang, C.H., Zhang, M.H., Wang, S.Y., Han, R.J., Cao, Y.F., Hua, W.Y., Mao, Y.J., Zhang,
X.J., Pang, X.Y., Wei, C.C., Zhao, G.P., Chen, Y., Zhao, L.P., 2010. Interactions between
gut microbiota, host genetics and diet relevant to development of metabolic syndromes in
mice. ISME J. 4, 232-241. Zhang, W., Aggrey, S.L., Pesti, G.M., Edwards, H.M., Bakalli, R.I., 2003. Genetics of phytate
phosphorus bioavailability: Heritability and genetic correlations with growth and feed
utilization traits in a randombred chicken population. Poult. Sci. 82, 1075-1079. Zhang, W., Aggrey, S.E., Pesti, G.M., Bakalli, R.I., Edwards, H.M., 2005. Genetic analysis
on the direct response to divergent selection for phytate phosphorus bioavailability in a
randombred chicken population. Poult. Sci. 84, 370-375. References Zhao, L., Wang, G., Siegel, P., He, C., Wang, H., Zhao, W., Zhai, Z., Tian, F., Zhao, J.,
Zhang, H., Sun, Z., Chen, W., Zhang, Y., Meng, H., 2013. Quantitative genetic
background of the host influences gut microbiomes in chickens. Sci. Rep. 3, 1163. Zhu, X.Y., Zhong, T., Pandya, Y., Joerger, R.D., 2002. 16S rRNA-based analysis of
microbiota from the caecum of broiler chickens. Appl. Environ. Microbiol. 68, 124-137. Zoetendal, E.G., Akkermans, A.D.L., Akkermans-van Vliet, W.M., deVisser, J.A.G.M.,
deVos, W.M., 2001. The host genotype affects the bacterial community in the human
gastrointestinal tract. Microb. Ecol. Health Dis. 13, 129-134. Zulkifli, I., Hashemi, S.R., Somchit, M.N., Zunita, Z., Loh, T.C., Soleimani, A.F., Tang, S.C.,
2012. Effects of Euphorbia hirta and virginiamycin supplementation to the diet on
performance, digestibility, and intestinal microflora population in broiler chickens. Arch. Geflugelkd. 76, 6-12.
|
https://openalex.org/W4386516251
|
https://drpress.org/ojs/index.php/ijeh/article/download/11578/11276
|
English
| null |
Research on the Development and Dissemination of Film Based on the Geopolitical Perspective
|
International journal of education and humanities
| 2,023
|
cc-by
| 4,143
|
1. Introduction Geopolitics is one of the important factors in the field of
film development and dissemination. It gradually exerted
influence in the film industry and promoted the reform of
early films. From the perspective of geopolitics, this paper
explores the interactive relationship between geopolitics and
film, and the impact of this relationship on the development
and dissemination of early film. The integration of geopolitics
and film has brought about innovations in content and
changes in production and dissemination modes, meeting the
diverse needs of audiences. At the same time, geopolitics has
also affected all aspects of film production, distribution, and
dissemination, such as user portraits, market trends, and
changes in the industrial chain, including research and
development, live marketing, and digitalization. International Journal of Education and Humanities
ISSN: 2770-6702 | Vol. 10, No. 2, 2023 International Journal of Education and Humanities
ISSN: 2770-6702 | Vol. 10, No. 2, 2023 Research on the Development and Dissemination of
Film Based on the Geopolitical Perspective Zinan Ye1, a 1Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu, 215028, China
azinanye02@163.com 1Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu, 215028, China
azinanye02@163.com Abstract: With the increasing integration of geopolitics in the film field, the development and dissemination of early films are
undergoing new changes. This paper explores this transformation from a geopolitical perspective and delves into the multilayered
interactions between geopolitics and film. First of all, geopolitics has brought new opportunities for film creation through
innovative content and production and dissemination methods to meet the diverse needs of audiences. Then, this paper reveals
the impact of geopolitical intervention in film production, distribution, and dissemination and discusses user portraits, market
trends, and changes in the industrial chain, including research and development, live marketing, and digitalization. However,
geopolitics has also brought cultural challenges, resulting in an imbalance in the film system, resulting in problems such as
homogenization and value conflicts. At the same time, transnational operations in geopolitics have also led to algorithmic
decision-making errors, cultural grievances, and security risks. Based on this, the paper proposes countermeasures, including
improving film quality, strengthening geopolitical regulation, optimizing collaborative governance, and restoring the cultural
value of films. Through these strategies, the film industry can achieve sustainable development and effectively respond to the
challenges posed by geopolitics. words: Geopolitical perspective, Film development, Dissemination strategy, Cultural challenge. Keywords: Geopolitical perspective, Film development, Dissemination strategy, Cultural challenge. changes to developing and disseminating early films. Although it also brought cultural challenges, the film industry
can achieve sustainable development through appropriate
coping strategies and effectively respond to various
challenges brought about by geopolitics. 2.1.1. The Meeting Point of Geopolitics and Film 2.1.1. The Meeting Point of Geopolitics and Film In the research process on film content innovation, we
mainly focused on factors such as creative technique and
narrative style, but ignored the potential fit between
geopolitics and film, leading to inaccurate and comprehensive
research on film content innovation. Based on this problem, a
comprehensive study is conducted on film content innovation,
focusing on the organic combination of geopolitics and film,
and by analyzing the key role of geopolitics in film creation,
it aims to enhance the effect of film content innovation. Firstly,
it reveals the potential value of geopolitical factors in film
creation by exploring the convergence of geopolitics and film. Secondly, through a detailed analysis of the influence of
geopolitics on film creation, the mechanism of geopolitical
elements in film content innovation is deeply excavated [1]. Then, by discussing the adjustment of creative techniques and
narrative styles, it shows how geopolitics brings new creative
opportunities for film content innovation. Finally, based on
summarizing the above analysis, it emphasizes the
importance of in-depth research on the relationship between
geopolitics and film content innovation, which provides a
new perspective and method for film creation. Based on this
research, we can better understand the integration of
geopolitics and film and realize new changes in film content
innovation. However, geopolitics has also triggered a series of cultural
challenges. Excessive geopolitical intervention may lead to an
imbalance in the film system, causing problems such as
homogenization
and
value
conflicts. Transnational
geopolitical operations can also lead to algorithmic decision-
making errors，cultural grievances, and security risks. This
paper proposes coping strategies to deal with these challenges. First of all, by improving the quality of films, we can achieve
high-quality dissemination of films, while emphasizing
locality and geopolitical conventions so that films can
respond to cultural challenges. Secondly, by strengthening
geopolitical regulation and supervision and governance,
optimizing decision-making and creator cultivation, and
realizing multi-dimensional sharing and collaborative
governance, thereby promoting the sustainable development
of the film industry. In the end, the paper emphasizes restoring
the cultural value of the film in order to maintain the diversity
and innovation of the film. To sum up, the geopolitical perspective brought new 120 and the film as an emotional interaction model, namely the
emotion-based audience model and the emotion-based film
model. The former focuses on audience emotion, and the
latter focuses on film emotion, that is, emotional interaction. 2.1.2. Multimodal Expressions of Geopolitics 2.1.2. Multimodal Expressions of Geopolitics The multimodal expression of geopolitics is a concept
developed in parallel with film, which is "soaked" with cross-
cultural concepts, highlights the interweaving orientation of
multiculturalism, and reflects the rich strategies of cultural
exchanges since globalization. However, it is still difficult to
get a clear definition when we try to construct the definition
and nature of multimodal expression with some traditional
standards. The intertwined multimodal expression of
geopolitics and film means that the film is a simple visual
presentation and covers multiple elements, such as sound,
language, and symbols, through which complex cultural,
political, and social connotations are interwoven [2]. As a
medium of cross-cultural communication, film's multimodal
expression conveys information, explores the interaction and
collision between different cultures in the context of
globalization, and presents unique geopolitical characteristics. Although the definition of multimodal expression is unclear,
this complexity and diversity endow film and geopolitics with
deeper meaning and further promote innovation and change
in the film field. 2.2.1. Film User Portrait and User Personalized Demand
Analysis y
Film user portraits and user personalized needs analysis
are important criteria for film production, distribution and
dissemination, and an in-depth expression of audience
characteristics. Different definitions of user portraits are
discussed from the perspectives of psychology and marketing
[5]. Some scholars believe that user portraits are the degree of
individualized needs of the audience or market segmentation. It is precisely because user portraits are more practical to
some extent and belong to market science aimed at audience
needs. The research history of user portraits can even be
traced back to the early days, and its main activities include
surveys and analysis. The concept and satisfaction of users'
personalized needs are closely related to the process of market
competition. Through user portraits, the film industry can
more accurately grasp the audience's characteristics, and
meeting the audience's diverse needs has become an
important industry responsibility. The main contribution of
market theory in the modern era is market segmentation. Therefore, the concept of user portraits initially focused on
audience
demand
measurement
based
on
market
characteristics. Through an in-depth understanding of
different user portraits, the film industry can better formulate
personalized production, distribution, and dissemination
strategies to meet audience needs accurately. The analysis of
film user portraits and user personalized needs based on the
perspective of geopolitics is shown in Figure 1. 2.1.1. The Meeting Point of Geopolitics and Film Although the interaction between geopolitical films and
audiences has experienced some practical failures, from the
perspective of emotional resonance, it can promote emotional
connections, and the shaping of audience emotions by
geopolitical films has gradually become a consensus in
research and practice. Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspectiv Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspective 2.1.3. Geopolitical Films and Audiences p
Compared with traditional films, geopolitical films
emphasize the relationship between film works and audiences
and
have
more
in-depth
emotional
communication
characteristics [3]. Although some scholars have questioned
that there may be no direct relationship between geopolitical
films and audiences, most scholars argue that geopolitical
films can positively impact audiences' emotions through
emotional resonance. Some researchers have proposed a
moving communication model that includes elements of
emotional expression, and this model has since become a
typical tool for studying the relationship between geopolitical
films and audiences, thus developing the concept of
emotional resonance. These scholars believe that emotional
resonance has emotional transitivity and is an "emotional
bridge". Only when the film can emotionally resonate with
the audience, the audience will be deeply touched [4]. Therefore, the emotional interaction between geopolitical
films and audiences results from emotional resonance. Some
scholars also summarize the interaction between the audience Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspective Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspective Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspective Figure 1 Analysis of film user portraits and user personalized needs based on a geopolitical perspective 121 an important subject of content creation. At this stage, in order
to strengthen the market control of films from a commercial
point of view, there are mainly three forms: One is the
application of the theme. Clarify the commercialization of
market hotspots between content creation and presentation. The second is standardization. Achieve standardized control
of film content by formulating content standards, reviewing
standards, and disclosing content standards to creators. The
third is internal process reengineering for process
optimization. Recently, film production and marketing have
used commercial means to improve efficiency and increase
commercial value. However, compared with artistic creation,
the creativity of current business needs to be further improved. 2.2.2. The Film Market Trend Promotes Film Decision-
making The practice of sensitive content filtering is often
influenced by geopolitics, making film productions censored
and restricted when they involve sensitive areas such as
politics, religion, and culture. However, such excessive
intervention may cause an imbalance in the film system and
limit the creative diversity and freedom of expression of films. 3.1.3. Value-oriented Conflict Superposition The fundamental difference between value-oriented
conflicts superimposed in the film industry lies in its cultural
attributes. Commercial value standards and cultural value
guidelines are aimed at commercial interests, and the
development of the film industry mainly reflects market
demand and profit orientation. In the development framework
of commercialization, an accurate grasp of the market,
innovation and creation, and improvement of output and
profit are the core values and highest commercial
development standards. The diversity of current film genres
and the differences in market demand have led to a
homogenization of film content. Although market orientation
has its positive significance, because the commercialization
mechanism is still not perfect, the commercialization itself
lacks a cultural guarantee mechanism. Therefore, this creates
a cultural "shortboard" that affects the creative diversity of the
film. 3.2.1. Mistakes in Algorithmic Decision-Making and
Mechanized Deviations in Management g
From the perspective of algorithmic decision-making,
existing algorithms cannot accurately provide the decision
support the film industry needs. The algorithm uses
satisfaction evaluation as the main form for the film market,
but the algorithm lacks comprehensive market information
and an accurate prediction mechanism. The core of this
problem may be the lack of data. In applying algorithms,
market
information
is
usually
described
as
"local
information", and its grasp of the film market directly reflects
the past situation. However, market dynamics are mostly
information about future trends, and long-term forecasts are
scarce. In general, future information is difficult to obtain or
measure. Information asymmetry and imperfect data directly
lead to obstacles in decision-making. 2.2.3. Extension of the Film Industry Chain The homogenization tendency of films is a phenomenon
that appears to meet market demand and obtain greater profits
under the trend of commercialization. However, too much
emphasis on commercialization may lead to a reduction in
creative innovation and affect the cultural diversity of films. In order to maintain the creative diversity of films, artistic
creation and cultural expression should be emphasized in the
business model, and more opportunities and support should
be provided to innovative filmmakers to promote the
sustainable development of films. The conceptual nature of film market trends driving film
decisions focuses on market information issues. A market
trend is the application of market thinking in actual operation. In order to overcome the defect of insufficient information, as
a new alternative model, the market information framework
has entered the research field. The basic idea of the
framework is that market participants should ensure the
effective realization of information; Set professional
standards for market output; "Capture" market signals
through techniques such as data analysis; Use market research
methods to measure market demand. The market information
framework reconstructs the decision-making process,
emphasizing the need to enhance decision-making accuracy
and build comprehensiveness, professionalism, pertinence
and forward-looking decision-making. In the film industry,
through an in-depth understanding of market trends, analysis
of audience interests and trends, and formulation of
corresponding
film
production,
distribution,
and
dissemination strategies to better meet market demand. The
market information framework makes film decision-making
more scientific and precise and helps promote the sustainable
development of the film industry. 2.1.3. Geopolitical Films and Audiences Therefore, to maintain social stability, it is necessary to
establish a more transparent, fair, and scientifically sensitive
content filtering mechanism to balance geopolitics and film
and promote the film industry's healthy development. 3.1.1. Filtering of Sensitive Content The filtering of sensitive content is an important part of
the cultural challenges brought about by geopolitics and film,
emphasizing the conflict between geopolitics and cultural
values, and directly reflecting the interweaving of politics and
culture through the censorship of film content. Some elements
of film production are gradually taking shape, and the filtering
of sensitive content and various evaluation systems are
gradually being paid attention to. However, from the
perspective of actual operation, some sensitive content
filtering practices are still at the superficial stage, and there
are still conflicts with the logical framework and generation
mechanism of film creation, which leads to problems such as
unclear review standards and unperfect review system. 3.2.1. Mistakes in Algorithmic Decision-Making and
Mechanized Deviations in Management 3.2.1. Mistakes in Algorithmic Decision-Making and
Mechanized Deviations in Management 3.3. The Birth of Derivative Risks Under
Geopolitical Domination In the context of geopolitical domination, derivative risks
cannot be avoided as challenges in the film industry. The
influence mechanism of geopolitics and political factors are
standard and effective influence tools and play an important
role in the film industry. This also makes geopolitics not only
a political concept but also a risk concept. Therefore, risk
prevention based on "political influence" has become an
important mechanism for dealing with challenges. 4.1.1. Films Quality Improvement Achieve High-quality
Migration of Films Geopolitical regulation and supervisory governance play
an important role in the film industry as an expression of
normative requirements for film production, distribution and
content. From different perspectives, geopolitical regulation
and supervisory governance have various definitions and
influences on the film industry. Some scholars believe that
geopolitical regulation, supervision, and governance are the
degree to which industry guidelines are formulated or the
means to ensure that the film industry is scientifically
managed according to specific purposes. The concept has
deep roots in film history, and its main activities include
formulating and implementing laws and regulations. In the
concept and practice of the film industry, geopolitical
regulation, supervision and governance are closely related to
the prosperity and development of the industry. Through
regulation and supervision, the film industry can fulfill its
social responsibilities. Especially in different historical
periods, the main contribution of the theory of the film
industry is to ensure that the film industry maintains creativity
and innovation while conforming to social values. Therefore,
geopolitical regulation and supervisory governance initially
focused primarily on evaluating the film industry based on
public policy. Film development aims to build mechanisms and optimize
systems to provide audiences with a film experience that
meets the expected standards, achieve high-quality transfer of
films, and eliminate cultural challenges. Although the
improvement of film quality is not a new topic, the current
development combines technology, creativity, and culture
innovatively, outlines the multi-dimensional characteristics of
the film industry, expands the technical connotation of the
film industry, and endows the value and significance of the
combination of film and technology, creation and market,
culture and entertainment. (2) Geopolitical Protocols and Content Censorship Compared with content censorship, geopolitical statutes
emphasize the interrelationships and interactions among
states and have obvious international characteristics. Although some scholars have questioned that there may be no
direct relationship between geopolitical regulations and
content censorship, most scholars maintain that geopolitical
regulations can rationally evaluate content censorship. Relevant researchers have proposed a classic geopolitical
protocol model that includes elements of international
cooperation, and this model has become an important tool for
analyzing international relations, from which the concept of
the international protocol was developed. These scholars
believe that geopolitical statutes promote international
cooperation and are a mediating factor in international
relations. Only when international statutes are effectively
implemented can international cooperation achieve good
results. It can be seen that international statutes play an
important role in international relations. As time passes, the
concept of international statutes has gradually become a
consensus in international research and practice. Although
international statutes have experienced some practical
difficulties, from the perspective of international relations,
they can promote international cooperation, and the concept
of international statutes has gradually become a consensus in
international research and practice. 3.1.2. Homogenization of Films From the perspective of film creation, the homogenization
tendency of films is the basic link of the film industry and the
core embodiment of film content presentation. The business
model is the main mode of operation of the film, and it is also 122 3.2.2. Cultural Grievances and Security Risks characteristics and cultural values in the film industry and
focuses on showing local social, historical and cultural
elements. This locality-oriented strategy can enhance a
country's or region's cultural identity while meeting the
audience's needs for a film experience closer to life and with
a greater sense of identity. 3.2.2. Cultural Grievances and Security Risks From the perspective of cultural grievances and security
risks, cultural differences have long restricted the ability of
international film cooperation. Since the 21st century,
international co-production projects with a global perspective
have reshaped film works through cultural diversity, but the
disadvantages of traditional cultural differences still restrict
cross-cultural cooperation. Not only due to cultural factors
such as language and habits but also due to the influence of
geopolitics, international cooperation has yet to be perfected. Under globalization, intercultural cooperation is regarded as
a direct way of cultural exchange. However, the actual role of
culturally focused international cooperation on cultural
differences is open to debate. At the same time, due to cultural
difficulties, there is a lack of consensus in international
cooperation. Therefore, cultural integration does not always
seem to achieve the goal of intercultural communication. It
can be seen that cultural integration is not only a technical
problem but also faces the problem of cultural compatibility. References [1] Hu Z, Lu D. Re-interpretation of the classical geopolitical
theories in a critical geopolitical perspective[J]. Journal of
Geographical Sciences, 2016, 26: 1769-1784. [2] Wang G, Gu X, Shen X, et al. A dual risk perspective of China's
resources market: Geopolitical risk and political risk[J]. Resources Policy, 2023, 82: 103528. [3] Blondeel M, Bradshaw M J, Bridge G, et al. The geopolitics of
energy system transformation: A review[J]. Geography
Compass, 2021, 15(7): e12580. 5. Conclusion The core of the cultural restoration strategy is to re-
examine the film industry's essential value to restore the film's
true appearance. In the basic links and core manifestations of
the film industry, cultural restoration emphasizes the
fundamental value of films and returns to the original
intention of films. At present, the film industry is facing a
balance between commercial value and cultural value, and the
cultural restoration strategy can be achieved through the
following three ways: First, clarify the relationship between
business and culture to ensure that the business model does
not erode the core of culture; Secondly, by formulating
cultural value standards and content norms, ensure that film
works convey positive and healthy cultural values; Finally,
the artistry and depth of film creation will be enhanced by
reengineering the internal process so that the film can better
reflect and convey cultural values. However, despite many
efforts in recent years, the commercial and cultural aspects of
the film still need to be further balanced, requiring the joint
efforts of all parties to restore the essence of the film. 4.2.2. Geopolitical Decision-Making and Creator
Cultivation can be achieved in the following three ways: First, clarify the
interrelationship between business and culture in the process
of film production and dissemination to ensure that the
business model does not erode the core of culture; Second, the
works convey positive and healthy cultural values; Finally,
through the reengineering of the internal processes of the film
industry, the artistry and depth of film creation are enhanced,
so that films can better reflect and convey cultural values. Although there have been many efforts in recent years,
compared with commerciality, the cultural nature of current
films needs to be further improved, and all parties need to
work together to restore the true colors of films. The essence of geopolitical decision-making and creator
cultivation focuses on effectively dealing with the cultivation
and development of creators in the context of geopolitics. This concept is an innovative model that applies geopolitical
thinking to the field of creator cultivation. It aims to overcome
the shortcomings of traditional cultivation methods. A new
alternative model of geopolitical decision-making and creator
cultivation framework is introduced. The basic idea of the
framework is to ensure the effective realization of creator
cultivation through geopolitical thinking; Set professional
standards for creators' output, "capture" creative inspiration
through technological means; Use multiple methods to
measure creative results. The emergence of geopolitical
decision-making and creator cultivation framework has
restructured the way of creator cultivation, emphasizing the
need to enhance the creativity and innovation of creators and
build the diversity, geography, culture, and sociality of
creation. This framework provides new perspectives and
methods for cultivating creators in the film industry in
response to changing geopolitical environments and cultural
challenges. 4.1.2. Film Upgrade under Geopolitical Domestication
(1) Indigenous" Emphasizes "Nativeness" is a concept developed parallel with
upgrading the film industry, which has a profound cultural
concept, highlights the geopolitical orientation of the film
industry, and reflects the upgrading strategy of the film
industry under geopolitical domestication [6]. However, it is
still difficult to reach a consistent conclusion when we try to
construct the definition and essence of "nativeness" with
some global standards. "Nativeness" emphasizes the creation based on regional 123 4.2.3. Multivariate Sharing and Collaborative
Governance Diversity sharing and collaborative governance are the
main strategies for the film industry to deal with geopolitical
and technological challenges. It embodies the principles of
multi-participation and cooperative governance and directly
reflects the sustainable development of the film industry
through the joint efforts of all parties. In this strategy, the
constituent elements of multiple shared responsibilities are
gradually being formed, including the joint participation of
governments at all levels, industrial organizations, creators,
audiences, etc., and various evaluation systems are gradually
being valued. However, from a practical point of view, some
multi-divisional sharing practices are still in their infancy, and
there are still conflicts with the logical framework and
generation mechanism of collaborative co-governance, which
leads to the issue of synergistic cooperation. These issues
need to be further resolved and improved regarding system
design, policy guidance, and resource allocation to promote
global cooperation and sustainable development of the film
industry. 4.3. Cultural Restoration: Rectifying Values
and Restoring Films [4] Bricout A, Slade R, Staffell I, et al. From the geopolitics of oil
and gas to the geopolitics of the energy transition: Is there a
role for European supermajors?[J]. Energy Research & Social
Science, 2022, 88: 102634. The cultural restoration strategy aims to re-examine the
value and essence of the film industry from multiple
dimensions to restore the true appearance of the film. From
the perspective of the basic links and core manifestations of
the film industry, cultural restoration must emphasize the
fundamental value of films and return to the original intention
of film creation. At present, while pursuing commercial value,
the film industry needs to strengthen its control of cultural
value to achieve a balance between business and culture. This [5] Qin M, Su C W, Umar M, et al. Are climate and geopolitics the
challenges to sustainable development? Novel evidence from
the global supply chain[J]. Economic Analysis and Policy,
2023, 77: 748-763. [6] Lemoine R T, Svenning J C. Nativeness is not binary—a
graduated terminology for native and non‐native species in the
Anthropocene[J]. Restoration Ecology, 2022, 30(8): e13636. 124
|
https://openalex.org/W2165827665
|
https://www.graphyonline.com/archives/IJJMC/2014/IJJMC-104/article.pdf
|
English
| null |
Three Years after the Great East Japan Earthquake
|
International journal of journalism & mass communication
| 2,014
|
cc-by
| 1,636
|
Three Years after the Great East Japan Earthquake Yasutaka Ueda*
Professor, Deparment of Mass Communication, Edogawa University, Japan Professor, Deparment of Mass Communication, Edogawa University, Japan Publication History:
Received: March 30, 2014
Accepted: May 10, 2014
Published: May 12, 2014 Publication History:
Received: March 30, 2014
Accepted: May 10, 2014
Published: May 12, 2014 The most powerful earthquake ever recorded in Japan occurred
at 2:46 in the afternoon of March 11, 2011 resulting in the loss of
numerous precious lives and the complete destruction of the towns
and villages that they called home. Although three years have passed
since that tragedy, 267,000 people still live in evacuation shelters. The Great East Japan Earthquake not only caused devastating social
and economic effects over these past three years, but also had a
significant effect on the media environment. The loss of confidence in
the mass media provided an opportunity for the Internet to become
increasingly prominent. Personally, I must confess that I have learnt
most of research tools from my education in the United States and still
keep absorbing them from new books and journals produced there. Yet it took some time to realize that such an apparently innocuous and
beautiful cabinet of expensive tools prove to be useless occasionally
in the real field work in my homeland. Why is it that? What am I
supposed to do in order to survive as a professional and produce a
meaningful research to the country where I live? From my own
experience, thus, I devised a temporary ‘solution’ that I need to be
more creative in a view of tools, and conceive ways to choose wisely
tools suitable for local needs. Although the Great East Japan Earthquake caused tremendous loss
and damage, in actuality, 90% of the Japanese population was not
directly affected. However, the perceptions of the Japanese people
clearly underwent a change since that day on March 11 three years
ago. A growing awareness and emphasis on "bonding" with the
victims that spread rapidly among the Japanese created a tendency
towards the need for communication. The free messaging application,
LINE, which began service in June 2011 following the disaster,
became firmly established as a communication tool used by a large
number of Japanese. Three Years after the Great East Japan Earthquake Although mixi, which initially became popular
as a social networking service (SNS) in Japan, appeared in 2004 and
was followed by similar jumps in popularity by Twitter in 2008 and
Facebook in 2010, LINE has continued to undeniably hold the top
spot since the disaster. In addition, the sentiment among residents that
"since no one knows what could happen tomorrow and the end could
come at any time, it is important to not to forget to live every day to
the fullest" resulted in the ascent of the popular girls' singing group,
AKB48 [1], to an exclusive rank that enabled them to have the top five
rated CD single sales for the years 2011 and 2012. This group ABK48
was characterized by containing numerous lyrics in their songs that
attempt to provide comfort and relief to young people exhausted by
recent events. In the face of social crises in the form of the Great East
Japan Earthquake and the Fukushima nuclear power plant accident,
economically, socially and psychologically discouraged young people
have supported this popular group as a result of sympathizing with
their lyrics based on a sense that the lyrics sung by its members are
targeted directly at them. At the time of the earthquake, 99.5% of the television sets in affected
areas were unable to be used due to power outages, and an average of
4.3 days was required to restore power. Since communication by cell
phone was also not possible, the only information available to family
members who had escaped to various locations and were unable to
contact each other was through bulletin boards installed at evacuation
shelters. As a result, persons in affected areas relied on information
provided on the radio, newspapers and portable Internet devices. Many of the victims recounted their experiences by indicating that,
even though land-based telephones were down, they were able to use
the Internet, or that even though cell phone e-mails had difficulty in
getting through, contacts were able to be made using Twitter on their
Smartphone's. The media strength of the Internet was also strongly
recognized with respect to two-way communication enabling persons
in affected areas to be heard directly or support for volunteers
delivering information on daily necessities. Analytical Data Keywords (consisting mainly of nouns) were extracted using text-
based data analysis software (IBM SPSS Text Analytics for Surveys
3.0.1J) [2]. Trends present in lyrics were determined by investigating
frequently appearing words among those keywords, paragraphs
containing certain keywords were extracted, and correspondence
analyses were conducted between those paragraphs and keywords to
analyze trends in their descriptive content. The targets of the analysis
consisted of a total of 281 songs consisting of 32 singles released by
AKB48 from February 2006 to August 2013, the songs contained On the other hand, the shortcomings of the nature of the Japanese
media industry structure of both television stations and newspaper
companies being divided into individual prefectures were clearly
exposed when news coverage was attempted to be provided on damage
over a wide area spanning multiple prefectures. Even after television
broadcasts were restored, there were regions where information on the
situation was provided and regions where it was not and there were
considerable discrepancies occurred in support activities, including
the collection of contributions, resulting in a sense of unfairness
among victims. At the time of the accident at the nuclear power plant
in Fukushima, the media was unable to provide any in-depth coverage,
and coverage essentially consisted only of reporting information on
statements made by government officials. Since the mass media per
se was unable to obtain accurate information, and since information
cannot be provided without knowing whether or not it is accurate, the
actual information provided to residents was limited, thereby creating
dissatisfaction and a feeling of distrust among residents. *Corresponding Author: Dr. Yasutaka Ueda, Professor, Deparment of Mass
Communication, Edogawa University, Japan, E-mail: y-ueda@edogawa-u.ac.jp
Citation: Ueda Y (2014) Three Years after the Great East Japan Earthquake. Int
J Journalism Mass Comm 1: 104 *Corresponding Author: Dr. Yasutaka Ueda, Professor, Deparment of Mass
Communication, Edogawa University, Japan, E-mail: y-ueda@edogawa-u.ac.jp Citation: Ueda Y (2014) Three Years after the Great East Japan Earthquake. Int
J Journalism Mass Comm 1: 104 Citation: Ueda Y (2014) Three Years after the Great East Japan Earthquake. Int
J Journalism Mass Comm 1: 104 Copyright: © 2014 Ueda. 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. Int J Journalism Mass Comm
ISSN: 2349-2635 IJJMC, an open access journal
Volume 1. 2014. Int J Journalism Mass Comm
ISSN: 2349-2635 Udea Y., Int J Journalism Mass Comm 2014, 1: 104
http://dx.doi.org/10.15344/2014/ijjmc/104 Udea Y., Int J Journalism Mass Comm 2014, 1: 104
http://dx.doi.org/10.15344/2014/ijjmc/104 Udea Y., Int J Journalism Mass Comm 2014, 1: 104
http://dx.doi.org/10.15344/2014/ijjmc/104 Udea Y., Int J Journalism Mass Comm 2014, 1: 104
http://dx.doi.org/10.15344/2014/ijjmc/104 Commentary
International Journal of
Journalism & Mass Communication Commentary
International Journal of
Journalism & Mass Communication Three Years after the Great East Japan Earthquake Analytical Data 104 Int J Journalism Mass Comm
ISSN: 2349-2635 s after the Great East Japan Earthquake. Int J Journalism Mass Comm 1: 104. DOI: http://dx.doi. Citation: Ueda Y (2014) Three Years after the Great East Japan Earthquake. Int J Journalism Mass Comm 1: 104. DOI: http://dx.doi. org/10.15344/2014/ijjmc/104 Citation: Ueda Y (2014) Three Years after the Great East Japan Earthquake. Int J Journalism Mass Comm 1: 104. DOI: http://dx.doi. org/10.15344/2014/ijjmc/104 Page 2 of 2 in 4 albums released from January 2008 to August 2012, and songs
performed at stage performances. All language data from the 281
target songs was input digitally and converted to text data followed
by carrying out text mining and categorizing the "headwords". Verification Results As a result of verifying the results of the analysis by text mining,
the headwords contained in songs performed by AKB48 were as
indicated in the table below (Table 1). Rank
Frequently Appearing
Word
Frequency
(No. of
Songs)
English
Japanese
1st
Dream
Yume
126
2nd
I (woman)
Watashi
119
3rd
Love
(formal)
Ai
112
4th
Heart
Kokoro
101
5th
Inside
Naka
91
6th
You (formal)
Anata
91
7th
I (man)
Boku
85
8th
Wind:
Kaze
83
9th
Sky
Sora
80
10th
You
(informal)
Kimi
78
10th
Myself
Jibun
78
12th
Love
(informal)
Koi
69
13th
Future
Mirai
68
14th
Road
Michi
61
15th
Hand
Te
56
Table 1: The results of the analysis by text mining, the headwords
contained in songs performed by AKB48. Table 1: The results of the analysis by text mining, the headwords
contained in songs performed by AKB48. Although words relating to falling in love such as "love (formal)",
"love (informal)", "you (formal)" or "you (informal)", which
frequently appeared in the lyrics of previous female artists, also
frequently appear in the lyrics of songs performed by AKB48 [3],
words indicating hope such as "dream" or "future", as well as words
invoking a sense of nostalgia for home such as "wind", "sky" or "road",
were also determined to appear at a high frequency. Int J Journalism Mass Comm
ISSN: 2349-2635 Int J Journalism Mass Comm References 1. Maxwell K (2011) Japan Goes Gaga Over a 92-Member Girl, The Wall Street
Journal. 2. Uchida O, Kawashima A, Isozaki S (2012) Text Mining by SPSS, Ohm. 3. Ueda Y, Hirota Y (2014) AKB48 eho-system which enables WTA in the
Japanese music market. Research Bulletin of the Edogawa University IJJMC, an open access journal
Volume 1. 2014. 104 Int J Journalism Mass Comm
ISSN: 2349-2635 ISSN: 2349-2635
|
https://openalex.org/W3178417266
|
https://journal.uokufa.edu.iq/index.php/kufa_arts/article/download/678/621
|
Latin
| null |
A legal and judicial investigation of the role of the multiplicity of the causes in determining the liability in driving accidents
|
Ādāb al-Kūfaẗ/Ādāb al-kūfaẗ
| 2,021
|
cc-by
| 4,033
|
Abstract
Humanﺍﳌﺴﺘﺨﻠﺺ Abstract
Humans
have
been
using
different vehicles for a long time to
transport goods and passengers. Given the growing number of
motor vehicles in recent years and
the subsequent increase in the
number of driving accidents, it is
not clear who should compensate
for losses incurred to the victim in
such accidents. Given that our laws
have been inspired by Islamic
jurisprudence, there are important
judicial rules including the rule of
destruction, causation, no-loss rule,
warning rule, etc. that have a
significant position in our current
regulations for determining the
liable person. Of these rules, the
theory of precedent cause has been
widely accepted. Key words : Liable Cause ,
ﺍﳌﺴﺘﺨﻠﺺ
َ ُنِمَ اَِِ ا
ٍت
ٍَُِ
وَ اَِ اَِ
ِاتَ ا َب، و
خا
رَةِ ی
ِ زََِی
ادَةِ
ذاتِ اَو
َا
ك
زّ َو
ی
ادَةِ
ورادِثِ اا
ِ ،
ُك
ي اول الَ اَ ٌض
یَ
ُِ
نأ یَ
ََذ
لَ هِی إاَبُ. ما
َلإ ی
ان نأ
یَ مِ اِ اِ ةَُ
ُك ِ ُِا
،فَة إِ ُِ
َی
ب،
َ ،َرَ ری و ... ا
َ
َم
ان یم
َی ال وَ دی
ا
َلُ م َ َول وا
ِ
َِا
ا اَِ ِ ی.ع
تا
ا ول ا: ا-
ّا
-
ّ او-
ادث اَ . ری ﺍﳌﺴﺘﺨﻠﺺ
َ ُنِمَ اَِِ ا
ٍت
ٍَُِ
وَ اَِ اَِ
ِاتَ ا َب، و
خا
رَةِ ی
ِ زََِی
ادَةِ
ذاتِ اَو
َا
ك
زّ َو
ی
ادَةِ
ورادِثِ اا
ِ
،
ُك
ي اول الَ اَ ٌض
یَ
ُِ
نأ یَ
ََذ
لَ هِی إاَبُ. ما
َلإ ی
ان نأ
یَم
ِ اِ اِ ةَُ
ُك ِ ُِا
،فَة إِ ُِ
َی
ب،
َ ،َرَ ری
و ... ا
َ
َم
ان یم
َی ال وَ دی
ا
َلُ م َ َول وا
ِ
َِا
ا اَِ ِ ی.ع
تا
ا ول ا: ا-
ّا
-
ّ او-
ادث اَ . ری Humans
have
been
using
different vehicles for a long time to
transport goods and passengers. Given the growing number of
motor vehicles in recent years and
the subsequent increase in the
number of driving accidents, it is
not clear who should compensate
for losses incurred to the victim in
such accidents. ﻓِﻘﻬ
ٌ
ﺔ
َ
ﺩِﺭﺍﺳ
ﻴ
ٌ
ﺔ
ﻘﻮﻗ
ُ
ﺣ
َ
ﻭ
ﻴ
ٌ
ﺔ
ﺓِ
َ
ﺩ
ﱢ
ﺪ
َ
ﻌ
َ
ﺘ
ُ
ﺳﺒﺎﺏِ ﺍﳌ
َ
ﻭﺭِ ﺍﻷ
َ
ﺩ
َ
ﻮﻝ
َ
ﺣ
ﺤﺪ
َ
ﰲ ﺗ
ﺪِ ﯾ
ﺴﺆﻭﻟ
َ
ﺍﳌ
ﺔِ ﻴ
ﱠ
ﻮﺍﺩِﺙِ ﺍﻟﺴ
َ
ﰲ ﺣ
ﺮِ ﯿ
A legal and judicial investigation of the role of
the multiplicity of the causes in determining the
liability in driving accidents
Leila Jahanbin
A faculty member of the Department of Theology and Islamic
Instructions (Islamic jurisprudence and Basics of Islamic law) , Payame
Noor University , Tehran – Iran
ljahanbin@gmail.com
ﻰﻠﯿﻟ ﺟﻬﺎﻥ ﺑ
ﯿﻦ
ُ
ﻀﻮ
ُ
ﻋ
ﻌﻠ
ﱠ
ﺓِ ﺍﻟﺘ
َ
ﺳﺮ
ُ
ﺍﻷ
ﯿﻤ
ﱠ
ﺔِ ﯿ
ﻠﻮﻡِ ﺍﻹِﺳﻼﻣ
ُ
ﻟِﻠﻌ
ﱠ
ﺔِ ﯿ
َ
ﺔِ ﺑ
َ
( ﰲ ﺟﺎﻣِﻌ
ُ
ﺻﻮﻝ
ُ
ﺍﻷ
َ
ﻭ
ُ
)ﺍﻟﻔﻘﻪ
ﺎﻡﯿ
ﻬﺮﺍﻥ ـ ﺇ
َ
ﻧﻮﺭـ ﻃ
ﺮﺍﻥﯾ ﻓِﻘﻬ
ٌ
ﺔ
َ
ﺩِﺭﺍﺳ
ﻴ
ٌ
ﺔ
ﻘﻮﻗ
ُ
ﺣ
َ
ﻭ
ﻴ
ٌ
ﺔ
ﺓِ
َ
ﺩ
ﱢ
ﺪ
َ
ﻌ
َ
ﺘ
ُ
ﺳﺒﺎﺏِ ﺍﳌ
َ
ﻭﺭِ ﺍﻷ
َ
ﺩ
َ
ﻮﻝ
َ
ﺣ
ﺤﺪ
َ
ﰲ ﺗ
ﺪِ ﯾ
ﺴﺆﻭﻟ
َ
ﺍﳌ
ﺔِ ﻴ
ﱠ
ﻮﺍﺩِﺙِ ﺍﻟﺴ
َ
ﰲ ﺣ
ﺮِ ﯿ
ﻰﻠﯿﻟ ﺟﻬﺎﻥ ﺑ
ﯿﻦ
ُ
ﻀﻮ
ُ
ﻋ
ﻌﻠ
ﱠ
ﺓِ ﺍﻟﺘ
َ
ﺳﺮ
ُ
ﺍﻷ
ﯿﻤ
ﱠ
ﺔِ ﯿ
ﻠﻮﻡِ ﺍﻹِﺳﻼﻣ
ُ
ﻟِﻠﻌ
ﱠ
ﺔِ ﯿ
َ
ﺔِ ﺑ
َ
( ﰲ ﺟﺎﻣِﻌ
ُ
ﺻﻮﻝ
ُ
ﺍﻷ
َ
ﻭ
ُ
)ﺍﻟﻔﻘﻪ
ﺎﻡﯿ
ﻬﺮﺍﻥ ـ ﺇ
َ
ﻧﻮﺭـ ﻃ
ﺮﺍﻥﯾ Leila Jahanbin
A faculty member of the Department of Theology and Islamic
Instructions (Islamic jurisprudence and Basics of Islamic law) , Payame
Noor University , Tehran – Iran
ljahanbin@gmail.com A legal and judicial investigation of the role..…………………. (776) Introduction In the modern world that is moving toward speed and
communication, vehicles play an effective role in speeding up goods and
human transportation. The increased use of vehicles has increased the
dangers associated with them. To fulfill civil liability in driving
accidents, three elements of loss, the harmful act, and the causation
relation should be established. The third element, i.e. the causation
relationship between the loss and the harmful act, which is the focus of
this study, is of special significance because this relation has many
complexities and ambiguities leading to the confusion of courts of law
dealing with civil liability claims. Accordingly, addressing these
methods, explaining their ambiguities, and providing practical solutions
for courts of law are of special importance. The most important question
is when the multiplicity of the causes lead to incurring the loss, i.e. there
is a causal relationship between each cause and incurred loss, which one
is the liable cause? Besides, in the cause of the accumulation of causes,
how the liability for the loss should be divided among them? These
questions are addressed in this paper. Abstract
Humanﺍﳌﺴﺘﺨﻠﺺ Given that our laws
have been inspired by Islamic
jurisprudence, there are important
judicial rules including the rule of
destruction, causation, no-loss rule,
warning rule, etc. that have a
significant position in our current
regulations for determining the
liable person. Of these rules, the
theory of precedent cause has been
widely accepted. Key words : Liable Cause ,
Cause , Vehicle , Driving
Accidents. Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ A legal and judicial investigation of the role..…………………. (777) The literal definition of cause Cause means excuse, reason, origin, proximity, kinship, and path
(Azartash, 2000: 274). It also means friendship, reason, and solution (Jar,
2001: 1164). In the Quran, cause means elevation as lifting water from
the well by a rope: “Let him extend a rope to the ceiling, then cut off [his
breath]” (Surah Hajj, Verse 15). Cause is not essentially of material nature and it refers to spiritual and
unseen reasons too. For instance, the holy Quran refers to "the ways into
the heavens” (Ghafir Surah, Verse 37). Judicial definition of cause The world cause in judicial terms refers to affairs that religion has
established an existential relationship between them and other affairs so
that they come to the existence or become nonexistence upon the
existence or nonexistence of the cause (Amid Zanjani, 2010: 78). According to al-Shahīd al-Awwal, “Every event/occurrence requires a
cause” (Al-Shahīd al-Awwal, 1991: 107). According to Imam Khomeini, the cause is any actions the produce an
effect that would not come to existence without the case such as drilling a
well (Imam Khomeini, 1963: 369). Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (778) The definition of cause in driving accidents Based on what was mentioned, the causing agent is not directly
involved in the occurrence of the accidents and is considered as only the
non-immediate agent. Therefore, cause in driving accidents means that
sometimes the accident is not the direct outcome of the driver’s actions,
as other factors may also be involved in the occurrence of the accident
and they are called causing agents. y
g g
The civil liability for the cause A legal and judicial investigation of the role..…………………. (778) The definitions of the cause provided by lawyers are influenced by
the definitions stated by jurists. According to Jafari Langroudi, “If two or
more persons cause a damage/loss to another person, the person
immediately linked to the resulting loss is called the agent and the other
person(s) who are connected to the incurred loss are called cause (Jafari
Langroudi, 2008: 352). In sum, cause refers to an act that is partially effective in the
occurrence of the effect. However, the cause by itself cannot result in the
effect but facilitates its occurrence (Law Research Group, Razavi
University of Islamic Sciences, 2011: 146). A legal and judicial investigation of the role..…………………. (779) 3. Theory of guaranty y
g g
The civil liability for the cause There are, generally, three types of civil liability that differ in terms
of the harmful act constituting one of the components of civil liability. The ordinary type of civil liability making up a general legal rule is the
liability resulting from a person’s action. This liability holds the
person(s) involving in the harmful act responsible for their actions. The
second type of liability results from another person’s action. Accordingly, a person is liable for compensating the loss incurred as the
result of another person’s act. The third type of civil liability is the
liability resulting objects/properties under which a person is responsible
for the losses incurred another person by an object possessed by the
person including an animal or an inanimate object. There are three elements for each of these liabilities that shall be
established to determine the person responsible for the incurred loss. These elements are: (1) The person incurring the loss, (2) The harmful
act, and (3) The losing party Depending on which element is emphasized, scholars have provided
different theories about the basis of civil liability. They can be divided
into three main theories: 1. Theory of fault 2. Theory of risk Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (779) A legal and judicial investigation of the role..…………………. (779) 3. Theory of guaranty According to Katouzian, “Although the theory of guaranty plays an
important role in creating civil liability, none of the mentioned theories
can exclusively be used as the basis for civil liability, because there is an
undeniable fact in these theories. According to them, the important thing
is to establish justice, and these logical tools (theories) only pave the way
for achieving this goal” (Katouzian, 1990: 128). These theories are discussed below:
The theory of fault These theories are discussed below: Fault in French is associated with guilt and blaming a person for his
actions or thoughts. Fault means a mistake, especially something for
which a person is to blame. A faulty behavior is blamable and indecent
(Badini, 2005: 52). Fault in Persian and Arabic means offense, guilt, negligence, etc. however, its most important connotation is negligence in doing
something (Anvari, 2002: 1834). Literally, a fault is a moral concept, and humans are morally
responsible for their actions and their faults must be assessed by referring
to their conscience. The real civil liability that was developed under the influence of the
beliefs of law scholars of the church was a fault-based system. Civil
liability is basically a technical tool for guaranteeing the moral
responsibility. The idea of compensation for losses is one of the oldest
human and moral ideals. This idea is highly valued in religious ethics. Repentance is accepted when losses resulting from guilt are
compensated. Humans are aware of their responsibilities and know that
they will be held for their mistakes and are relieved when they can
compensate for evils resulting from their actions (Katouzian, 1975: 66). According to the theory of fault, the standard for determining the
liability in driving accidents is the behavior of the vehicle owner. The
owner is held liable when he/she commits a fault; otherwise, he/she will
be not responsible. Based on the general principle of civil liability, the
owner can also disclaim responsibility by establishing some legal reasons
and excuses. Therefore, the subjective standard of fault is no longer to
meet the requirements of the modern industrial society because the
development of industrial life, new inventions, and the growing increase
of accidents and their complexities make it difficult to prove the fault of
the vehicle’s manufacturer, owner, or driver. Therefore, most legal Adab Al-Kufa Journal
No. A legal and judicial investigation of the role..…………………. (780) A legal and judicial investigation of the role..…………………. (780) systems use objective standards to recognize the person who commits a
fault including the standard of “the good father family” in the French law
and “a rational human” in the Common Law. However, according to the
objective and social standards, fault in the realm of civil liability is
defined as “a mistake/error committed by a caution person when he/she is
exposed to the same physical conditions surrounding the person who
commits the fault” (Badini, 2005: 73). 3. Theory of guaranty 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (780) Theory of risk This theory maintains that a person who engages in activity and
creates a dangerous environment for others shall compensate for the
losses incurred to others as a result of the activity. Investors and factory
owners who make a profit from their activities shall compensate damages
and losses resulting from these activities even if the resulting accidents
occurred due to workers' negligence or it was not possible to predict them
(Katouzian, 1995). However, the opponents of the theory of risk severely attack
materialistic interpretations of the proponents of the theory, and they
believe that the denial of fault in civil liability claims and the systematic
replacement of fault by the risk that means the dominance of the material
over the soul is not acceptable, because laws have established for humans
to regulate interpersonal relations. In fact, laws originating from human
thinking cannot ignore their creator and objectify a human being who
possesses spirit and will. Objects are addressed by law only for their
relationships with humans. Accordingly, properties are subject to law as
they are considered as personal belongings (Badini, 2005: 266). Besides, the theory of risk results in the underdevelopment of the
community because it discourages creative human resources and
suppresses the innovative power of individuals and they lose motivation
to invent new things when they know that they will be blamed even if
they do not commit a fault (Badini, 2005: 346). The omission of the concept of fault in civil liability claims not only
did not solve the problems faced by the losing party but also complicated
such claims. It also makes it difficult for the losing party to prove the
cause(s) leading to the loss. Theory of guaranty According to the theory of guaranty proposed by Boris Starck in
France, everybody has the right to a healthy and safe life in society and
making profits from their properties. This right has been protected and Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (781) A legal and judicial investigation of the role..…………………. (781) supported by laws by stipulating the aggressor’s civil liability: All people
should respect others’ rights and do not endanger other people’s safety. When a right is spoiled, the person spoiling it shall compensate it and the
requirement for compensation is called civil liability. This enforcement is
nothing but compensation for the loss incurred (Katouzian, 1995: 209). This theory effaced the concepts of fault and liability and emphasized
the support for the losing party. It should be mentioned that if the theory
of guaranty is used as a basis for legislation, it is stipulated in the
compulsory insurance law as follows: All owners of vehicles are required to ensure their vehicle for
physical and financial damages incurred to third parties as results of
accidents caused by the vehicle”. This stipulation severs as the basis for
the Compulsory Insurance Law enacted in 2008. The criteria for determining multiplicity of the causes in driving
accidents The criteria for determining multiplicity of the causes in driving
accidents Sometimes damages are incurred by two or more persons; an issue
known as the multiplicity of the causes. In this case, of the multiplicity of
the causes leading to the damage, the one which deserves to bear such
liability is identified. For the accumulation or overlap of the causes, three
conditions shall be established : 1. The multiplicity of the causes or the accumulation of two or more
causes in a single case. 2. None of the causes is influenced or diminished by other causes. 3. The effects of one of the causes do not distance the two causes (Jafari
Langroudi, 2009: 438). The assessment of the new Islamic punishment law shows that the
legislators seeking to find a rational solution to multiplicity of causes has
provided three distinct assumptions: 1. The unity of the cause and agent 2. The unity of concurrent causes 3. The unity of sequential causes The unity of the cause and the agent means the agent's action creates
a barrier between the cause and the loss and thus disconnecting the direct
relationship between them so that the cause must result in the loss or a
precondition for its impact. However, according to Article 332 of the
Civil Law and Article 363 of the former Islamic Punishment Law, in the
unity of the cause and the agent, the latter shall bear the responsibility for
the loss unless the cause is stronger than the agent and thus the loss is Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (782) A legal and judicial investigation of the role..…………………. (782) attributed to the former. Even Islamic scholars believe that this principle
is one of the most valid juridical rules and there is consensus on it
(Mohaghegh Damad, 2008: 121). However, according to the new Islamic Punishment Law, a person is
responsible for the offense if the offense is attributed to him/her. In
contrast, if the offense is attributed to all agents, all of them will be
equally held accountable. Besides, if each of the agents has contributed
differently to the damage/loss, they will be held accountable based on
their contribution. The criteria for determining multiplicity of the causes in driving
accidents Accordingly, it can be suggested that under the current
legal regulations, the theory of fault serves as the basis for the driver's
civil liability. Therefore, the causes of the accident must be recognized
based on the faults committed by each driver. A legal and judicial investigation of the role..…………………. (783) A legal and judicial investigation of the role..…………………. (783) existence of the other cause. According to Article 535 of the Islamic
Punishment Law, "If two or more persons are involved sequentially in
committing a crime, the person whose action precedes the occurrence of
the crime shall be held accountable". For instance, if a person digs a hold
and another person places a stone next to the hole and a passerby collides
with the stone and falls into the hold, the person placing the stone next to
the hole shall be held accountable unless all the persons intended to
commit the crime and thus their participation is considered as complicity. According to the current judicial procedure, if multiple vehicles are
involved in the occurrence of an accident, all of them shall equally pay
compensation to the victims (Jamshidi, 2011: 178). The unity of concurrent causes Concurrent causes are those that act at the same time and their
simultaneous interaction results in damage or loss and they cannot be
distinguished chronologically (Abedi, 2013: 10). According to the
Islamic Punishment Law, “When two or more persons take part in
committing a crime or incurring damage to another person in a manner
that the crime or the damage is attributed to both or all of them, they all
will be equally held accountable". The provisions of this article apply to
the collision between two land, sea, and air vehicles. For instance, if two
or more persons roll down a stone from the mountain slope and it hits a
car passing the road leading to the driver's injury, the involved persons
will be held accountable. However, it should be noted that since all the
persons have been involved in rolling down the stone and they are
considered as the single cause of the stone moving down they will be
responsible for compensating the resulting down based on their
contribution to rolling down the stone. Nevertheless, determining the
exact contribution of each person is difficult and sometimes impossible. Therefore, the effect of each cause does not depend on other causes as
each cause has contributed to the incurrence of the loss independently. In
this case, the simultaneity of the effects of the causes is taken into
account as the main requirement (Sadeghi, 1997: 78) and each person is
responsible for compensating the damage following their contribution. Sequential causes q
Sequential causes are those factors resulting in a loss sequentially
with the loss occurring upon the fulfillment of the last factor. The causes
are arranged in a manner that the effect of each cause depends on the Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020 (783) Conclusion When multiple causes are involved in incurring damage, establishing
the causation between the causes and the damage will be a challenging
task. When several persons are sequentially involved in committing an
illegal act, the person whose action precedes the occurrence of the
harmful accident shall be held accountable. Besides, concerning the
multiplicity of concurrent causes, the legislator adhered to the scholars’
consensus and accepted the theory of fault. In the unity of the cause and
the agent as an instance of sequential causes, an agent is responsible for
the offense if the offense is attributed to him/her. In contrast, if the
offense is attributed to all agents, all of them will be equally held
accountable. Besides, if each of the agents has contributed differently to
the damage/loss, they will be held accountable based on their
contribution and their role in committing the crime. This criterion
corresponds to the principle of the subjectivity of liabilities. References 1. The holy Quran 2. Azartash, A. (2000). The modern Arabic-Persian dictionary. Tehran: Ney
Publisher. 3. Anvari, H. (2002). The unabridged dictionary of speech. Tehran: Maharat
Publishing Center. 4. Badini, H. (2005). The philosophy of civil liability. Tehran: Publishing
Corporation. 5. Jafari Langroudi, M. J. (2008). Legal terminology. Tehran: Ganje Danesh. 6. Jafari Langroudi, M. J. (2008). Annotated legal terminology. Tehran: Ganje
Danesh. 7. Jamshidi, A. (2011). The civil liability of owners of land motor vehicles. Tehran: Javdaneh Press, Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢ (784) A legal and judicial investigation of the role..…………………. (784) 8. Jar, K. (2001). Larousse dictionary. Translated by S. H. Tabibian. Tehran:
Amir Kabir Press. 9. Al-Shahīd al-Awwal (1991). Al-ghavaed va al-Favaed. Translated by S. M. Sanei. Mashhad: Ferdowsi University Press. 10. Sadeghi, M. H. (2005). Crimes against individuals. Tehran: Mizan Press. 11. Katouzian, N. (1990). Civil law: Automatic guarantees of civil liability. Tehran: Tehran University Press. 12. Katouzian, N. (1995). Non-contractual requirements (automatic guarantees). Tehran: Tehran University Press. 13. Law Research Group of Razavi University of Islamic Sciences, (20
Rules of penal jurisprudence. Mashhad: Astan Quds Razavi Press. 14. Abedi, M. (2013). The enigma of the multiplicity of causes in the Islamic
punishment law with a focus on civil liability in the multiplicity of causes in
medical malpractice. The 4th Conference on Medicine and Judgment, Shiraz. 15. Amid Zanjani, A. (2010). Causes of guaranty. Tehran: Mizan Press. 16. Mousavi Khomeini, R. A. (1963). Tahrir al-Wasilah. Translated by A. Eslami. Islamic Publications Office affiliated with Qom Seminary Teachers
Association. 17. Mohaghegh Damad, S. M. (2008). Jurisprudential rules. Tehran: Islamic
Sciences Publishing Center. Adab Al-Kufa Journal
No. 46
Rabeea Althane 1442 / December 2020
آداب ا
: دا
٦٤
رما
٢٤٤١
/ نم
ولا
٠٢٠٢
|
https://openalex.org/W2011403054
|
https://europepmc.org/articles/pmc3092418?pdf=render
|
English
| null |
Antimicrobial resistance surveillance in the AFHSC-GEIS network
|
BMC public health
| 2,011
|
cc-by
| 6,874
|
* Correspondence: william.meyer6@us.army.mil
1Armed Forces Health Surveillance Center, 11800 Tech Rd, Silver Spring, MD
20904, USA
Full list of author information is available at the end of the article REVIEW Open Access © 2011 Meyer et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Introduction and background developed diverse surveillance programs that often
involve studies of microbial sensitivities to antibiotics. Many of these projects include outcomes that not only
direct appropriate use of antimicrobials for individual
patients and regional health planners, but are also
aligned with DoD efforts of medical diplomacy with
capacity building and investments in outbreak detection
and response. These activities often involve collabora-
tions with the host-nation civilian and military clinics
and hospitals, as well as university systems. Other anti-
microbial resistance-related programs represent colla-
borations within DoD for identification and tracking of
infections at military medical facilities. Taken together,
these activities are beneficial to both force health pro-
tection and the host nation in many interconnected
levels. The following is a synopsis of selected partner
activities from 2009. Antimicrobial resistance is a growing threat to the con-
trol of infectious disease globally and within the United
States. Lethal organisms once thought to be on the
decline are re-emerging with resistance to commonly
used antimicrobials. Resistant organisms once acquired
exclusively in hospital settings are now widely circulat-
ing in communities. Infections with resistant organisms
not only result in greater severity and higher rates of
morbidity and mortality, but also increase health care
treatment costs and long-range expenses related to
research and development of new drugs. Since its inception, AFHSC-GEIS has a legacy of anti-
microbial resistance surveillance. This activity continues
as one of the five key focus areas for AFHSC-GEIS. Although few of the annual AFHSC-GEIS-funded pro-
posals are singularly devoted to antimicrobial resistance,
over several years the organization’s partners have Antimicrobial resistance surveillance in the
AFHSC-GEIS network William G Meyer1*, Julie A Pavlin2, Duane Hospenthal3, Clinton K Murray3, Kurt Jerke4, Anthony Hawksworth5,
David Metzgar5, Todd Myers6, Douglas Walsh7, Max Wu7, Rosa Ergas8, Uzo Chukwuma8, Steven Tobias9,
John Klena10, Isabelle Nakhla10, Maha Talaat10, Ryan Maves11, Michael Ellis12, Glenn Wortmann12, David L Blazes1,
Luther Lindler1 Abstract International infectious disease surveillance has been conducted by the United States (U.S.) Department of Defense
(DoD) for many years and has been consolidated within the Armed Forces Health Surveillance Center, Division of
Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) since 1998. This includes activities that
monitor the presence of antimicrobial resistance among pathogens. AFHSC-GEIS partners work within DoD military
treatment facilities and collaborate with host-nation civilian and military clinics, hospitals and university systems. The goals of these activities are to foster military force health protection and medical diplomacy. Surveillance
activities include both community-acquired and health care-associated infections and have promoted the
development of surveillance networks, centers of excellence and referral laboratories. Information technology
applications have been utilized increasingly to aid in DoD-wide global surveillance for diseases significant to force
health protection and global public health. This section documents the accomplishments and activities of the
network through AFHSC-GEIS partners in 2009. Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Cholera surveillance Investigators at the U.S. Armed Forces Research Institute
of Medical Sciences (AFRIMS) responded to a large out-
break of severe diarrhea that affected over 70,000 people
in 2009. They found 52 percent of the 158 samples were
positive for Vibrio cholera strain O1, 18 percent
were heat-labile toxin-expressing ETEC and 13 percent
were heat-stable toxin-expressing ETEC. All V. cholerae
strains isolated from the outbreak were resistant to nali-
dixic acid and trimethoprim-sulfamethoxazole, but they
were sensitive to tetracycline, ciprofloxacin, norfloxacin
and ampicillin [4]. The V. cholerae and rotavirus reference center for the
Middle East and Africa established by the U.S. Naval
Medical Research Unit Number 3 (NAMRU-3) per-
formed antibiotic sensitivity testing of 303 archived
V. cholerae samples. The center’s studies demonstrate
widespread resistance to streptomycin, trimethoprim-
sulfamethoxazole and nalidixic acid. Resistance to ampi-
cillin and chloramphenicol was observed only in isolates
from Somalia, and resistance to tetracycline was limited
to single isolates from Qatar and Somalia [5]. Another NAMRU-2 effort, the Indonesian Pediatric
Diarrhea Surveillance program, links six distinct geo-
graphic sites on five islands of the Indonesian archipe-
lago. The ongoing multi-year collaborative effort has
collected more than 12,000 specimens from patients
with acute diarrhea symptoms. Bacterial pathogens were
identified in 1,142 cases (11 percent), with Campylobac-
ter and Shigella species being the most prevalent etiolo-
gies isolated. Vibrio cholerae was the most common
Vibrio species identified. Shigellosis was identified as the
cause of diarrhea in 300 (2 percent) of the cases. Anti-
microbial susceptibility testing of Shigella samples
demonstrated very high levels of resistance to trimetho-
prim-sulfamethoxazole, often used as the first-line anti-
biotic to treat children with diarrhea in Indonesia [3]. Pathogens in Southeast Asia Pathogens in Southeast Asia Pathogens in Southeast Asia Over the last several years, a dramatic rise in antibiotic
resistance of enteric pathogens, including enterotoxigenic Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 2 of 8 Escherichia coli (ETEC), Shigella, Salmonella and Cam-
pylobacter has been documented in Southeast and South
Asia [1]. Many hospitals and laboratories in this region
lacked the essential infrastructure and capability to iso-
late and identify enteric pathogens and to reliably test
antimicrobial resistance. Several U.S. Naval Medical
Research Unit Number 2 (NAMRU-2) projects involved
laboratory-based microbiologic surveillance of patients
presenting for the care of febrile illnesses in host-nation
civilian and military clinics and hospitals scattered
around the regions. These efforts not only help deter-
mine the etiology of febrile illnesses and antimicrobial
sensitivity patterns in these areas but also provide essen-
tial host-nation laboratory capacity building and training. patterns were observed for isolates of C. coli. An increase
in minimum inhibitory concentrations (MIC) for several
antibiotics, including ciprofloxacin, was found when ana-
lyzing results over the time span of specimen collections. NAMRU-2 implemented a real-time polymerase chain
reaction (PCR) assay to discriminate between Campylo-
bacter isolate wild-type and mutant alleles that can con-
fer resistance to fluoroquinolones. The study found the
majority of resistant isolates possessed this mutation. Several strains that were negative by PCR but resistant by
MIC testing are being further characterized by sequence
analysis of the quinolone resistance-determining region
in an attempt to identify other novel mutations that may
confer resistance [3]. y
p
y
g
g
In an effort to determine the epidemiology and etiolo-
gies of acute febrile illness of unknown origin among
persons seeking medical care in Cambodia, NAMRU-
2 established a five-year hospital-based surveillance
study commencing in December 2006. Depending on
the patients’ presenting complaints, nasal/throat swabs,
serum samples, blood cultures, malaria smears and stool
samples were obtained from 4,751 patients with fever at
as many as nine different health care centers over the
years. Although the most prevalent pathogens isolated
among Cambodian patients were influenza, dengue and
malaria, the studies also identified diseases such as lep-
tospirosis, hantavirus and hepatitis A, B and E, as well
as diseases caused by rickettsial infections. Bacterial
organisms collected from these studies were analyzed
for antimicrobial resistance. The surveillance also identi-
fied the first cases of multidrug-resistant Salmonella
typhi with reduced susceptibility to fluoroquinolones in
Cambodia [2]. Health care-associated pathogens g
Health care-associated infections and antimicrobial
resistance have emerged as important public health pro-
blems in both developed and resource-poor countries,
as well as among DoD personnel. Increased surveillance
in hospitals in developing countries may determine risk
factors (e.g., overuse of antibiotics, counterfeit drugs or
deficiencies in local patient management guidelines and
policies) that are different from those in developed
countries. This may also define high-risk medical prac-
tices and enable the hospitals to tailor and implement
intervention plans to reduce infection rates in resource-
limited settings. These surveillance activities may
become examples that other developing countries can
follow in their attempts to reduce nosocomial infection
rates and increase the safety of health care systems in Campylobacter species were identified in 314 cases of
children presenting with diarrhea to participating hospi-
tals and health clinics in Jakarta, Makassar and Mataram. Antimicrobial susceptibility testing to C. jejuni identified
rates of ciprofloxacin resistance as high as 65 percent
with several cases exhibiting macrolide resistance. Similar Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 3 of 8 Page 3 of 8 minocycline. Antimicrobial resistance testing using the
Etest® strip method indicated production of metallo-
beta-lactamase in 18 (14 percent) of isolates. regions where U.S. personnel and travelers visit fre-
quently. Furthermore, knowledge about the genetic
determinants of antimicrobial resistance can be very
important for tailoring antibiotic policies and tracing the
nosocomial spread of pathogens. Molecular characteriza-
tion studies are essential tools that may help reduce
nosocomial infection mortality rates and the cost of
treating antibiotic-resistant infections, and limit inap-
propriate antibiotic use. The NAMRU-3 surveillance of health care-acquired
infections and antimicrobial resistance demonstrated that
gram-negative organisms constituted the majority of iso-
lates in both countries (61 percent in Egypt and 65.2 per-
cent in Jordan) [8]. In Jordan, Klebsiella species were the
most commonly isolated gram-negative pathogens
(17.4 percent of all isolates), whereas in Egypt, Klebsiella
species and A. baumannii were equally prevalent among
the gram-negative bacilli with each organism accounting
for 15.4 percent of the isolates. Not surprisingly, high rates
of antimicrobial resistance were reported in hospitals in
both countries. Extended-spectrum beta-lactamase produ-
cer rates among E. coli isolates were 64.3 and 66.7 percent
in Egypt and Jordan, respectively. Klebsiella species
showed extended-spectrum beta-lactamase producer rates
of 57.1 percent in Egypt and 75 percent in Jordan. Health care-associated pathogens In Egypt, resistance of Pseudomonas aeruginosa to imipe-
nem was 58.8 percent, and almost 88 percent of Staphylo-
coccus aureus isolates were resistant to methicillin. The higher rates of antimicrobial resistance in specific
intensive-care units (ICUs) encourage the establishment of
better-informed antimicrobial use policies and implemen-
tation of stricter infection control practices. These mitiga-
tions help decrease antimicrobial resistance rates and
reduce the risk of spread to the community. More than 30,000 U.S. military personnel have been
wounded in action while serving in Iraq or Afghanistan
[6], and many of these patients are at risk for serious
complications. Wound infections have been a common
complication of these injuries and, as in previous wars,
are often caused by gram-negative organisms. Minimal
information is available about the mechanism of antimi-
crobial resistance of bacterial infections in Middle
Eastern countries. Working with various regional host-
nation partners, the laboratories have been able to docu-
ment the geographic spread of antimicrobial resistance
in common organisms. This vital information is used to
effectively advise local and national health care leaders
about necessary changes to antimicrobial formularies
and provides important information on appropriate anti-
biotic treatment for troops deployed in the Middle East
[7]. Extended spectrum beta-lactamase-producing gram-
negative rods and Acinetobacter baumannii are major
causes of infections in health care settings. Many of
these nosocomial infections are difficult to treat with
antibiotics, and the antibiotic-resistant organisms that
cause them are increasingly being seen in community-
acquired infections [8]. Acinetobacter infections
A
b l Some samples processed by
each of the partner laboratories were sent to BAMC and
WRAIR for analysis, generating a systematic collection
of PFGE patterns-identified strains infecting DoD per-
sonnel. This enhanced the capabilities of DoD to deter-
mine the initial geographic location of contamination as
well as the spread and prevention of virulent strains of
gram-negative organisms originating from wound infec-
tions. Specific isolate information coupled with patient
outcomes led to the identification of specific virulence
factors within particularly virulent strains and guides the
use of specific or novel antibiotic treatments [14]. from hospitalized injured personnel have been multi-
drug-resistant, limiting the use of some empiric antibio-
tics for treatment of wound infections [11]. Continuous surveillance with genetic characterization
of Acinetobacter is the ideal method to direct infection-
control measures or altering of antimicrobial regimens. Molecular genotyping of these isolates enhances infec-
tion control of personnel wounded in the Middle East
and the possible nosocomial transmission of these
organisms within the DoD hospital system. Characteri-
zation of the multi-drug resistant organisms via PFGE
genotyping will help identify possible sources of infec-
tion and lead to strategies for employing appropriate
antibiotics and isolation practices. p
Landstuhl Regional Medical Center (LRMC) has an
active surveillance protocol for A. baumannii. All
incoming patients from Operation Iraqi Freedom,
Operation Enduring Freedom and Africa are screened
for colonization of Acinetobacter. Between October
2008 and March 2009, more than 500 A. baumannii iso-
lates were processed. Three main clusters of 90 percent
similarity were identified and account for 66 percent of
the strains examined. This comparison of Acinetobacter
genotypes significantly benefits infection control efforts
by helping identify potential sources of spread, particu-
larly those that occur during the evacuation chain from
the battlefield to U.S.-based hospitals. This genotyping
data has been used for infectious disease surveillance
and infection control at LRMC and thereby improved
total patient care. Drug resistance studies at Brook Army Medical Center
The primary objective of the Center of Excellence for
Leptospirosis at BAMC is developing reliable molecular
diagnostic techniques based on PCR for the disease. The
center has also assessed the antimicrobial therapies for
leptospirosis using in vitro and in vivo models. Acinetobacter infections
A
b l After
developing a calorimetric system to test antimicrobial
resistance by an in vitro model to various leptospiral
strains from around the world, the center has continued
to collect novel strains from around the world to test
while assessing a broad array of serovars against older
and newer antimicrobial agents. Antimicrobial agents that have been tested to date
include older agents such as amikacin, cefazolin, ceftazi-
dime, cephalexin, colistin, fosfomycin, gentamicin,
metronidazole, minocycline, polymyxin, rifampin, sulfa-
methoxazole, tobramycin, trimethoprim, imipenem, and
vancomycin [15-18]. Newer and novel agents including
tigecycline, doripenem, cethromycin, CEM-101 and
CEM-102 also have been tested. In addition, the more
active in vitro antimicrobial agents have been character-
ized further in a lethal hamster model for in vivo activ-
ity. This work has enabled the center to respond to
specific requests by clinicians in the developing world to
determine if older more commonly used antimicrobials
not used in the U.S., such as chloramphenicol, have
activity. In January 2009, an increase in the number of
A. baumannii infections with similar antibiograms in
the LRMC ICU was observed [13]. The three strains
were shown to be genotypically similar. While the actual
source was not determined, the infection control team
adopted additional measures, and further spread did not
occur. The data from the Acinetobacter studies, includ-
ing the PFGE, antibiotic susceptibility and plasmid pro-
filing, will provide valuable information regarding the
epidemiology and evolution of A. baumannii from the
onset of Operation Iraqi Freedom. Collection of this data allows researchers to identify
strains of interest for further detailed molecular work
(e.g., genome sequencing). The PFGE data collected at
LRMC is shared with other military treatment facilities. The DiversiLab and PFGE data generated at LRMC
allow AFHSC-GEIS and DoD to make more informed
funding decisions by comparing the two systems in
terms of cost, ease of use and quality of data. Additionally, BAMC has maintained a referral labora-
tory at the San Antonio Military Medical Center (Texas)
to support DoD outbreak investigations and other epide-
miological investigations and research. This center pro-
vides
centralized
molecular
biology
support
to
characterize multidrug-resistant bacteria pathogens and
has been involved in multiple studies. BAMC developed
a collaborative research relationship with Fort Sill
(Oklahoma) to support a methicillin-resistant Staphylo-
coccus aureus (MRSA) colonization study supported A unique three-year collaborative effort championed
by Dr. Acinetobacter infections
A
b l Antimicrobial-resistant strains of bacteria threaten U.S. military personnel deployed in the Middle East and
Afghanistan from combat- and non-combat-related
infections caused by these highly resistant pathogens
[10]. Acinetobacter baumannii-calcoaceticus complex,
P. aeruginosa, Klebsiella and E. coli are common
pathogens, but, compared to past wars, the acquisition
of multidrug-resistant isolates appears to be signifi-
cantly increased [11]. A. baumannii is a common
nosocomial challenge in Egypt and has emerged as one
of the important opportunistic pathogens in hospita-
lized patients throughout the world. Additionally, these
infections plague DoD and Veterans Affairs medical
treatment facilities and contribute to prolonged hospi-
tal stays. Outbreaks of Acinetobacter infections are
becoming increasingly common among patients in
ICUs, surgical units and burn units [12]. In Egypt and Jordan, NAMRU-3 has been engaged in
surveillance of health care-acquired infections and anti-
microbial resistance with emphasis on intensive care
units [9]. Researchers analyzed 124 highly resistant
gram-negative rods that were collected as part of this
surveillance network. Isolates were characterized pheno-
typically by standard bacteriological procedures and
antimicrobial susceptibility profiles were obtained using
Clinical and Laboratory Standards Institute (CLSI) cri-
teria. Major mechanisms of antimicrobial resistance in
isolates confirmed as either extended-spectrum beta-
lactamase-producing bacteria or Acinetobacter bauman-
nii were further characterized by pulsed-field gel
electrophoresis (PFGE), plasmid profiling, PCR amplifica-
tion and DNA sequencing. The resulting antimicrobial resistance profile classified
46 (37 percent) of the isolates as multidrug-resistant
organisms. Nineteen isolates (15 percent) were resistant
to imipenem and 20 (16 percent) were resistant to mer-
openem. Sixteen isolates (13 percent) were resistant to
both carbapenems. All isolates were sensitive to colistin
and were also sensitive or intermediately sensitive to The severe impact of an Acinetobacter outbreak on
hospital operations requires a quick assessment of the
potential spread of these infections. Comparison of
A. baumannii PFGE patterns from Egypt with isolates
collected at military treatment facilities in the U.S. showed high levels of genetic variability among collec-
tions. The majority of the Acinetobacter isolates cultured Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 4 of 8 Walter Reed Army Institute of Research (WRAIR),
Walter Reed Army Medical Center and LRMC. Some of
the samples processed were sent to BAMC and WRAIR
for comparison with the samples of all participating
military treatment facilities. Acinetobacter infections
A
b l Luther Lindler, the GEIS Acinetobacter Surveil-
lance Initiative, established standard operating proce-
dures for PFGE of Acinetobacter species among
laboratories at Brooke Army Medical Center (BAMC), Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 5 of 8 aminoglycosides and, to a lesser extent, carbapenems. Identifying the most prevalent resistance mechanisms,
optimal susceptibility testing and judicious use of anti-
microbial agents may help preserve the last remaining
agents with activity against multidrug-resistant bacteria. Overall these projects have enabled BAMC to leverage
its various programs to collaborate with internal and
external partners to improve treatment of combat-
related injury infections and multi-drug resistant infec-
tions by assessing local delivery of antibiotics in animal
models and characterizing novel resistant bacteria. It has
also enabled better characterization of multi-drug-
resistant infection rates throughout the military health
care system from point of injury through tertiary care
referral hospitals in the U.S. through AFHSC-GEIS and an Infectious Disease Clinical
Research Program-sponsored project of chlorhexidine-
impregnated cloths to prevent skin and soft tissue infec-
tions in U.S. Marine officer candidates. Additionally, they
performed resistance and virulence factor gene analysis
for Klebsiella pneumonia and developed and analyzed
numerous multidrug-resistant pathogens and possible
outbreaks including analysis of MRSA isolates recovered
from the U.S. Army Institute of Surgical Research burn
unit during the past 25 years [19,20]. aminoglycosides and, to a lesser extent, carbapenems. Identifying the most prevalent resistance mechanisms,
optimal susceptibility testing and judicious use of anti-
microbial agents may help preserve the last remaining
agents with activity against multidrug-resistant bacteria. Overall these projects have enabled BAMC to leverage
its various programs to collaborate with internal and
external partners to improve treatment of combat-
related injury infections and multi-drug resistant infec-
tions by assessing local delivery of antibiotics in animal
models and characterizing novel resistant bacteria. It has
also enabled better characterization of multi-drug-
resistant infection rates throughout the military health
care system from point of injury through tertiary care
referral hospitals in the U.S. Further studies at BAMC evaluated the resistance
mechanisms of Enterobacteriaceae as well as extended-
spectrum beta-lactamase-producing Klebsiella isolates
and their impact within a burn unit. They also studied
the efficacy of topical agents for Klebsiella, Pseudomo-
nas, MRSA, and Acinetobacter baumannii-calcoaceticus
complex in the burn unit [21,22]. These projects
included the study of multidrug-resistant Klebsiella,
Acinetobacter baumannii-calcoaceticus, and Pseudomo-
nas infections over time within a patient and between
patients. Acinetobacter infections
A
b l A major focus of the BAMC group is to further
characterize antimicrobial activity against Acinetobacter
baumannii-calcoaceticus complex [23]. Antimicrobial resistance in military trainee populations
Another ongoing AFHSC-GEIS-supported activity at
Naval Health Research Center (NHRC), San Diego,
characterizes the clinical isolates of Streptococcus pyo-
genes from U.S. military basic trainees [24]. Group A
S. pyogenes (GAS) infections are common in young
adults and may present clinically as pharyngitis, scarlet
fever or invasive disease. GAS is also associated with
post-infectious sequelae, including rheumatic heart dis-
ease and glomerulonephritis. Acute GAS infections
remain susceptible to penicillin but resistance to macro-
lide antibiotics has been noted in recent years. Clinical laboratory testing methods and broth microdi-
lution were used to define the susceptibility phenotypes
of 107 single-patient isolates from blood and wound
infections to 15 antimicrobial agents. Genetic relation-
ships were determined by PFGE, and isolates were
screened for selected resistance determinants. The iso-
lates were resistant to an average of nine agents and
four antimicrobial classes, with 92 percent meeting a
definition of multidrug-resistance. Antibiotics are frequently used for prophylaxis of
recruits against infections; therefore characterization of
GAS isolates is necessary in these populations. Ongoing
surveillance since 1998 has demonstrated continued sus-
ceptibility to penicillin and low-level resistance to
macrolides and other antibiotics in GAS isolates col-
lected at nine recruit training sites. The most active agents were colistin (MIC90 0.5 µg/mL,
99 percent susceptible) and minocycline (MIC90 4 µg/mL,
90 percent susceptible). Carbapenems, traditionally reserved
for multidrug-resistant infections, were relatively inactive
(imipenem MIC90 0.5 µg/mL, 38 percent S). Fifty-two per-
cent of isolates carried the OXA-23 carbapenemase, which
substantially degraded the activity of imipenem (78.4 per-
cent S without, versus 1.8 percent S with OXA-23 present). Rifampin has promising in vitro activity (MIC90 4 µg/mL);
however, no susceptibility breakpoints have been defined. Aminoglycosides also had very limited activity (amikacin
MIC90 ≥256 µg/mL, 16.8 precent S; gentamicin MIC90
≥32 µg/mL, 4.7 percent S; tobramycin MIC90 ≥32 µg/mL,
27.1 percent S). Aminoglycoside-modifying enzymes were
heterogeneous in these isolates, and poorly predictive of the
aminoglycoside susceptibility phenotype. Macrolide resistance is of particular concern because
this class of antibiotics is often used for prophylaxis and
treatment of individuals who are allergic to penicillin. NHRC tested 2,837 GAS isolates from recruits since the
study’s inception in 1998. Acinetobacter infections
A
b l Among 240 isolates collected
in 2009, in comparison with previous annual studies,
lower resistance was seen with erythromycin (6.6 per-
cent), while higher resistance was seen for tetracycline
(7.0 percent), clindamycin (4.3 percent), and levofloxacin
(6.5 percent). Higher levofloxacin resistance was seen in
2009 at Marine Corps Recruit Depot, Parris Island
(South Carolina) and higher clindamycin, erythromycin
and tetracycline resistance was seen at Fort Benning
(Georgia). Additionally, M protein gene (emm) typing of
S. pyogenes performed by NHRC has demonstrated asso-
ciations of certain emm types to resistance and viru-
lence. Other emm types have been shown to be more
likely associated with outbreaks among U.S. military
trainees. Nearly half (49.5 percent) of the isolates carried the
class 1 integron, a marker for the acquisition of cassettes
containing multiple antimicrobial resistance genes. Of
107 isolates, 106 (99 percent) carried at least one resis-
tance determinant. Significant inaccuracies were found
in some clinical testing methods for tetracyclines, Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 6 of 8 member medical encounters between October 2006 and
May 2008. Monitoring high-risk populations such as recruits for
emergence of potentially virulent strains could lead to
early interventions that may prevent outbreaks and
reduce morbidity. The most common emm gene types
among trainees were 3, 5, 44, 6 and 75. Emm type
75 was associated with increased levels of erythromycin
resistance, but without apparent increased virulence. Emm type 5 was less common but is one of the poten-
tially more virulent strains and was implicated in several
outbreaks in 2006 and 2007. A high degree of correla-
tion exists in the temporal distribution of strain patterns
between multiple sites suggesting a concerted strain
turnover pattern occurring on a larger scale. Seventy-four percent of cultured skin and soft tissue
infection cases were associated with S. aureus, followed
by coagulase-negative Staphylococcus (6 percent), E. coli
(2 percent), P. aeruginosa (1 percent) and Proteus mir-
abilis (1 percent). More than half of the S. aureus iso-
lates tested for oxacillin sensitivity were defined as
MRSA. MRSA isolates were not only resistant to oxacil-
lin but also to erythromycin (90 percent). Methicillin-
sensitive S. aureus (MSSA) isolates were sensitive to
other commonly-used antibiotics, including trimetho-
prim-sulfamethoxazole and vancomycin. Electronic surveillance of antimicrobial resistance A completely different approach using electronic data
sources for antimicrobial resistance surveillance was
undertaken by the Navy Marine Corps Public Health
Center (NMCPHC). NMCPHC developed algorithms
and tools to interpret Health Level 7 (HL7) data derived
from the DoD Composite Health Care System for sur-
veillance of diseases significant to public health [25]. Use of this data for the surveillance of antibiotic resis-
tance feeds data into BacLink and WHONET, tools
developed by the World Health Organization (WHO). Using its experience with inpatient and outpatient
encounter records, laboratory and pharmacy data, and
other medical and personnel databases, NMCPHC
explored trends in disease burden and antibiotic-
resistant microorganisms. The importance of electronic surveillance is demon-
strated further in the NMCPHC analysis of the study,
Acinetobacter species Infections: Trends in Active-Duty
Servicemembers. The investigation identified more than
6,300 Acinetobacter isolates found in 2,467 DoD active-
duty servicemembers between 2005 and 2008. Acineto-
bacter species isolates from wound specimens made up
34 percent of active-duty servicemember isolates
(n=2,138) and showed levels of susceptibility between
45 percent and 80 percent to all of the commonly pre-
scribed antibiotics reviewed throughout the study time
period; 95 percent of these isolates were Acinetobacter
baumannii-calcoaceticus complex [25]. The DoD-wide HL7 electronic microbiology labora-
tory data were restructured to rapidly identify and
monitor emerging antimicrobial resistance in organisms,
such as A.baumannii, K. pneumoniae, P. aeruginosa and
other pathogens of public health concern. The HL7 data
stream provides an opportunity for active surveillance of
trends in antibiotic resistance and near-real-time
response to significant health threats, especially in high-
risk populations. Isolates identified from blood specimens made up
6.5 percent of all active-duty service member isolates
(n=409). Overall susceptibility of these isolates to amika-
cin was 36 percent, imipenem susceptibility was 57 per-
cent and colistin susceptibility was 33 percent. Meropenem results for these isolates were quite limited,
with only 15 isolates tested and an overall susceptibility
of 7 percent [25]. These capabilities were used to enhance surveillance
and understanding of trends in emerging pathogens and
antibiotic resistance as well as to answer requests for
information about invasive MRSA and S. pneumoniae in
the Military Health System beneficiary population and
describe skin and soft tissue infections among DoD
members. More than 175,000 skin and soft tissue cases
were identified among DoD active-duty military service A similar electronic study of upper respiratory infec-
tions and antibiotic resistance among U.S. Acinetobacter infections
A
b l HL7 outpatient
pharmacy data showed that the antibiotics used to treat
both types of infection were very similar; trimethoprim-
sulfamethoxazole was prescribed frequently for both
MRSA (67 percent) and MSSA (52 percent). These results demonstrated the sensitivity and specifi-
city of a recently developed, rapid, high-throughput
strain identification technology and provided the basis
for a method of rapid inferential prediction of clinically
relevant characteristics using rapid strain typing meth-
ods. This surveillance also provided information on cir-
culating strains for GAS vaccine development initiatives. p
p
The first iteration of an NMCPHC antimicrobial-
resistant organism surveillance website displays antibio-
grams and high-profile organism counts, as well as similar
data broken out by service and region [26]. The project
includes the use of WHONET to generate facility-specific,
DoD-wide and regional antibiograms for comparison and
assessment of trends external to their own patient popula-
tions. The methodology used to restructure antimicrobial
data was validated by comparing the sensitivity profiles
prepared by military treatment facilities to the NMCPHC
sensitivity profiles from the restructured data. The valida-
tion process is ongoing. Electronic surveillance of antimicrobial resistance Navy recruits
with upper respiratory infection-associated medical
encounters
and
microbiology
records
document Page 7 of 8 Page 7 of 8 Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 isolation of Streptococcus in 92 percent and P. aerugi-
nosa in 3 percent of 1,022 laboratory specimens [25]. analyzed for antimicrobial resistance. The rate and
spread of antimicrobial resistance can be tracked and
helps to direct patient care, antibiotic prescribing prac-
tices and national policy. Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Published: 4 March 2011 Conclusion Infectious diseases have always been a major threat to
U.S. military forces and global public health. Antimicro-
bial resistance surveillance has been a pillar of military
force health protection and global public health for
AFHSC-GEIS since its creation in 1998. AFHSC-GEIS
funding has enhanced the ability of partner laboratories
to maintain robust infectious disease surveillance. Acknowledgements
Th
h
i h The authors wish to thank the numerous individuals who perform
surveillance as part of the AFHSC-GEIS global network, including all
individuals in the Ministries of Health and Ministries of Defense of partner
nations whose efforts have contributed to the success of the network. The
opinions stated in this paper are those of the authors and do not represent
the official position of the U.S. Department of Defense. This article has been published as part of BMC Public Health Volume
11 Supplement 1, 2011: Department of Defense Global Emerging Infections
Surveillance and Response System (GEIS): an update for 2009. The full
contents of the supplement are available online at http://www. biomedcentral.com/1471-2458/11?issue=S2. The creation of an Antimicrobial-Resistant Organism
Steering Committee is planned for fiscal year 2011 and
will help develop a unified global surveillance plan to
combat this common enemy. Increased and sustained
surveillance capabilities that can rapidly identify genetic
and phenotypic patterns of resistance are essential tools
for surveillance in the ever-changing field of microbiol-
ogy and antimicrobial resistance. Author details
1 1Armed Forces Health Surveillance Center, 11800 Tech Rd, Silver Spring, MD
20904, USA. 2Armed Forces Research Institute of Medical Sciences, 315/
6 Rajavithi Road, Bangkok, Thailand 10400. 3Brooke Army Medical Center,
3871 Roger Brooke Drive, Fort Sam Houston, TX 78234-6200, USA. 4Landstuhl
Regional Medical Center, Department of Immunology, CMR 402, Box 483,
APO AE 09180, USA. 5Naval Health Research Center, 140 Sylvester Road, San
Diego, CA 92106, USA. 6National Naval Medical Center, 8901 Wisconsin
Avenue, Bethesda, MD 20889, USA. 7U.S. Army Medical Research Unit-Kenya,
U.S. Embassy, ATTN: MRU, United Nations Avenue, Post Office Box 606,
Village Market, 00621 Nairobi, Kenya. 8U.S. Navy and Marine Corps Public
Health Center, 620 John Paul Jones Circle, Suite 1100, Portsmouth, VA 23708,
USA. 9U.S. Naval Medical Research Unit Number 2, Kompleks Pergudangan
DEPKES R.I., JI. Percetakan Negara II Number 23, Jakarta, 10560, Indonesia. 10U.S. Naval Medical Research Unit Number 3, Extension of Ramses Street,
Adjacent to Abbassia Fever Hospital, Postal Code 11517, Cairo, Egypt. 11U.S. Naval Medical Research Center Detachment, Centro Medico Naval “CMST,”
Av. Venezuela CDRA 36, Callao 2, Lima, Peru. 12Walter Reed Army Medical
Center, 6900 Georgia Avenue Northwest, Washington, DC 20307, USA. The ability to track antimicrobial resistance in organ-
isms causing disease in DoD beneficiaries is essential for
infectious disease and public health leaders to formulate
policy and determine appropriate actions for mitigation. AFHSC-GEIS will continue to fund studies that increase
its knowledge of the forces and mechanisms that create
resistant organisms so that the battle against antimicro-
bial resistance can be waged more effectively. Future direction and initiatives To better understand the global picture of infectious
disease, it is essential to use standardized nomenclature
and laboratory procedures in order to correctly identify
the causative microorganisms. Additionally, the various
reports must be collated if the data is to be relevant to
DoD populations spread around the world. AFHSC-
GEIS-funded partners have continued to contribute
greatly to essential research and development of techni-
ques that further the investigation of disease causing
organisms. Rapidly changing technologies and computer
applications speed the process of analysis of the organ-
isms and allow more educated and informed policies
and interventions to better treat and prevent infectious
disease threats. This report has provided a synopsis of
recent antimicrobial resistance surveillance accomplish-
ments achieved by AFHSC-GEIS partners. Since the inception of DoD-GEIS and now AFHSC-
GEIS, multiple partners have studied various aspects of
antimicrobial resistance with a multitude of methods. This section documents the 2009 partner accomplish-
ments and activities. The increasing prevalence of emerging antimicrobial-
resistant infections remains one of the greatest threats to
global health and will continue to be a major concern for
AFHSC-GEIS. To successfully counter this threat, the
global network will need to be united and coordinated. The three-year GEIS Acinetobacter Surveillance Initiative
demonstrated the value of standardized operating proce-
dures and central specimen archives in expanding the
knowledge base for these unique, but all too common,
infections. AFHSC-GEIS-funded partners will need to
develop closer collaborations to best understand the var-
ious components involved with global antibiotic-resistant
organism surveillance. AFHSC-GEIS has begun initiatives
such as subject matter expert steering committees to bet-
ter direct and coordinate funded proposals as a means of
identifying surveillance gaps, avoiding redundancies and
assuring state-of-the art technologies. References
d NAMRU-3 Annual Report: Establishment of a Vibrio cholerae and rotavirus
microbiology and molecular biology reference center for the Middle
East and Africa. GEIS Ops Proposal C0071_09_N3 . 6. OEF, OIF: [http://siadapp.dmdc.osd.mil/personnel/CASUALTY/oefwia.pdf],
h
// i d
d d
d
il/
l/CASUALTY/ if
d d
l df p
p
6. OEF, OIF: [http://siadapp.dmdc.osd.mil/personnel/CASUALTY/oefwia.pdf],
http://siadapp.dmdc.osd.mil/personnel/CASUALTY/oif-wounded-total.pdf. 24. Metzgar D, Baynes D, Hansen CJ, McDonough EA, Cabrera DR, et al:
Inference of Antibiotic Resistance and Virulence among Diverse Group A
Streptococcus Strains Using emm Sequencing and Multilocus
Genotyping Methods. PLoS ONE 2009, 4(9):e6897. 7. Talaat M, Hafez S, Saied T, Elfeky R, El-Shoubary W, Pimentel G: Surveillance
of catheter-associated urinary tract infection in 4 intensive care units at
Alexandria university hospitals in Egypt. Am J Infect Control 2010,
38(3):222-228, Epub 2009 Oct 17. 25. NMCPHC Annual Report: Methods for characterization of antimicrobial
resistance using electronic databases. GEIS Ops Proposal C0095_09_NE . p
8. NAMRU-3 Annual Report: Project Title: Molecular characterization of
Extended Spectrum Beta-lactamase (ESBL) Gram-negative rods and
Acinetobacter baumannii from Egypt and Jordan. GEIS Ops Proposal
C0041_09_N3 . 26. NMCPHC Annual Report: Rapid Response System for Early Identification
of Emerging Pathogens and Antimicrobial Resistant Organisms. GEIS Ops
Proposal C0044_09_NE . doi:10.1186/1471-2458-11-S2-S8
Cite this article as: Meyer et al.: Antimicrobial resistance surveillance in
the AFHSC-GEIS network. BMC Public Health 2011 11(Suppl 2):S8. 9. NAMRU-3 Annual Report: Project Title: Surveillance of Healthcare-
acquired Infections (HAI) and Antimicrobial Resistance (AMR) in Egypt
and Jordan. GEIS Ops Proposal C0002_09_N3 . 10. Murray CK, Yun HC, Griffith ME, Thompson B, Crouch HK, Monson LS,
Aldous WK, Mende K, Hospenthal DR: Recovery of multi-drug resistant
bacteria from combat personnel evacuated from Iraq and Afghanistan
at a single military treatment facility. Mil Med 2009, 174:598-604. 11. Akers KS, Mende K, Yun HC, Hospenthal DR, Beckius ML, Murray CK:
Tetracycline susceptibility testing and resistance genes in isolates of
Acinetobacter baumannii-calcoaceticus complex from a U.S. military
hospital. Antimicrob Agents Chemoth 2009, 53:2693-2695. 12. Keen EF 3rd, Murray CK, Robinson BJ, Hospenthal DR, Co EM, Aldous WK:
Changes in the incidences of multidrug-resistant and extensively drug-
resistant organisms isolated in a military medical center. Infect Control
Hosp Epidemiol 2010, 31(7):728-732. p p
13. LRMC Annual Report: Multi-Drug Resistant Organism (MDRO) Surveillance
in the EUCOM AOR. GEIS Ops Proposal C0055_09_MC . 14. References
d Huang XZ, Frye JG, Chahine MA, Cash DM, Barber MG, Babel BS, Kasper MR,
Whitman TJ, Lindler LE, Bowden RA, Nikolich MP: Genotypic and
Phenotypic Correlations of Multidrug Resistant Acinetobacter
baumannii- calcoaceticus Complex Strains Isolated from Patients at the
National Naval Medical Center. J Clin Microbiol 2010, [Epub ahead of print]. 15. Ressner RA, Griffith ME, Beckius ML, Pimentel G, Miller RS, Mende K,
Fraser SL, Galloway RL, Hospenthal DR, Murray CK: Antimicrobial
susceptibilities of geographically diverse clinical human isolates of
Leptospira. Antimicrob Agents Chemother 2008, 52:2750-2754. 16. Murray CK, Pimentel G, Parker T, Beckius ML, Samir A, Rhman BA, Mende K,
Galloway RL, Hospenthal DR: Antimicrobial susceptibility of clinical human
isolates of Leptospira from Egypt. In Am J Trop Med Hyg. Volume 79. 57th
Annual Meeting of the American Society for Tropical Medicine and
Hygiene: 7-11 December 2008; New Orleans, LA; 2008:(Suppl):78. 17. Hinkle MK, Green JA, Martin GJ, Kochel TJ, Hall ER, Villaran M, Garcia J,
Scott P, Bautista CT, Sateren WB, Gray M, Murray CK, Hospenthal DR,
Maves RC: Serosurvey of leptospirosis in Peruvian military personnel
deployed to Haiti. In Am J Trop Med Hyg. Volume 79. 57th Annual Meeting
of the American Society for Tropical Medicine and Hygiene: 7-11 December
2008; New Orleans, LA; 2008:(Suppl):79. Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at
www.biomedcentral.com/submit Submit your next manuscript to BioMed Central
and take full advantage of: 18. Robertson JL, Becker SJ, Yu X, Hawley JA, Griffith ME, Beckius ML,
Hospenthal DR, Mende K, Murray CK: Detection of leptospiral DNA from
inoculated blood and urine samples using five PCR primers. In Am J Trop
Med Hyg. Volume 79. 57th Annu Mtg Am Soc Trop Med Hyg, New Orleans,
LA, 7-11 December 2008; 2008:(Suppl):80. 19. References
d A significant spin-off of these efforts is the acquisition
of multiple isolates of microorganisms infecting patients
from various regions of the world which can be further 1. Tjaniadi P, Lesmana M, Subekti D, Machpud N, Komalarini S, Santoso W,
Simanjuntak CH, Punjabi N, Campbell JR, Alexander WK, Beecham HJ 3rd,
Corwin AL, Oyofo BA: Antimicrobial Resistance Of Bacterial Pathogens Meyer et al. BMC Public Health 2011, 11(Suppl 2):S8
http://www.biomedcentral.com/1471-2458/11/S2/S8 Page 8 of 8 Associated With Diarrheal Patients In Indonesia. Am J Trop Med Hyg 2003,
68(6):666-670. methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at a
burn center. Burns 2009, 35(8):1112-1117, Epub 2009 May 27. methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered at a
burn center. Burns 2009, 35(8):1112-1117, Epub 2009 May 27. p
y
21. Keen EF 3rd, Robinson BJ, Hospenthal DR, Aldous WK, Wolf SE, Chung KK, 2. Kasper MR, Sokhal B, Blair PJ, Wierzba TF, Putnam SD: Emergence of
multidrug-resistant Salmonella enterica serovar Typhi with reduced
susceptibility to fluoroquinolones in Cambodia. Diagn Microbiol Infect Dis
2009, Epub. 21. Keen EF 3rd, Robinson BJ, Hospenthal DR, Aldous WK, Wolf SE, Chung KK,
Murray CK: Prevalence of multidrug-resistant organisms recovered at a
military burn center. Burns 2010, 36(6):819-825, Epub 2010 Jan 18. Murray CK: Prevalence of multidrug-resistant organisms recovered at a
military burn center. Burns 2010, 36(6):819-825, Epub 2010 Jan 18. 22. Glasser JS, Guymon CH, Mende K, Wolf SE, Hospenthal DR, Murray CK:
Activity of topical antimicrobial agents against multidrug-resistant
bacteria recovered from burn patients. Burns 2010, [Epub ahead of print]
PMID: 20542641. 3. NAMRU-2 Annual Report: The identification of enteropathogens among
pediatric patients with acute diarrhea. GEIS Ops Proposal C0063_09_N2. 3. NAMRU-2 Annual Report: The identification of enteropathogens among
pediatric patients with acute diarrhea. GEIS Ops Proposal C0063_09_N2. 4. AFRIMS Annual Report: Outbreak and Contingency Fund. GEIS Ops
Proposal C0065_09_AF . pediatric patients with acute diarrhea. GEIS Ops Proposal C0063_09_N2. 4. AFRIMS Annual Report: Outbreak and Contingency Fund. GEIS Ops
Proposal C0065_09_AF . 23. Akers KS, Mende K, Yun HC, Hospenthal DR, Beckius ML, Murray CK:
Tetracycline susceptibility testing and resistance genes in isolates of
Acinetobacter baumannii-calcoaceticus complex from a U.S. military
hospital. Antimicrob Agents Chemoth 2009, 53:2693-2695. 5. NAMRU-3 Annual Report: Establishment of a Vibrio cholerae and rotavirus
microbiology and molecular biology reference center for the Middle
East and Africa. GEIS Ops Proposal C0071_09_N3 . 5. References
d BAMC Annual Report: Continued development of a multiply-drug
resistant (MDR) bacteria molecular epidemiology referral laboratory. GEIS
Ops Proposal C0096_09_MC . 19. BAMC Annual Report: Continued development of a multiply-drug
resistant (MDR) bacteria molecular epidemiology referral laboratory. GEIS
Ops Proposal C0096_09_MC . 20. Murray CK, Holmes RL, Ellis MW, Mende K, Wolf SE, McDougal LK,
Guymon CH, Hospenthal DR: Twenty-five year epidemiology of invasive 20. Murray CK, Holmes RL, Ellis MW, Mende K, Wolf SE, McDougal LK,
Guymon CH, Hospenthal DR: Twenty-five year epidemiology of invasive
|
https://openalex.org/W4319211063
|
https://www.tu.edu.ye/journals/index.php/arts/article/download/258/170
|
Arabic
| null |
مسائل الاشتغال في النحو لابن هشام الأنصاري (761 هـ) - دراسة وتحقيق
|
Al-ādāb. Li-l-dirāsāt al-luġawiyyaẗ wa-al-ādābiyyaẗ
| 2,021
|
cc-by
| 30,732
|
مسائل االشتغال في النحو ال
بن هشام األنصاري (
761
هـ)
دراسة وتحقيق .د حسان بن عبد هللا الغنيمان*
halghonaiman@ksu.edu.sa مسائل االشتغال في النحو ال
بن هشام األنصاري (
761
هـ)
دراسة وتحقيق
.د حسان بن عبد هللا الغنيمان*
halghonaiman@ksu.edu.sa *
رحهاذ اللغق الند ا اوك-
ُسر اللغق العتبيق آداب ا-
كليق اآلدا-
باتعق ا لك حع د–
.ا مل ق العتبيق السع ديق * Associate Professor of Language and Syntax - Department of Arabic Language and Literature - College of Arts - King Saud
University - Saudi Arabia. امللخص: امللخص: امللخص: يتنااا ه ااالا البدااسة دواحااقَ سدئياااَ تساااالت اأوااهغاه ال ااف ر وداااا اباانة ع اا و(
669
ه) فاا
( آخااااااااات باااااااااا اأواااااااااهغاه تااااااااان هابااااااااا ا ئااااااااات ت وَ اااااااااتَحَ َا ابااااااااانة ا اااااااااا ا ااااااااااو761
ه) فااااااااا اااااااااال
ْالتحالق. سضااام نَ ُسااارة الدواحاااق ةً اااا دواحاااقْ تااا بنِْ فل َ ًو َااااش ابااانت ع ااا و ابااانت ا اااا دواحاااق
ت لق للتحالق.تَ و تَحة ال التحالقة ال ُّ َوَ ال ف يأتي علً ا ال اغ لة تا يناهةُة عه اا تان تغٍُّاي فا ح ارت
حَ مو االت ا ااغ ه عنااا عغاا ال اااغل تاااا يحيس ااع علياا تااان ح اار ُعتاِااي للم اااغ ه عناا تة بهدت اَااقْ بماااا
كان ةي ال اغلة ويئْا احدْ ا ثر تا كان ةي ال اغل ويئٍن عغ سن ُّع ما بٍن حَ بَبٍَونت ر ضامٍيَيونت
ر ضاامٍي حَ اابٍَّت ف عغاا
سناا ُّا الضاامٍي تااا بااٍن ته اال تن اال تااا بااٍن تتةاا ا تن اا . ا ه ماا
ااال ال اا و فاا ع اات تساااال سضاامُّ ا-بدسااع ح اار ا عماال عغاا ال اااغل- تجم عهااان تتسااا :هانش
خمس
لك ا خياو ف ا عمل عغ ر ال اغلٍَونت ا لك وَ :ونت عغ اخهالة ماا خماس يجاع ةً اا ا عمال
عغ رحد ال اغلٍَونت
.ا لك وَ :ونت :الكلمات املفتاحية تساال
؛ اأوهغاه
؛
ا ئت ت ؛ الند. 40 Questions of 'AL-Ishtighaal' in Grammar fessor of Language and Syntax - Department of Arabic Language and Literature - College of Arts - King Saud University - Saudi Arabia. Abstract: This research deals with an investigational study of the manuscript on the topic of
(Ishtighaal) issues reported by Ibn Asfour (669 AH) in his book (Al-Muqarreb) and
elucidated by Ibn Hisham (761 AH.) here in this manuscript/letter. A section of the work
includes a brief study on the authors, Ibn Asfour and Ibn Hisham, and a detailed study
on the manuscript, explaining the forms the occupier/subject element (Al-Shaaghil)
takes, the change that occurs due to treating the occupied/object element (Al-
Mashghoul) as an occupier/subject pronoun (Al-Shaaghil), and its syntactic case. We
first examined the case in which the occupier/subject pronoun was one element, and
then the case in which the occupier/subject pronoun consisted of two elements, taking
into account different types of cases: pronoun vs. causal, free-morpheme pronouns vs. bound morpheme, and nominative vs. accusative. The ten forms have included ten
issues that can be divided into two equal groups; in five of which you can assign the
syntactic case to either occupier equally regardless of the differences between the two
types, while in other five of which you must assign the syntactic case to only one
occupier. Key Words: Issues, Topic, Al-Muqarreb, Al-Shaaqil, Grammar. 41 املقدمة: املقدمة: لئد اي أ هللا خدتق لغق هاب دين علماءَ رةلاذْا عغ تَ ت ت الع و ةبلل ا ب دار
ِ ر س ر ف الهعلير الهأليف ةأل ا ة هةبْا ذات تناهج تهعد ت د وحاال ذات ت ض عات
( تهن تعق كان تن بٍن افأء العلماء ابنة ع و669
ه) الل رل ف هاب الند ا و
( "ا ئت ت " ابنة ا ا ا او761
ه) الل واوك ف سآليف تهن عق كان تن بيه ا ال
.التحالق ال ف رةَُد ت ت ا الي للئة ت اء سَتَنا ه ال التح
الق ت
سألق تن تساال اأوهغاه ا
البا الند ا و الل
تعد دت اآلواء ح ل َ ثةيَ ا جدهة ةي تن ع و الند ا هئد ت تق ُل ي تنا ا عاضت. ال ا سألق
ر وداا ابن ع و ف آخت با اأوهغاه تن هاب ا ئت ت ذ ت ةً ا ال و ال ف يأتي علً ا
ال اغل ةَمَ نو عتف ا ل ال و رَلَر ب ا رحاط ببا اأوهغاه. Abstract: ُد رسئن ابن ع و سألي ا
ستسيب ا سن يم ا؛ تما يةنٍّئ عن عئلي ه الت:اضي ق ال ل ِ لللك دو تَ نو صاغ ال ا سألق عغ
تن ال ُد باء بعد ابن ا ا ةأَ وضَ عَ ا ةَ لَ ا رَبَانَ َا ةختب بدة ل ق وائعق ت د ب بياعق
تفل ا َ وئ ف العلر ُد ُد تو ة لهدئيا ال التحالق بدواحق تة َ لَق عه ا ت عق بدواحق ت بنِ فل ا
ابن ا ا صاحع ا حن ابن ع و. اأ لا رُد ت ت ا للئاوئ ال ت:ر وابيا رن سناه احهدسا
ةما كان ةً ا تن ص ا ةمن هللا تا كان ةً ا تن خَ لَل
ةمن س ف ال يطان حسٍّف رني
.ابت دو تة ف سدئيئ ا بللو ة ُ اوى ب د ف ذلك 42
لر يخلة سدئيا ال التحالق تن صع بات غٍي تن ا عماه كان تن ربتز ال ع بات
ال ف
اب هنف
صع بق ا ع ه عغ نسخ ال التحالق اأخهالةات ال ثٍيِ بٍن نةسَ خت ال
التحالق؛ تما يسهلن الحييُّس ط ه البدس لل ص ه ُل اخهياو ا فلف ةً ا ُضاةق ُل ُتل ق عَتوض لر يخلة سدئيا ال التحالق تن صع بات غٍي تن ا عماه كان تن ربتز ال ع بات
ال ف
اب هنف
صع بق ا ع ه عغ نسخ ال التحالق اأخهالةات ال ثٍيِ بٍن نةسَ خت ال
التحالق؛ تما يسهلن الحييُّس ط ه البدس لل ص ه ُل اخهياو ا فلف ةً ا ُضاةق ُل ُتل ق عَتوض 42 ال ا سألق ف هع النداِ عغ ال وِ ال ف عتض ا ابن ع و. Abstract: ب ضل تتنَ هللا تغل بو ة عغ
ال ال ع بات ةخَ تَبَ ت التحالق هلل ا عمد–حسع تتي-
.ب وِ بميلق تَ توضت ي ق خهاتا رو ت كل تن رعا نف عغ سدئيئ ا ُد ل يد الع ن ا ساعدِ رخصُّ بال ت
الد ه و بابت بن عبد هللا الستي تع الل روودني ُل ال التحالق ز دني ببعض نسخ ا ا حهاذ
عم ا و سمال
الباحس ف تت ن ا لك ةي ل الل ز دني ببعض نسخ التحالق أ رنس ى غٍيار
تمن كان ل ر ال ضلة ف ُعا ف ف سدئيا ال التحالق ةللجميع تنف بن:ل ال ت الهئديت
ْ رحأه هللا الع ير رن يجعل عمغ خال ْا ل ب ال ت:ر ا عمد هلل و ت العا ٍن ر أْ آخت.ا :تعريف موجز بابن هشام ا ال يخة اإلتا ة بماهة الد ت ين رب تدمد عبد هللا بن بماه الدين ية حف بن عبد هللا بن
ية حف بن رحمد بن عبد هللا بن ا ا الن د ا او ال اةع ثر ا عنبغ(1)
ِ لد ف الئاات
ف ا خاتس تن ذ الئعدِ حنق708
ه سئت:با. ب ا ن أ ط لع العلر ةً ا تنل صغت ةالز
( و ا الدين عبد اللطيف بن عبد العن:ن بن الامة تَح ت ل744
ه) دَوَسَ عغ ساج الدين عمت بن
( عغ ال ا ت َانتي731
( ه) تدمد بن ُبتااير بن حعد بن بماعق ال ت نَانتي733
ه) ومس الدين
( تدمد بن تدمد بن تدمد بن مٍي بن الستاج747
ه
) ساج الدين عغ بن عبد
هللا ا ودبيغ
( اله بويينت746
( ه) ويخ اإلحال سئي الدين عغ بن عبد الكاف السُّ بوكتي755
.)ه
رسئن العتبيق ة اق ا ُتان كان ُتاتْ ا ةً ا ل ا عتةق الهاتق ف الئتاءات ا عديس
ال ئ . دَو س رةاد سخت ج ب بماعق تن رال الدياو ا ت:ق تن رال ت ق َ ا با و ب ا رُتر
. ها حيب : عد ِ تتات
(
)
(
) ا ال يخة اإلتا ة بماهة الد ت ين رب تدمد عبد هللا بن بماه الدين ية حف بن عبد هللا بن
ية حف بن رحمد بن عبد هللا بن ا ا الن د ا او ال اةع ثر ا عنبغ(1)
ِ لد ف الئاات
ف ا خاتس تن ذ الئعدِ حنق708
ه سئت:با. ب ا ن أ ط لع العلر ةً ا تنل صغت ةالز
( و ا الدين عبد اللطيف بن عبد العن:ن بن الامة تَح ت ل744
ه) دَوَسَ عغ ساج الدين عمت بن
( عغ ال ا ت َانتي731
( ه) تدمد بن ُبتااير بن حعد بن بماعق ال ت نَانتي733
ه) ومس الدين
( تدمد بن تدمد بن تدمد بن مٍي بن الستاج747
ه
) ساج الدين عغ بن عبد
ُهللا ا ودبيغ (تج
ٍي بن
بن
بن
بن
بن ب
ين
) ج هللو بي
( اله بويينت746
( ه) ويخ اإلحال سئي الدين عغ بن عبد الكاف السُّ بوكتي755
.)ه
رسئن العتبيق ة اق ا ُتان كان ُتاتْ ا ةً ا ل ا عتةق الهاتق ف الئتاءات ا عديس
ال ئ . :تعريف موجز بابن هشام دَو س رةاد سخت ج ب بماعق تن رال الدياو ا ت:ق تن رال ت ق َ ا با و ب ا رُتر
. ها حيب : عد ِ تتات ةصت ف بأ ة يحة زتا حيب : ريات(2). ُاه عن ابن حجت(3) ش"
َا و َ تَدَ بال ااد الغت:قب
ا باحس الدُيئق اأحهدواكات العجيبق َاله دو ئتيا الوبَالتغ اأط ت الا ا تتط اأ ُهداو عغ
اله َتُّف ف الو َ الَ ا و َلَ َ ق ال ف كَانَ يهَمَ ن ب ا تن اله عوبتٍي عن تَ ئو ة د بما يةتت:د تة سو تبْا تة بت نْا 43 تَ عَ اله َاضةع َالوبتي ت ال َ ئَ ق دتاثق ا خة لةا َوتُ ق الئلع. ُاه لنا ابن خلد نش تا زلنا دن با غت
نسمع ر : ظ ت بم ت عالر بالعتبيق يئاه ل ابن ا ا ر ح تن حيب"
. َكَانَ تة و ثتيْا تن الد ت يَا َق
.َِ َالوعتبَاد تَ عَ اله َاضةع َالوبتي ت ال َ ئَ ق دتاثق ا خة لةا َوتُ ق الئلع. ُاه لنا ابن خلد نش تا زلنا دن با غت
نسمع ر : ظ ت بم ت عالر بالعتبيق يئاه ل ابن ا ا ر ح تن حيب". َكَانَ تة و ثتيْا تن الد ت يَا َق
.َِ َالوعتبَاد ( كَانَ ثٍي ا و ةخَ ال َ ق للعال تق رِي حَ ي ان ا دلس ف745
ه) وَ دت يد اأ دتاف عَن ة لعل الا
( يع د ُل تا ذ ت ال كاني1250
)ه(4) ش"
تتنو رن رَبا حَ ي ان كَانَ تة نو َ تتدْا بت َلَ ا الو َ ن ت ف ذلك الوعَ و ت
غٍي تة دَ اةَع عن الس با ةي ثر كَانَ ا و ةنو َ تتدة بعد اة َ صَاحع الح يوبَ مَ ق–
ر ش ابن ا ا-
َ َ ثتٍيْا تَ ا
يةنَاةتسة التبة لة تَ نو كَانَ ُبل ف وسبه ال ف صاو ُلً ا؛ ُظ اوْا ل ضل س باأُهداو عغ تناحمه
ن كَانَ ُبل رَ بالهم ن تن الوبلة غ ُل تَ ا لر يبلغ ُلي ُأ ةَأَبة حَ ي ان اة َ تن اله مَ ُّ ن تن اَ لَ ا
الو َ ن بمَ كَان َلر ي ن للمهأخت:ن تثلَ ة َتثلَ صَاحع الح يوبَ مَ ق" . :تعريف موجز بابن هشام صَن فَ ابنة ا ا ف العتبيق غٍياا تفل ات
اةعق ته اش تغنى اللبيع عن هع ا عاو:ع
ا ها ت يد لر ية َ ن ف ف الند تثل اوت ت ف حياس ف ال ا ت ت اوهغل ب رال
الع ت صَن فَ ر ضح ا سالك ُل رل يق ابن تالك اله عولتيا عغ ت كل رل يق ابن تَ الك
حاويق عغ تس يل ال ااد ول و اللاع وتح ُطت الندى وتح اإلعتا
عن
ُ اعد اإلعتا وَ توحة الل مو دَ ق البَدو وت: ق ف علر العتبيق ل وحاال تهعد ت دِ ف ت ض عات ته ت تُق
غٍياا ثٍي(5). بميع ال ا ن ات طةبع تدئ ئق تا
عدا الهعليا عغ ت كل رل يق ابن تالك
.ةإ لر يةطبع حاويه عغ التس يل لر سةدئ ا . ل وعت ر
كا رحتسة ة رحتِْ علمي ق ةل ابنان تن العلماء بالعتبيق غٍيااش عبد التحمن
تدمد (
799
ه) َكَانَ رَ وحَ دَ ع ت ف
سدئيا الن دو يئاهش كان ر ح تن ربي(6). ل رح اد
للك ذ تار تدئ تا ا ج ات ا نضد(7). 44 ت بماه الدين بن ا ا
س ف –
وحم هللا- بعد عمت حاةل بالعطاء ف لَيولَق ا و جة مة عَق خاتس
ذ الئعدِ حنق761
.ه :تعريف موجز بابن عُصْفُ ور ُّ ا العال تق رب ا عسن عغ
بن تفتن بن تدمد بن عغ ا عضتتي اإلوبيغ ا عت ف بابن
عة و و(8)
.. ةلتدَ حنق حبع تسعٍن خمسمااق ف ُتوو بتيلتيَق با دلس
رخَ ل العتبي ق ا دَ عن ركابت العلماء تثل ويخ ا دلس العال تق رِي ا عسن عغ بن بابت
( الد ب اج اإلوبيغ646
ه) ثر عن ا( حهاذ رِي عغ عمتَ بن تة دَ م د اإلوبيغ ال لَ وبٍن645
)ه ُّ ا العال تق رب ا عسن عغ
بن تفتن بن تدمد بن عغ ا عضتتي اإلوبيغ ا عت ف بابن
عة و و(8)
.. ةلتدَ حنق حبع تسعٍن خمسمااق ف ُتوو بتيلتيَق با دلس
ا
غ
ا
ر
ال ال
ا ل
ل
ركا ال ل ا
ا
ال
ر َ رخَ ل العتبي ق ا دَ عن ركابت العلماء تثل ويخ ا دلس العال تق رِي ا عسن عغ بن بابت
( الد ب اج اإلوبيغ646
ه) ثر عن ا( حهاذ رِي عغ عمتَ بن تة دَ م د اإلوبيغ ال لَ وبتٍن645
)ه
أزت ُتابق ع تِ رع ا ةد ل تن تا لر يةد ت ل غٍي ،
كان تن ر جع تَ نو ُتر علي
. ربتع ر ربل ت ر ا حاتل ل اء العتبيق با دلس ف زتا بدتْا ةً ا وَ: ان تن ا د . كان ُتاتا حاة ا
تة هوئتنْا ة يدا أ ية َ اُّ غةباو أ يةجاوى يةئتتئ ال ة هةع ال باو ف العتبيق كان رصبي الن اس عغ
.ا طالعق أ يَمَ لُّ تن ذلك ِ اوسدل ُل عدِ تدن ف ا دلس ا غت ا جناات س نس س د و للهدويس ةً ا تد
ةأَُوبل عَلَيو ت الط لبَق ُتر علي خَ لوا ث ٍي ا ه ع ا ب ،
. كان يةمو غت تن صدو 45
ويس ً
ت جن ت س و
س
ن
و ل ُ
ةأَُوبل عَلَيو ت الط لبَق ُتر علي خَ لوا ث ٍي ا ه ع ا ب ،
. :تعريف موجز بابن عُصْفُ ور كان يةمو غت تن صدو
كان ويخَ السلطانت ا ستن تت باهلل رِي عبد هللا تدمد بن ز ت:ا ا ع ص ف ت صاحع س نس
.ثر بليس حينما كان ليًّا للع د ثر حلطا ا
ل سآليف تةعَدُّ تن رحسن اله ا يف التة ة ةً ا حَ ول تة نوسَ بتك تة دَ ل ته اش
ا و ةئَ ت ت ف الند ا تن رار آثاو ال ف حازت و تِ وَ توحة لر يةهتم ا مهع ف
ال تف ضتاات ال عت وَ توحة ها َ حت يب َ:و،
ل ثالثق وت ح عغ بمل النباج وَ توحة ربيات
اإليضاح تخه ت الغةت ِ أبن الد ا ان تخه ت ا دتسع أبن بنف. ل وت ح لر ية و مت لو ا ته اش كان ويخَ السلطانت ا ستن تت باهلل رِي عبد هللا تدمد بن ز ت:ا ا ع ص ف ت صاحع س نس
.ثر بليس حينما كان ليًّا للع د ثر حلطا ا
ل سآليف تةعَدُّ تن رحسن اله ا يف التة ة ةً ا حَ ول تة نوسَ بتك تة دَ ل ته اش كان ويخَ السلطانت ا ستن تت باهلل رِي عبد هللا تدمد بن ز ت:ا ا ع ص ف ت صاحع س نس
.ثر بليس حينما كان ليًّا للع د ثر حلطا ا 45
ل سآليف تةعَدُّ تن رحسن اله ا يف التة ة ةً ا حَ ول تة نوسَ بتك تة دَ ل ته اش
ا و ةئَ ت ت ف الند ا تن رار آثاو ال ف حازت و تِ وَ توحة لر يةهتم ا مهع ف
ال تف ضتاات ال عت وَ توحة ها َ حت يب َ:و،
ل ثالثق وت ح عغ بمل النباج وَ توحة ربيات
اإليضاح تخه ت الغةت ِ أبن الد ا ان تخه ت ا دتسع أبن بنف. :تعريف موجز بابن عُصْفُ ور ل وت ح لر ية و مت لو ا ته اش 45 وَ توحة اإليضاح لل اوس ف وَ توحة ا جن ليق وَ توحة ا وعاو السهق ا جااليق وَ توحة ا عماحق وَ توحة
.دي ان ا هنٍّف غٍي ذلك :نسبة الرسالة إلى ابن هشام كان ابن ا ا ا او-وحم هللا- صاحع س ا يف تهعد ت دِ وحاال ثٍيِ؛
تما بعل
ت ن ت
ي
هعت الحيابر أ يل ت ن كل وحاال ال ف رل ا ما ه عادت ر ته ا وحاله ف تساال
اأوهغاه يةفَ: تد الا تا ُال صاحع ها السعع ال ابلق ف ستبمه أبن ا ا ا او بعد
ذ ت تفل اس(9)ش "
تن س ا ي ريضاش«ر ضح ا سالك...» تن التحاال الض ابط ال ا اد
ش فء ثٍي
ح ىُ
ن تتاحالس ُل رصعاب أ يةخو لتً ا تن ة اادَ د : ق غت:بق ل رب بق ف العتبي ق
أ سةدص ى". 46
ذ ت ال يخ خالد ا زات ف اله ت:ح(
10
)
ِ السي طي ف بغيق ال عا(
11
)
ابن العماد
ا عنبغ ف ولوات اللاع(
12
)
."عددا تن تفل ات ابن ا ا ثر ُال اش "... غٍي ذلك
( وري اإلتا عمت بن َُدت يد بن عبد هللا الئَ لَمو طا الئاات ا عن ي856
)ه (
13
)
َنَسَ ع
ال التحالق أبن ا ا ف ستبمه ل ف حاويه عغ ر ضح ا سالك(
14
) . للك ُ عغ نسخق تن تغنف اللبيع(
15
)
نسخق تن ر
ضح ا سالك(
16
)
ذة تت ةً ما
ستبمق أبن ا ا ُد سضم ن ااسان الحيبمهان نسبق ال التحالق أبن ا ا ا او(
17
) . ُد رثبت الدواحق صع ق نسبق ال التحالق ال ف بٍن ريدينا أبن ا ا ا او ؛ لألدلق
اآلسيقش ذ ت ال يخ خالد ا زات ف اله ت:ح(
10
)
ِ السي طي ف بغيق ال عا(
11
)
ابن العماد
ا عنبغ ف ولوات اللاع(
12
)
."عددا تن تفل ات ابن ا ا ثر ُال اش "... غٍي ذلك ذ ت ال يخ خالد ا زات ف اله ت:ح(
10
)
ِ السي طي ف بغيق ال عا(
11
)
ابن العماد
ا عنبغ ف ولوات اللاع(
12
)
."عددا تن تفل ات ابن ا ا ثر ُال اش "... :تعريف موجز بابن عُصْفُ ور غٍي ذلك ( وري اإلتا عمت بن َُدت يد بن عبد هللا الئَ لَمو طا الئاات ا عن ي856
)ه (
13
)
َنَسَ ع
ال التحالق أبن ا ا ف ستبمه ل ف حاويه عغ ر ضح ا سالك(
14
) . للك ُ عغ نسخق تن تغنف اللبيع(
15
)
نسخق تن ر
ضح ا سالك(
16
)
ذة تت ةً ما
ستبمق أبن ا ا ُد سضم ن ااسان الحيبمهان نسبق ال التحالق أبن ا ا ا او(
17
) . ُد رثبت الدواحق صع ق نسبق ال التحالق ال ف بٍن ريدينا أبن ا ا ا او ؛ لألدلق
اآلسيقش 46 آ 1. نسبتة ا ل تع وحالق (ا ب بق عن ا ه ا "لغق ةضال
" د ا
م ا) ذلك عغ غالف
( نسخق ا زات:ق ذات التُر132295
د ) للك نسبت ا عغ غالف نسخق ا زات:ق
( ا ختى ذات التُر66565
د ) ُن كا النسبق ف ال النسخق ت ه بق بخط
.حديس 1. نسبتة ا ل تع وحالق (ا ب بق عن ا ه ا "لغق ةضال
" د ا
م ا) ذلك عغ غالف
( نسخق ا زات:ق ذات التُر132295
د ) للك نسبت ا عغ غالف نسخق ا زات:ق
( ا ختى ذات التُر66565
د ) ُن كا النسبق ف ال النسخق ت ه بق بخط
.حديس 2. ( ب د ال التحالق ف كل نسخق تن نسخ ا تا عدا نسخق ا زات:ق ذات التُر66565
د . ) ف تجلد تجم ا سضم ن عدِ وحاال أبن ا ا 3. اس اق نسخ ال التحالق ف تئدتت ا عغ اله ت:ح باحر تفل ا ابن ا ا ا او
نسبق سألي ا ل ةئد ود ةً اش"
ُاه ال يخ اإلتا ا دئ ت ا ا دُ تا بماه ال صعاء رب
تدمد عبدة هللا بماهة الدين بنة ال يخ ا بل ي حفَ بنت
ا ا ا او ت-
وض ف هللا
تعال عن-ش
الا ة ل عئدسة ة بد ه هللا تعال له يل الئ ه ف تساال اأوهغاه
ا لك وِ ف ر اخت البا تن ها ا ئت ت". كل ال ا دلق سةثب صعق نسبق ال التحالق ال ف بٍن ريدينا فل ا بماه الدين بن
. ا ا او . ا ا ا او . ا ا ا او تحقيق اسم
الرسالة
ش 47
لااار ية َااات تح ابااان ا اااا فااا تئدتاااق اااال التحاااالق باحااار ل اااا ُ ماااا ُااااهش"
اااالا ة ااال عئدسُّااا ة
بد ه هللا تعال له يل الئ ه ف تساال اأوهغاه ا لك وِ ف ر اخت البا تن ها ا ئت ت". لر ية ضع ل ا عن ان عغ غالف نسخق داو ال هع ا ت:ق أ نسخق ال اات:ق؛ ن ما
ة هبها ضمن تجم عٍَونت يهضم نان وحاال أبن ا ا ة هب التحاال ةً ما تههاليق ةإذا ا ت
وحالق ابهدرت التحالق الهاليق ف السطت الل يلي(
18
) . ود احم ا عغ غالف نسخق ت هبق الك جتس ا تت:كي
نسخق ت هبق ا خنا ق العاتق
بهط ان ف ا غت
ش "س يل ال ئ ه ف تساال اأوهغاه ا لك وِ ف آخت البا تن ها ا ئت ت" . 47 الا عن ان ط :ل تأخ ذ تما ذ ت ابن ا ا ف تئدتق ال التحالق السابا
الل ُّ و ت؛ تما يده
.عغ ر ليس تن ضع ابن ا ا بخاصق رن ااسٍن النسخهٍن ت ه بهان حديثا كاسب ما احد
( ود احم ا عغ غالف نسخق ا زات:ق ذات التُر132295
د ) "ص ا ا ئاه ف
."تساال اأوهغاه للك ذة تت التحالق ف ستبمق ابن ا ا ف نسخق تن تغنف اللبيع(
19
)
نسخق تن
ر ضح ا سالك(
20
) باحر
ش
.""ص ا ا ئاه ف تساال اأوهغاه تن ب ق ت روى رن الا العن ان
ليس دُيئا؛ ليس اناك خطأ ف تساال
اأوهغاه ال ف ذ تاا ابن ع و ف آخت با اأوهغاه تن ها ا ئت ت ة ل ا ابن ا ا ف
ال التحالق أ ف ال العلماء علً ا ح ى ية َ تما يةعن تز الا خطأة الناسخ ف هابق لئع
ابن ا ا ةئد هب ا لاش "باله الدي ُّن بن ا ا " بدأْ تن "بماه الدين بن ا ا "؛ تما يده
.عغ ر ليس طالع علر
رتا ستبمها ابن ا ا ا لك وَ سَانت ف نسخق تن تغنف اللبيع رختى تن ر ضح ا سالك ةال
يةعولَرة كاسب ما ة ما تنس خهان بخط تة خهلتف عن خط ت ا خط طهٍن الغالع رن ما تن ُضاةق
رحدت تة ال كت ا خط طهٍَونت
ما ه عادِ تة ال ك ا خط طات ةهف ستبمق تج لق ةال يةسَ ل ر بكل تا
. تحقيق اسم
الرسالة
ش لعد و عغ غالف ر نسخق تن نسخ ال التحاال
تج ء "تساال" ف العن ان تجم عقْ يه اةا تع تعبٍي ابن ا ا عه ا با جمع ف تئدتق
ال التحالق :تناحع تع تضم ن ال التحالق؛ أوهمال ا عغ ع ت تساال صت ح ابن ا ا
.بل ت عدداا ف ا ئدتق ( عغ غالف نسخق ا زات:ق ذات التُر66565
) د ا
"تساال اأوهغاه ف الند "؛
عن ان ت بَ ن داهٌّ عغ تضم ن التحالق ا ُن كان تنس خْ ا بخط حديس ُأ ر يف: تد اأحر
الل ر ود اإلتا عمت بن َُدت يد ف سَتوبَ مَ ه أبن ا ا ا
"ال ال عغ تساال اأوهغاه". لر
رخحي العن ان الل ذ ت ابن َُدت يد
؛
ن كلم ف "ال ال عغ ..." تما يةستبعد ضعة ة ف عنا :ن
التحاال. لعد و عغ غالف ر نسخق تن نسخ ال التحاال تج ء "تساال" ف العن ان تجم عقْ يه اةا تع تعبٍي ابن ا ا عه ا با جمع ف تئدتق
ال التحالق :تناحع تع تضم ن ال التحالق؛ أوهمال ا عغ ع ت تساال صت ح ابن ا ا
.بل ت عدداا ف ا ئدتق ُخباوة ابنت ا ا عن ال التحالق بأن ا ةَ ول حينما ُاه ف تئدتت اش"
الا ةَ و ل عئدس
بد ه هللا تعال له يل الئ ه ف تساال اأوهغاه ا لك وِ ف
ر اخت البا تن ها ا ئت ت"
أ
يعنف رن ا تجم عق تن ا ساال تَغٍَ يت رَحو كَاتة َا بالن تسو بَق ُل البا الل ُبل ا ُ ما ا ئ د
بال ل انا اة َ الئَ وهة ال َاضت حة البٍَ تن ال ل يَنو َ ت ل ب ا و ةتَاد عن غٍي(
23
)
. اإلخباوة عن التحالق بأن ا
ةَ ول ر تت
احهخدت ابنة ا ا ف بعض وحاال(
24
). تحقيق اسم
الرسالة
ش ود ةً ا تن ب ق ت روى رن الا العن ان
ليس دُيئا؛ ليس اناك خطأ ف تساال
اأوهغاه ال ف ذ تاا ابن ع و ف آخت با اأوهغاه تن ها ا ئت ت ة ل ا ابن ا ا ف
ال التحالق أ ف ال العلماء علً ا ح ى ية َ تما يةعن تز الا خطأة الناسخ ف هابق لئع
ابن ا ا ةئد هب ا لاش "باله الدي ُّن بن ا ا " بدأْ تن "بماه الدين بن ا ا "؛ تما يده
.عغ ر ليس طالع علر رتا ستبمها ابن ا ا ا لك وَ سَانت ف نسخق تن تغنف اللبيع رختى تن ر ضح ا سالك ةال
يةعولَرة كاسب ما ة ما تنس خهان بخط تة خهلتف عن خط ت ا خط طهٍن الغالع رن ما تن ُضاةق
رحدت تة ال كت ا خط طهٍَونت
ما ه عادِ تة ال ك ا خط طات ةهف ستبمق تج لق ةال يةسَ ل ر بكل تا
. ود ةً ا ( ود احم ا عغ غالف نسخق ا زات:ق ذات التُر66565
د ) بخط حديس "تساال
( اأوهغاه ف الند ". وري اإلتا عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي856
ه) حينما
سَتوبَ رَ أبن ا ا ف حاويه عغ ر ضح ا سالك(
21
)
."حم ااا "ال ال عغ تساال اأوهغاه تعدُّ د احر ال التحالق يدلُّنا عغ رن ابن ا ا لر يضَعو ل ا عن ا ا ما ه عادس ف
بعض وحاال(
22
)؛ للا يهعٍ ن اخهياو العن ان ا نسع ل ا. روى رن رنسع ال عنا :ن ل ا ا تا ود 48 ( عغ غالف نسخق ا زات:ق ذات التُر66565
) د ا
"تساال اأوهغاه ف الند "؛
عن ان ت بَ ن داهٌّ عغ تضم ن التحالق ا ُن كان تنس خْ ا بخط حديس ُأ ر يف: تد اأحر
الل ر ود اإلتا عمت بن َُدت يد ف سَتوبَ مَ ه أبن ا ا ا
"ال ال عغ تساال اأوهغاه". لر
رخحي العن ان الل ذ ت ابن َُدت يد
؛
ن كلم ف "ال ال عغ ..." تما يةستبعد ضعة ة ف عنا :ن
التحاال. :منهج املؤلف في الرسالة ر ضح ا فل تفة ابنة ا ا-وحم هللا-
ال دفَ تن سألي ال التحالق ف تئدتت ا حينما
ُاهش"
الا ة ل عئدس بد ه هللا تعال له يل الئ ه ف تساال اأوهغاه ا لك وِ ف ر اخت
البا تن ها ا ئت ت"
. ال ا ساال بمع ةً ا ابن ع و ال و ال ف يأتي علً ا ال اغل تا
ينهُ عه ا تن تغٍُّي ف ح ر حَ مو لت ا غ ه عن عغ ال اغل تا يحيس ع علي تن ح ر ُعتاِي
للم غ ه عن تبهداا بما كان ةي ال اغل ويئا احدا ثر تا كان ةي ال اغل ويئٍن عغ
ُّ سن ع ما بٍن حببٍن ر ضمٍيين ر ضمٍي حبٍّف عغ سن ُّا الضمٍي تا بٍن ته ل تن ل تا 49 بٍن تتة ا تن تع بمع ال و ال ف يك ن ةً ا ح ر ا غ ه عن تة ه تئْ ا ةيما يةدمل
علي(
25
) . :منهج املؤلف في الرسالة ُد كان ُيتاد ابن ع و ال ال و دُيئا بدسع ال اغل الل يةدمل علي ا غ ه
عن
تا يحيس ع علي تن تغٍُّي ف ا ع ر اإلعتاِي للم غ ه عن الل كان تئ دَ ابن ع و
تن ذ ت ال ال و؛ ُذ بعل تداو ا عديس علي ةئاه ف بدايت اش"
اأحرة الامة و هَغَلة عن ف الا
...البا ُن كان ل ضمٍي احد"؛ للا حاو ابن ا ا ف عتض تساال ال التحا لق عغ ستسيع
ابن ع و سماتا د ن اخهالف ُد ة ل ابن ا ا ال ال و حتداا ف ع ت تساال
َُس مَ َا–
حسع وؤ:ه-
ُل تجم عهٍن تتسا :هٍن بدسع ح ر ا عمل عغ ال اغل تا يحيس ع
علي تن ح ر ُعتاِيش
خمس
ٍلك ا خياو ف ا عمل عغ ر ال اغل ن ا لك و:ن عغ اخهالة ما
خمس
.يجع ةً ا ا عمل عغ رحد ال اغلٍن ا لك و:ن لر يةدالف اله ةياة ابنَ ا ا ف الا الهئسير؛ ن ابن ع و ذ ت رن ل ل ا ساال
ثالثق رحكا(
26
) ةهف سندوج سد ثالث تجم عات أ اثنهٍن ما صَنَعَ ابن ا ا ةا سأل
هان
ا ل الثا يق تن ا جم عق ا ل ليس
ًة ما
ُأ واغل احد ةإذا رود ا رن جعل
م ا تن با
اأوهغاه بع ا عمل عغ ال اغل ا لك و ليس ًام ة
ب آخت ُأ التةع عغ اأبهداء ا
التاجح ةيما ُذا كان ال اغل تن با ما ف د ش ز:د ضتبهة ة ز:د ضتب ة رخا التةع يةخو تتجة
ا سألهٍن تن با اأوهغاه ؛ للا ةا عمل عغ اأوهغاه ةً ما رتت ب ِيٌّ أ ب از ٌّ حسع تا
ذ ت ابن ا ا ةكان علي رن يةئَ س ت مَ َا ُل
ثالث تجم عات
؛
ةئا لألحكا ال ف ر وداا ابن
.ع و 50
لعل أبن ا ا العلوَ ف الا؛ ن طالع العلر يجد ف با اأوهغاه صع بق؛ لبناء
غالع تساال عغ
رتثلق اةحياضيق(
27
)
؛ الا تا بعل ابن ا ا ف آخت ال التحالق يئ هش"
ال
ُّ ا ساال ر ل بأن سةلَئ عَ با ساال العَ و ت ا و ةهوعتبَق للع ت أ ا ساال ال ف ر وداا رب ناو البغداد 50 ِا لئ عة بتمَ لتكت النُّدا"
ةأواد ابن ا ا باخه او الهئسير سئت:ع تساال اأ وهغاه تس يل ا عغ
طال العلر ذلك بد تاا ف ُ اعد ُليلق؛ بدليل ر رواو ُل ح ر ا عمل عغ اأبهداء ف ثٍي
تن ا ساال(
28
). :منهج املؤلف في الرسالة ف ا سألق
التابعق تن تساال ا جم عق الثا يق لر يل ت س يالت ا سئديتْا ثال ا بعد ا عمل
؛
ْا ه اء
باإلحالق عغ و دت تماثتلت ت ف ا سألق ال ف ُبل ا. للك لر يل ت ف ا سألق الثا يق تن تساال
ا جم عق الثا يق عتل قَ تنع ا عمل عغ الضمٍي ا ن ل ا ن حٍن ب د الضمٍي ا ه ل للك ذ ت-عَتَضْا-
"تَ لتكَ النُّداِ ربا ناو البغداد هابَ ة "ا ساال العَ و ت ا و ةهوعتبَق للع ت
حينما ورى رن تساال اأوهغاه ال ه ا ل بهلوئتيب ا با ساال العَ و ت أ .ِتساال تَ لتكت النُّدا وغبقْ تن ابن ا ا ف اأخه او
؛ ةإ أ ي
تجح
ا ب
عغ آخت للك تا أ يل ت بعض
ا حكا
؛
ا ه اءْ باإلحالق ُل و داا ف ت ضع آخت تن التحالق ةنيا لر يل ت ح ر ا سألق ا ل
تن ا جم عق ا ل ُ ما رواو ف ح ر ا سألق الثا يق ُل
دخ ه ح ر ا ل ةً ا. ف ا سألق
التابعق تن تساال ا جم عق الثا يق لر يل ت س يالت ا سئديتْا ثال ا بعد ا عمل
؛
ْا ه اء
باإلحالق عغ و دت تماثتلت ت ف ا سألق ال ف ُبل ا. :منهج املؤلف في الرسالة للك لر يل ت ف ا سألق الثا يق تن تساال
ا جم عق الثا يق عتل قَ تنعت ا عمل عغ الضمٍي ا ن ل ا ن حٍن ب د الضمٍي ا ه ل
.ا تة ا ُحالقْ عغ تا ذ ت تن تعليلت ف ا سألق ال ف ُبل ا
تن ص و اأخه او ال ف ا ههج ا ابن ا ا ف ال التحالق ُوباؤ ا عديس عن ح ر
ال علٍن
ش (
َ ةَئَ دَ عَدت)
ُل ا سألق ا خٍيِ تن تساال التحالق عغ التغر تن رن ابن ع و ذ ت تن ا ن ات الند :ق ح ى وتح ا ئت ت أبن ع و حينما ر ود سئديتَ ابنت ع و ةي ثاهت
ا سأل
هٍن
التابعق ا خاتسق تن تساال ا جم عق ا ل
؛
.لكي يةبٍَ تن حَ و َ ة ف سئديتَ:و :ة َ تب ما
للك ذ ت عا ا د ن س ت:ح باحم
ةئاهش"ا لا َُد وَ بعض ر"
نَسَ عَ ل سئديتْا ثاه ا سألق
.ا خاتسق تن تساال ا جم عق ا ل ثر رسبع باأعحياض علي ف ذلك الهئديت تن ا ن ات الند :ق ح ى وتح ا ئت ت أبن ع و حينما ر ود سئديتَ ابنت ع و ةي ثاهت
ا سأل
هٍن
التابعق ا خاتسق تن تساال ا جم عق ا ل
؛
.لكي يةبٍَ تن حَ و َ ة ف سئديتَ:و :ة َ تب ما
للك ذ ت عا ا د ن س ت:ح باحم
ةئاهش"ا لا َُد وَ بعض ر"
نَسَ عَ ل سئديتْا ثاه ا سألق
.ا خاتسق تن تساال ا جم عق ا ل ثر رسبع باأعحياض علي ف ذلك الهئديت
للك ذ ت-عَتَضْا-
"تَ لتكَ النُّداِ ربا ناو البغداد هابَ ة "ا ساال العَ و ت ا و ةهوعتبَق للع ت
حينما ورى رن تساال اأوهغاه ال ه ا ل بهلوئتيب ا با ساال العَ و ت أ .ِتساال تَ لتكت النُّدا
وغبقْ تن ابن ا ا ف اأخه او
؛ ةإ أ ي
تجح
ا ب
عغ آخت للك تا أ يل ت بعض
ا حكا
؛
ا ه اءْ باإلحالق ُل و داا ف ت ضع آخت تن التحالق ةنيا لر يل ت ح ر ا سألق ا ل
تن ا جم عق ا ل ُ ما رواو ف ح ر ا سألق الثا يق ُل
دخ ه ح ر ا ل ةً ا. :منهج املؤلف في الرسالة ريضا ُد جد أبن ا ا العلو؛ إلتكان حمل صنيع عغ اله َحُّ ع؛ تا دا رن ا سألق
ةً ا ب ان- ُن لر ي ن رحداا تن با اأوهغاه–
ةهف سدخل تع تا بعداا تن تساال ذات
. ب ٍن سَ َحُّ عْا ثر حَ تَدَ ابنة ا ا ا ساالَ تسألقْ تسألقْ تن غٍي ذ ت لنص ت عباوِت ابن ع و ا ت حق
ر سئيُّد ب ا. ُد اس بَعَ تنهجْ ا احدا ف ال التحالق ذلك بل ت ر أْ تااي ق ال اغلش
رضمٍي ا ر
ٌّحبٍّف
؟ ثر
ع بدسع ح م اإلعتاِي ثر ية ود تثاأ للمسألق يةتوبتعة ة بل تت تا يةدمل علي
ا غ ه عن ثر يَلو ة تة سئديتَ تثاهت ا سألقت بعد ا عمل ُذا احهلن الهئديتة تعليال ذ ت له ضيح
ا ع ر الا اود ف كل ا ساال تا عدا ا سألهٍن ا ل الثا يق تن ا جم عق ا ل ؛ تا ذاك ُأ
.ل ض ح الهئديت ةً ما سَنَ اَ الهعليل الل يل ت ابن ا ا تا بٍن تعليل للهئديت تعليل للعمل عغ رحد
ال اغلٍن(
29
) تعليل لل وِ ال ف ودت علً ا ا سألق(
30
) . ُد يسهدع بيانة ا ع رت تن:دَ ُيضاح س يل تما يسهلن تع اإلطالق ف الهعليل الا
تا جد ظااتْا ف ا سألق التابعق تن ا جم عق ا ل ؛ حتصا تن عغ رن سك ن تساالة ال
التحالق اضعقْ ت تقْ؛ للا ورينا يل ت اإلوكاه ال اود عغ بعض س اصيل ا سألق ثر ية جيع
. :منهج املؤلف في الرسالة عن ما ف ا سألق التابعق تن ا جم عق ا ل ُد ات سَ ر تنهج ابن ا ا ف ال التحالق باأخه او ةال جد لدي خت با عم ا سضم ن
صُّ ابن ع و أ تى لدي ذت و تْا آلواء العلماء ر ت ن ات ر أ للخالةات الند :ق ةلر يل ت 51 تن ا ن ات الند :ق ح ى وتح ا ئت ت أبن ع و حينما ر ود سئديتَ ابنت ع و ةي ثاهت
ا سأل
هٍن
التابعق ا خاتسق تن تساال ا جم عق ا ل
؛
.لكي يةبٍَ تن حَ و َ ة ف سئديتَ:و :ة َ تب ما
للك ذ ت عا ا د ن س ت:ح باحم
ةئاهش"ا لا َُد وَ بعض ر"
نَسَ عَ ل سئديتْا ثاه ا سألق
.ا خاتسق تن تساال ا جم عق ا ل ثر رسبع باأعحياض علي ف ذلك الهئديت
للك ذ ت-عَتَضْا-
"تَ لتكَ النُّداِ ربا ناو البغداد هابَ ة "ا ساال العَ و ت ا و ةهوعتبَق للع ت
حينما ورى رن تساال اأوهغاه ال ه ا ل بهلوئتيب ا با ساال العَ و ت أ .ِتساال تَ لتكت النُّدا
وغبقْ تن ابن ا ا ف اأخه او
؛ ةإ أ ي
تجح
ا ب
عغ آخت للك تا أ يل ت بعض
ا حكا
؛
ا ه اءْ باإلحالق ُل و داا ف ت ضع آخت تن التحالق ةنيا لر يل ت ح ر ا سألق ا ل
تن ا جم عق ا ل ُ ما رواو ف ح ر ا سألق الثا يق ُل
دخ ه ح ر ا ل ةً ا. ف ا سألق
التابعق تن تساال ا جم عق الثا يق لر يل ت س يالت ا سئديتْا ثال ا بعد ا عمل
؛
ْا ه اء
باإلحالق عغ و دت تماثتلت ت ف ا سألق ال ف ُبل ا. :منهج املؤلف في الرسالة ال اخه اوات تعت ةق
( لدى النُّس اخ حم ااا ا خضت1287
ه) َدو هْا ف ا خط ت(
32
) . عغ ل حق الغالف ة هتعَ عن انة ا
ا ش
)(ص ا ا ئاه ف تساال اأوهغاه
عن انة
التحالق ال ف سلً اش (ا ب بق عن ا ه ا "ةضال
) لغق" د اما تن ا ل اظ ال
.وحالق أبن ا ا ا او طةبع ر ثي تن تتِ بهدئيئات رحماء تخهل ق
ة هع عغ يساو العن ان
ش
." "تما تن ب هللا عغ عبد ال ئٍي رحمد ا وزاد عة تيَ عن
سد عن ان التحالق روِعق رخها لر ي ت تا ةً ا بدُ ق ة ي العةلو ت ت ته ا عباوِة
ش
" ُف هلل
تعال " لر رسبٍ ن الباق ف الل رح ل تن بملق
ش
"ت هبق تع د دتياط..." ف ا خهومَ ٍونت
ِالل لين عغ يساو عباو
ش
."..."ت هبق ا زات ال ت:ف
ُد َُد تو ة ال النسخق عغ غٍياا؛ لئل ق رخطائ ا
ن ا سكاد سك ن النسخق السليمق تن ا سأل
هٍن ا ل الثا يق تن ال التحالق-
لر يتد ل ا و ااد عن العت ُ ما ه تبني ق عغ
ِرتثلق صَنَعَ َا الندا(
31
) . وصف النسخ الخطية املعتمدة في التحقي:ق ُ ل ل التحالق-حسع علمف- حت ُّ نةسَ خ ف العالر احهطع ا ع ه علً ا كل ا
هلل
ا عمد. ُلي ر ا عديس عه اش 1 –
نسخق ت هبق ا زات:ق ه تد ظق ةً ا ضمن تجم ا سد وُرش8374
د132295
عا . ه تك ق تن ثالث ل حات ف ف كل ص دق23
حطتا ة هب بخط النس خ
ةً ا تعئيع؛ ُذ ية هع ف ن ايق كل ل حق الكلمق ال ف سبدر ب ا الل حق ال ف سلً ا. ال
النسخق تتابعق تصح عَ ق ةما حئط ته ا تن كلمق ر حتف ة هتعَ ف ااتش الل حق رةسبع
)بتتن (صح
. ل حات ا تتُ مق بخط حديس كان كاسب ا يتتن لكلمق "حينئل" با"ح" لكلمق "ريضا" با"ريض". ال اخه اوات تعت ةق
( لدى النُّس اخ حم ااا ا خضت1287
ه) َدو هْا ف ا خط ت(
32
) . :منهج املؤلف في الرسالة للك لر يل ت ف ا سألق الثا يق تن تساال
ا جم عق الثا يق عتل قَ تنعت ا عمل عغ الضمٍي ا ن ل ا ن حٍن ب د الضمٍي ا ه ل
.ا تة ا ُحالقْ عغ تا ذ ت تن تعليلت ف ا سألق ال ف ُبل ا تن ص و اأخه او ال ف ا ههج ا ابن ا ا ف ال التحالق ُوباؤ ا عديس عن ح ر
ال علٍن
ش (
َ ةَئَ دَ عَدت)
ُل ا سألق ا خٍيِ تن تساال التحالق عغ التغر تن رن ابن ع و ذ ت
ح م ما ف ا سألق ا خٍيِ ا سألهٍن اللهٍن ُبل ا؛ تا ذاك ُأ وغبق تن ابن ا ا ف اأخه او
؛
ِ ورى رن ال ا ةعاه الثالثق اس ددَ تو ف ح ر احد لتعتل ق بمع بيه ا ورى رن العل ق ظاات
.ِب وِ ر بي ف ا سألق ا خٍيِ ةأَخ ت ا عديس عن ح ر الين ال علٍن ُل ا سألق ا خٍي اعهمد ابن ا ا ف س ضيح تساال ال التحالق عغ رتثلق ت ن عق ةلر ية ود ر تثاه
تسم ا عن العت ُد يك ن أبن ا ا العةلو وة ف الا؛ ن بميع تساال اأوهغاه-
تا عدا 52 ا سأل
هٍن ا ل الثا يق تن ال التحالق-
لر يتد ل ا و ااد عن العت ُ ما ه تبني ق عغ
ِرتثلق صَنَعَ َا الندا(
31
) . وصف النسخ الخطية املعتمدة في التحقي:ق
ل ل التحالق-حسع علمف- حت ُّ نةسَ خ ف العالر احهطع ا ع ه علً ا كل ا
هلل
ا عمد. ُلي ر ا عديس عه اش
1 –
نسخق ت هبق ا زات:ق ه تد ظق ةً ا ضمن تجم ا سد وُرش8374
د132295
عا . ه تك ق تن ثالث ل حات ف ف كل ص دق23
حطتا ة هب بخط النس خ
ةً ا تعئيع؛ ُذ ية هع ف ن ايق كل ل حق الكلمق ال ف سبدر ب ا الل حق ال ف سلً ا. ال
النسخق تتابعق تصح عَ ق ةما حئط ته ا تن كلمق ر حتف ة هتعَ ف ااتش الل حق رةسبع
)بتتن (صح
. ل حات ا تتُ مق بخط حديس
كان كاسب ا يتتن لكلمق "حينئل" با"ح" لكلمق "ريضا" با"ريض". :منهج املؤلف في الرسالة عغ ل حق الغالف ة هتعَ عن انة ا
ا ش
)(ص ا ا ئاه ف تساال اأوهغاه
عن انة
التحالق ال ف سلً اش (ا ب بق عن ا ه ا "ةضال
) لغق" د اما تن ا ل اظ ال
.وحالق أبن ا ا ا او طةبع ر ثي تن تتِ بهدئيئات رحماء تخهل ق 53
ة هع عغ يساو العن ان
ش
." "تما تن ب هللا عغ عبد ال ئٍي رحمد ا وزاد عة تيَ عن
سد عن ان التحالق روِعق رخها لر ي ت تا ةً ا بدُ ق ة ي العةلو ت ت ته ا عباوِة
ش
" ُف هلل
تعال " لر رسبٍ ن الباق ف الل رح ل تن بملق
ش
"ت هبق تع د دتياط..." ف ا خهومَ ٍونت
ِالل لين عغ يساو عباو
ش
."..."ت هبق ا زات ال ت:ف
ُد َُد تو ة ال النسخق عغ غٍياا؛ لئل ق رخطائ ا
ن ا سكاد سك ن النسخق السليمق تن
بٍن باق النسخ
؛ للا وُ م النص ا دئ ا طبئا لل حات ا
." وتنت ل ا با عتف "ر ة هع عغ يساو العن ان
ش
." "تما تن ب هللا عغ عبد ال ئٍي رحمد ا وزاد عة تيَ عن
سد عن ان التحالق روِعق رخها لر ي ت تا ةً ا بدُ ق ة ي العةلو ت ت ته ا عباوِة
ش
" ُف هلل
تعال " لر رسبٍ ن الباق ف الل رح ل تن بملق
ش
"ت هبق تع د دتياط..." ف ا خهومَ ٍونت
ِالل لين عغ يساو عباو
ش
."..."ت هبق ا زات ال ت:ف 53 2 –
نسخق ت هبق ا زات:ق ه تد ظق ةً ا سد وُرش4393
د66565
عا . ه
تنس خق ف خمس ل حات ف كل ص دق19
حطتا ة هب بخط النسخ ا خهلط بخط
التُعق ةً ا تعئيع. :منهج املؤلف في الرسالة ة هب
بخط نسخ تع وَحو رت بعضت ا عت ف بخط التُعق ةً ا تعئيع
ص دات ا تتُ مق بخط
حديس ي مل ا جم ا كل ف غالف الا ا جم ا ة هع
ش
"ش فء ف اللغق" ة ت توت هابه
روِع تتات ة هع سد ال هابق الس غ تن
ش
"ال ح ق ف اللغق للعالتق ابن ا ا
"وحم هللا ف رح ل الغالف سملُّك باحر ا .لسيد حعد بن جل عبد التحمن
." ُد وتنت ل ل النسخق با عتف "ظ
كل ال النسخ ت ه بق بعد ع ت ا فلف؛ لحيحُّ م ر علي تغم دَ ة هللا ب احع وحمه . كل ا
غة ل تن احر اسخ ا؛ ُتا لعد ذ ت ف آختاا ما ف نسخق ا زات:ق ذات التُرش4393
د ُتا ن ا ضمن تجم ا احر الناسخ ية هع عادِ ف الل حق ا خٍيِ تن ليس
. الل حق ا خٍيِ بٍن يد
5 –
نسخق ت هبق الك جتس ا تت: يق ه تد ظق ةً ا سد وُرش6101
PJ
. ه تنس خق
ف ثالث ل حات سده
كل ص دق عغ
25
حطتا. ة هب بخط وُعق حديس ةً ا
تعئيع
ال النسخق تتابعق تصح عق ةالكلمات الساُطق سة هع ف ااتش الل حق
.) سةتبع بتتن (صح
عغ ل حق الغالف ة هتعَ عن انة ا
ا ش
"س يل الئ ه ف تساال اأوهغاه ا لك وِ ف
."آخت البا تن ها ا ئت ت للعالتق بماه الدين بن ا ا وحم هللا آتٍن
ف رح ل الل حق
خَ هورة ت هبق الك جتس
ه نسخق حديثق هب ا عبد العن:ن عطيق
حم دِ ا تن علماء ا زات ا و ةدو دَ ثتٍن ةئد نَسَ خَ وحالقَ ا ئاتقت اللفلف:ق للسي طي ف14
وعبان1347
ه التحالق ت ب دِ ف باتعق ا لك حع د سد وُرش1161
. . ا هبئ تي تن ال التحالق تنس خ ف ل حهٍن سده
كل ص دق عغ
19
حطتا. :منهج املؤلف في الرسالة ة هب
بخط نسخ تع وَحو رت بعضت ا عت ف بخط التُعق ةً ا تعئيع
ص دات ا تتُ مق بخط
حديس ي مل ا جم ا كل ف غالف الا ا جم ا ة هع
ش
"ش فء ف اللغق" ة ت توت هابه
روِع تتات ة هع سد ال هابق الس غ تن
ش
"ال ح ق ف اللغق للعالتق ابن ا ا
"وحم هللا ف رح ل الغالف سملُّك باحر ا .لسيد حعد بن جل عبد التحمن
." ُد وتنت ل ل النسخق با عتف "ظ . ا هبئ تي تن ال التحالق تنس خ ف ل حهٍن سده
كل ص دق عغ
19
حطتا. ة هب
بخط نسخ تع وَحو رت بعضت ا عت ف بخط التُعق ةً ا تعئيع
ص دات ا تتُ مق بخط
حديس ي مل ا جم ا كل ف غالف الا ا جم ا ة هع
ش
"ش فء ف اللغق" ة ت توت هابه
روِع تتات ة هع سد ال هابق الس غ تن
ش
"ال ح ق ف اللغق للعالتق ابن ا ا
"وحم هللا ف رح ل الغالف سملُّك باحر ا .لسيد حعد بن جل عبد التحمن
." ُد وتنت ل ل النسخق با عتف "ظ
كل ال النسخ ت ه بق بعد ع ت ا فلف؛ لحيحُّ م ر علي تغم دَ ة هللا ب احع وحمه . كل ا
غة ل تن احر اسخ ا؛ ُتا لعد ذ ت ف آختاا ما ف نسخق ا زات:ق ذات التُرش4393
د ُتا ن ا ضمن تجم ا احر الناسخ ية هع عادِ ف الل حق ا خٍيِ تن ليس
. الل حق ا خٍيِ بٍن يد
5 –
نسخق ت هبق الك جتس ا تت: يق ه تد ظق ةً ا سد وُرش6101
PJ
. ه تنس خق
ف ثالث ل حات سده
كل ص دق عغ
25
حطتا. :منهج املؤلف في الرسالة ال النسخق تتابعق تصح عق ةالكلمات الساُطق سة هع ف
ااتش الل حق سةتبع بتتن (صح) الكلمات ا و ةلوبتس قة
ُتاءت ا ة هب ف ااتش الل حق ة هع
ِة ُ ا "ن" تمد د ل حات ا تتُ مق بخط حديس
ف حط ل حات ا كل ا وكل بمال عغ
ايئق تة ضَل ع وباع تة نه ت ر ف حط االه ت ه ح ُل ا ع غ داخل تناوِ سد الئ س
ة هع بخط ط :ل
ش
.""ا زات ال ت:ف ف رعغ ل حق الغالف ة هع احر التحالق "تساال اأوهغاه ف الند " بجا ب
ا
ا يست
ة هع
ش
" ُف حعادِ واغع باوا" رة مل ف السطت الل رح ل
ثر ف با ب ا ا يست ة هع
بخط حديس روُات ا
ف رح ل الل.حق خَ هور ليس اضعا كان كاسب ا يتتن لكلمق "حينئل" با"ح" لكلمق "ريضا" با"ريض". لر ي هع است خة َا احمَ ة ف
.ن ايق التحالق ." " ُد وتنت ل ل النسخق با عتف 3 –
نسخق داو ال هع ا ت:ق ه تد ظق ةً ا ضمن تجم ا اوهمل عغ وحاال أبن
( ا ا وُم ش102
د ). ه تنس خق ف ثالث ل حات سحيا ح رحطت ص دات ا تا بٍن28
ُل32
حطتا ة هب بخط وُعق تمن ج بخط النسخ ةً ا تعئيع. ص دات ا تتُ مق
بخط حديس ي مل ا جم ا كل
. ليس ف ت وت ا ال ف بٍن يد ل حق الغالف 4 –
نسخق داو ال هع ال اات:ق ف دت ا ه تد ظق ةً ا ف تجم ا سضم ن وحاال أبن
( ا ا وُم ش9304
.عا ). ف ال النسخق حَ ئو ط تن ر ل ا بمئداو ص دق حطت:ن 54 . ا هبئ تي تن ال التحالق تنس خ ف ل حهٍن سده
كل ص دق عغ
19
حطتا. :منهج املؤلف في الرسالة ة هب بخط وُعق حديس ةً ا
تعئيع
ال النسخق تتابعق تصح عق ةالكلمات الساُطق سة هع ف ااتش الل حق
.) سةتبع بتتن (صح
عغ ل حق الغالف ة هتعَ عن انة ا
ا ش
"س يل الئ ه ف تساال اأوهغاه ا لك وِ ف
."آخت البا تن ها ا ئت ت للعالتق بماه الدين بن ا ا وحم هللا آتٍن
ف رح ل الل حق
خَ هورة ت هبق الك جتس
ه نسخق حديثق هب ا عبد العن:ن عطيق
حم دِ ا تن علماء ا زات ا و ةدو دَ ثتٍن ةئد نَسَ خَ وحالقَ ا ئاتقت اللفلف:ق للسي طي ف14
وعبان1347
ه التحالق ت ب دِ ف باتعق ا لك حع د سد وُرش1161
. ." ُد وتنت ل ل النسخق با عتف "ظ كل ال النسخ ت ه بق بعد ع ت ا فلف؛ لحيحُّ م ر علي تغم دَ ة هللا ب احع وحمه . كل ا
غة ل تن احر اسخ ا؛ ُتا لعد ذ ت ف آختاا ما ف نسخق ا زات:ق ذات التُرش4393
د ُتا ن ا ضمن تجم ا احر الناسخ ية هع عادِ ف الل حق ا خٍيِ تن ليس
ق ا خٍ ِ ٍن د
الل 5 –
نسخق ت هبق الك جتس ا تت: يق ه تد ظق ةً ا سد وُرش6101
PJ
. ه تنس خق
ف ثالث ل حات سده
كل ص دق عغ
25
حطتا. ة هب بخط وُعق حديس ةً ا
تعئيع
ال النسخق تتابعق تصح عق ةالكلمات الساُطق سة هع ف ااتش الل حق
.) سةتبع بتتن (صح عغ ل حق الغالف ة هتعَ عن انة ا
ا ش
"س يل الئ ه ف تساال اأوهغاه ا لك وِ ف
."آخت البا تن ها ا ئت ت للعالتق بماه الدين بن ا ا وحم هللا آتٍن
ف رح ل الل حق
خَ هورة ت هبق الك جتس
ه نسخق حديثق هب ا عبد العن:ن عطيق
حم دِ ا تن علماء ا زات ا و ةدو دَ ثتٍن ةئد نَسَ خَ وحالقَ ا ئاتقت اللفلف:ق للسي طي ف14
وعبان1347
ه التحالق ت ب دِ ف باتعق ا لك حع د سد وُرش1161
. 55 نةسو خَ قة وحالق ابن ا ا ال تنئ لق ت ن نسخق ت هبق ا زات:ق ا د ظق ةً ا ضمن
( تجم ا سد وُرش4393
د ) ا لك وِ آ ْ ا سد وُر2. :منهج املؤلف في الرسالة ." ُد وتنت ل ا با عتف "ك
6 –
نسخق ت هبق ا خنا ق العاتق بهط ان ف ا غت ه تد ظق ةً ا ضمن تجم ا سد
وُرش360 ه نسخق حديثق هب ا عبد العن:ن عطيق حم دِ كا سعة نسخقت ت هبقت
الك جتس السابئق ه تهطابئق تع ا ف كل ش فء ح ى ف ت اضع الكلمات تن كل حطت؛
. للا لر رعهمد علً ا ف سدئيا ال التحالق نةسو خَ قة وحالق ابن ا ا ال تنئ لق ت ن نسخق ت هبق ا زات:ق ا د ظق ةً ا ضمن
( تجم ا سد وُرش4393
د ) ا لك وِ آ ْ ا سد وُر2. ." ُد وتنت ل ا با عتف "ك
6 –
نسخق ت هبق ا خنا ق العاتق بهط ان ف ا غت ه تد ظق ةً ا ضمن تجم ا سد
وُرش360 ه نسخق حديثق هب ا عبد العن:ن عطيق حم دِ كا سعة نسخقت ت هبقت
الك جتس السابئق ه تهطابئق تع ا ف كل ش فء ح ى ف ت اضع الكلمات تن كل حطت؛
. للا لر رعهمد علً ا ف سدئيا ال التحالق نةسو خَ قة وحالق ابن ا ا ال تنئ لق ت ن نسخق ت هبق ا زات:ق ا د ظق ةً ا ضمن
( تجم ا سد وُرش4393
د ) ا لك وِ آ ْ ا سد وُر2. ." ُد وتنت ل ا با عتف "ك ." ُد وتنت ل ا با عتف "ك 6 –
نسخق ت هبق ا خنا ق العاتق بهط ان ف ا غت ه تد ظق ةً ا ضمن تجم ا سد
وُرش360 ه نسخق حديثق هب ا عبد العن:ن عطيق حم دِ كا سعة نسخقت ت هبقت
الك جتس السابئق ه تهطابئق تع ا ف كل ش فء ح ى ف ت اضع الكلمات تن كل حطت؛
. للا لر رعهمد علً ا ف سدئيا ال التحالق :عملي في التحقيق 56
ق
ي ي
الغايق تن الهدئيا ه ن ت ا خط طق صعيدق ما ضع ا تفل ت ة ا ُد حَ عَيو ة ف
سدئيئي للع اظ عغ ص ت ا خط طق ةَست توتة ف الهدئيا حسع ا حس اآلسيقش
1 -
لر رس خت لو ُحدى النسخ ا خط طق رصالْ ؛ لهأختاا كل ا عن ع ت ا فلف ُ ما رثب ُّ النص
ا ص روتت ُل اخهالف النسخ ا ختى ف ا عاويق تع سئديمف نسخق ت هبق ا زات:ق
( ذات التُرش8374
د )؛ لئل ق رخطائ ا ن ا سكاد سك ن النسخق ا حلر تن بٍن باق
النسخ؛ ل .] [ لا وُ مو ة النص ا دئ ا طبئْ ا لل حات ا ة ضع روُات ا بٍن تعئ ةٍن
2 -
الحزت ف الهدئيا با داة ق عغ ص وِ النص ا صغ ةلر رسدخل ةي ُأ بالئدو اليسٍي
الل أ يمسُّ ب ات تثل هابه ةا الئ اعد اإلتالايق ا عت ةق اآلن َ ضوع عالتات
.الحيُير
3 -
. :عملي في التحقيق ث ئ ا ساال الند :ق تن ت ان ت ا ف ال هع الند :ق خت ب آواء العلماء تن هب ر
4 -
.ضبطو ة كل تا يدهاج ُل ضَ بوط ف النص ت
5 -
.ستبَ مو ة لألعال اللين ذ تار ا فلف تعهمدا عغ هع الحيابر ا هخ ت َ ق
6 -
.ذَي لو ة البدس ب تس ا ادو ا تابع الغايق تن الهدئيا ه ن ت ا خط طق صعيدق ما ضع ا تفل ت ة ا ُد حَ عَيو ة ف
سدئيئي للع اظ عغ ص ت ا خط طق ةَست توتة ف الهدئيا حسع ا حس اآلسيقش
1 -
لر رس خت لو ُحدى النسخ ا خط طق رصالْ ؛ لهأختاا كل ا عن ع ت ا فلف ُ ما رثب ُّ النص
ا ص روتت ُل اخهالف النسخ ا ختى ف ا عاويق تع سئديمف نسخق ت هبق ا زات:ق
( ذات التُرش8374
د )؛ لئل ق رخطائ ا ن ا سكاد سك ن النسخق ا حلر تن بٍن باق 56 ص وِ ل حق الغال"ف تن نسخق ت هبق ا زات:ق "ر ص وِ ل حق الغال"ف تن نسخق ت هبق ا زات:ق "ر 57 "ص وِ ال دق الثا يق تن نسخق ت هبق ا زات:ق "ر "ص وِ ال دق الثا يق تن نسخق ت هبق ا زات:ق "ر 58 58 "ص وِ ال دق ا خٍيِ تن نسخق ت هبق ا زات:ق "ر "ص وِ ال دق ا خٍيِ تن نسخق ت هبق ا زات:ق "ر 59 " " ص وِ ص دق الغالف تن نسخق ت هبق ا زات:ق " " ص وِ ص دق الغالف تن نسخق ت هبق ا زات:ق 60 60 " " ص وِ الل حق ا ل تن نسخق ت هبق ا زات:ق 61 ص وِ ال دق ا خٍيِ تن" " نسخق ت هبق ا زات:ق ص وِ ال دق ا خٍيِ تن" " نسخق ت هبق ا زات:ق 62 62 "ص وِ ال دق ا ل تن نسخق ت هبق داو ال هع ا ت:ق "د "ص وِ ال دق ا ل تن نسخق ت هبق داو ال هع ا ت:ق "د 63 "ص وِ ال دق ا خٍيِ تن نسخق ت هبق داو ال هع ا ت:ق "د 64 64 "ص وِ ص دق الغالف تن نسخق داو ال هع ال اات:ق "ظ "ص وِ ص دق الغالف تن نسخق داو ال هع ال اات:ق "ظ 65 "ص وِ ال دق ال ف سبدر ب ا نسخق داو ال هع ال اات:ق "ظ "ص وِ ال دق ال ف سبدر ب ا نسخق داو ال هع ال اات:ق "ظ 66 66 ِص و"ال دق ا خٍيِ تن نسخق داو ال هع ال اات:ق "ظ 67 "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ""ك 68 68 "ص وِ ال دق الثا يق تن نسخق ت هبق الك جتس "ك "ص وِ ال دق الثا يق تن نسخق ت هبق الك جتس "ك "ص وِ ال دق الثا يق تن نسخق ت هبق الك جتس "ك 69 "ص وِ ال دق ا خٍيِ تن نسخق ت هبق الك جتس "ك " "ك 70 [1
] بسر هللا التحمن التحير
صغ هللا عغا حايد ا تدماد عغا آلا صاعب حالر. ُااه ال ايخ(
33
)
اإلتاا ا دئ تاا ا ادُ تا
بماااه ال صااعاء رباا تدمااد عباادة هللا بماااهة الاادين باانة ال اايخ ا باال ي حاافَ باانت ا ااا ا اااو ت-
وضااااا ف هللا تعاااااال عنااااا(
34
)-
ش اااااالا ة ااااال عئدسُّااااا ة(
35
) بدااااا ه هللا تعاااااال له ااااايل الئااااا ه فااااا تسااااااال(
36
)
اأوهغاه ا لك وِ ف ر اخت(
37
)
البا تن ها الامة ئَ ت ت(
38
). اعلاااار رن ااااا ع اااات تساااااال رن ااااا(
39
) سنئساااار اااا ٍن(
40
) تتسااااا :ٍنش خمسااااق(
41
)
يجاااا ز لااااك ةً ااااا
ا عمااال عغااا ال ااااغل ا ااالك و احااادا كاااان ر ر ثاااي حاااببيًّا كاااان ر ضااامٍيا ته اااال كاااان الضااامٍي(
42
)
ر(
43
)
.تن ااال. خمسااق يجااع ةً ااا ا عماال عغاا بعااض ال اااغل ا االك و د ن بعااض فأمااا الخمسااة
:األولى [1
] بسر هللا التحمن التحير
صغ هللا عغا حايد ا تدماد عغا آلا صاعب حالر. ُااه ال ايخ(
33
)
اإلتاا ا دئ تاا ا ادُ تا
بماااه ال صااعاء رباا تدمااد عباادة هللا بماااهة الاادين باانة ال اايخ ا باال ي حاافَ باانت ا ااا ا اااو ت-
وضااااا ف هللا تعاااااال عنااااا(
34
)-
ش اااااالا ة ااااال عئدسُّااااا ة(
35
) بدااااا ه هللا تعاااااال له ااااايل الئااااا ه فااااا تسااااااال(
36
)
اأوهغاه ا لك وِ ف ر اخت(
37
)
البا تن ها الامة ئَ ت ت(
38
). اعلاااار رن ااااا ع اااات تساااااال رن ااااا(
39
) سنئساااار اااا ٍن(
40
) تتسااااا :ٍنش خمسااااق(
41
)
يجاااا ز لااااك ةً ااااا
ا عمااال عغااا ال ااااغل ا ااالك و احااادا كاااان ر ر ثاااي حاااببيًّا كاااان ر ضااامٍيا ته اااال كاااان الضااامٍي(
42
)
ر(
43
)
.تن ااال. "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك خمسااق يجااع ةً ااا ا عماال عغاا بعااض ال اااغل ا االك و د ن بعااض فأمااا الخمسااة
:األولى صغ هللا عغا حايد ا تدماد عغا آلا صاعب حالر. ُااه ال ايخ(
33
)
اإلتاا ا دئ تاا ا ادُ تا
بماااه ال صااعاء رباا تدمااد عباادة هللا بماااهة الاادين باانة ال اايخ ا باال ي حاافَ باانت ا ااا ا اااو ت-
وضااااا ف هللا تعاااااال عنااااا(
34
)-
ش اااااالا ة ااااال عئدسُّااااا ة(
35
) بدااااا ه هللا تعاااااال له ااااايل الئااااا ه فااااا تسااااااال(
36
)
اأوهغاه ا لك وِ ف ر اخت(
37
)
البا تن ها الامة ئَ ت ت(
38
). فإحااااداها ش رن يكاااا ن ال اااااغل ضاااامٍيا احاااادا ااااالا الضاااامٍي ُااااد يكاااا ن تتة عااااا ُااااد يكاااا ن
تن با تثال ماش ز:دْ ا ضتبهة ة(
44
) رز:د ُا ؟ الثانياااااة
ش رن يكااااا ن حاااااببيًّا احااااادا(
45
) :نئسااااار ريضاااااا ُلااااا تتةااااا ا تن ااااا تثال مااااااش ز:ااااادْ ا
ضااااااتب ة رخااااااا(
46
)
رز:ااااااد ُااااااا رباااااا ؟ ةااااااال ُوااااااكاه ر ااااااك ُذا لاااااار سَجو عَاااااالت اأحاااااارَ تبهاااااادرْ سَدو متاااااالة(
47
)
فاااااا
ال اااا وسٍن بميعااااا(
48
) عغاااا ذلااااك ال اااااغل ةااااإن(
49
) كااااان ذلااااك(
50
)
َ ال اااااغل تن اااا بْا اااابو(
51
)
عغاااا
ُضماو ذلك ال عل ُن كان ال اغل(
52
)
ضمٍيْا ر عغ ُضماوت أزتت ت ُن كان ال اغل حببيًّا. "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ُن كاان
تتة عاااااا وةعااااا َ كااااااان ال اااااال حينئاااااال تداااااهمال لالبهاااااداء للعماااااال(
53
) عغااااا ال عاااااال(
54
)
الهئاااااديت فاااااا
ا لاااا(
55
)
َ ش رَضَااااتَبو(
56
)
ز:اااادْ ا ضَااااتَبوهَ ة(
57
)
؟ رُااااا(
58
) ز:ااااد ُااااا ؟ الهئااااديت فاااا الثا يااااق(
59
)
ش رَاَ نواااا ة(
60
)
ز:اااادْ ا
ضتب ة رخا رَأَ بَسَ ز:د ُا رب(
61
) ؟ الثالثااة
ش رن يكاا ن ال اااغل حَ اابَبٍَونت داا ش رز:ااد(
62
)
َ َضَاات(
63
)
رخاا غالتَ اا ة ؟ ةهَدو متاالة عغاا ر مااا
وااااا(
64
) :كااااا ن اإلضاااااماو ةً ماااااا تااااان(
65
)
ا عناااااى د ن الل ااااامل :كااااا ن ا ئاااااد وة(
66
) -
ُذا وةعااااا-
َةعااااال 71 ا عاااااا هت حاااااادَ ة-
ُذا ااااااب- ةعاااااالَ ال اعاااااالت تااااااع ال اعاااااال(
67
)
َ َ ةيةئَ ااااااد وة فاااااا "رز:ااااااد ضَاااااات(
68
)
رخاااااا
َغالتَ اا ة؟"ش رَرةات ااٍن(
69
)
[ ز:ااد ضَااتَ َ رخاا غالتَ اا ة؟2
ر] :ةئَ ااد وة ُذا ااب ش رَرَاااان ز:اادْ ا رخاا ضَااتَ َ رخاا
غالتَ ة(
70
) ؟ ُ ما َُد ووتَ ال اعلَ انا ماا َُد ووسَا ة فا ا ساألق ا
لا تان تثااه(
71
)
اأواهغاه با ن ا
رعنف(
72
)
""ز:دْ ا ضَ تَبوهة ة(
73
). رتاا تساألق اأواهغاه باا تة ا ةالاامة ئَ د و(
74
)
ةً اا ةتعوال د ن ةاعلا ماا
ِتَ ت ؛ ن ال اعل ا الامة هَغَلة عن ا دل ف بالضت و(
75
)
.ةعل حدَ ة "ةإن ُيلش ُن ا ئد و ف "ز:دا ضَتَبوهة ة(
76
)
ةاعل تضمت(
77
)
. أ ةاعل ظااات . ة الا ةَاتوق أ رَثَاتَ لا
َ ااالا الئاادو ااا الاال بَ عَاال(
78
) فاا ااس بعااض الطلبااق اا وْ ا عاان ااالا الهئااديت؛ ن اار ُ مااا رَلت ة اا ا(
79
)
سئديتَ ال اعل تن(
80
)
ِحيس ا كا جنء تن ال عل أ تن حيس ا احر ظاات تساهئلٌّ االا أ عباي
ب . ُ ما ُد ووسة ة تةفَخ تْا عن ا ع ه
؛
. ليع د الضمٍي عغ تة هئد ت الرابعااااة(
81
)
ش رن يكاااا ن ال اااااغلة ضاااامٍيين تن اااالٍن داااا ش رز:اااادْ ا ُيااااا(
82
)
. "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ً ةااااإن احااااتن تسر(
112
)
َسئااااديت ال عاااال(
113
) ت ع لَاااا ة تاااان حيااااس ُن
ا عاااا ه لاااايس كااااا جنء تاااان
ال عال ةاللك أز للم انف(
114
) ريضاا رأ سااتى رن الهئاديت(
115
) عناد اللك بدالة ما(
116
)
ُأ ر اا
لاااار يةئَ ااااد ت و ح ااااتْا ةيَ و سة اااادة ال ااااال ة بعااااد سئااااديت(
117
)
ُيااااا ؛ ُذ صاااااو يْااااا تدضْااااا ةدَ َاااالَ الهخااااالةفة
بٍن(
118
)
.بمل ف اله سٍي ذلك خطأ اضح ما بي ن ا ةاااااااإن ُيااااااالش ُن(
119
) ا ع ااااااات فااااااا ا جملاااااااق ا ااااااالك وِ فااااااا با اااااااع ال اعااااااال
ة ياااااااف ح لوهَااااااا ة فااااااا
سئااديتك(
120
) ا ه ُلاا با ااع ا عاا ه؟ ُلاا ش ال اعاال ا عاا ه فاا ااال ا سااألق لااامة سَ مًّ ى(
121
)
احااد ةاااااااإن ُيااااااالش ُن(
119
) ا ع ااااااات فااااااا ا جملاااااااق ا ااااااالك وِ فااااااا با اااااااع ال اعااااااال
ة ياااااااف ح لوهَااااااا ة فااااااا
سئااديتك(
120
) ا ه ُلاا با ااع ا عاا ه؟ ُلاا ش ال اعاال ا عاا ه فاا ااال ا سااألق لااامة سَ مًّ ى(
121
)
احااد
.ةال ةتق ف ا عنى ةاااااااإن ُيااااااالش ُن(
119
) ا ع ااااااات فااااااا ا جملاااااااق ا ااااااالك وِ فااااااا با اااااااع ال اعااااااال
ة ياااااااف ح لوهَااااااا ة فااااااا
سئااديتك(
120
) ا ه ُلاا با ااع ا عاا ه؟ ُلاا ش ال اعاال ا عاا ه فاا ااال ا سااألق لااامة سَ مًّ ى(
121
)
احااد ُ مااااا لااااار رةَُااااد ت وو(
122
)
رداِ ا ع ااااات داخلاااااق عغاااا ال اعااااال ااااا أ سةداااالف رداِ اأحاااااه ناء :بئااااا
ا ستثنى باس اق(
123
) . "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك لاااار يَضوااااتت و ُأ ااااا
ُ ما ُد تنا ا ع ه(
83
)
.لين ل ُ ما ح ت ا ال اعل لللك الرابعااااة(
81
)
ش رن يكاااا ن ال اااااغلة ضاااامٍيين تن اااالٍن داااا ش رز:اااادْ ا ُيااااا(
82
)
. لاااار يَضوااااتت و ُأ ااااا
ُ ما ُد تنا ا ع ه(
83
)
.لين ل ُ ما ح ت ا ال اعل لللك لاااك فااا اااال(
84
)
ريضاااا ُذا لااار سةئَ اااد ت و اأبهاااداء رن َ سَدو متااالَ عغااا ر ت ماااا وت ااا و(
85
)؛ ن ا ن ااال(
86
)
كال اات(
87
) ةا ن الن(
88
) كال ااات:ن(
89
)
الس ابَبتي ٍونت(
90
) ُاد ت ا ى ر اك(
91
)
ةً ماا سدمال عغا ر ت ماا
َ وت ااا و(
92
)
ةاااإن حملاااا عغااا ا ن ااا ااااب بهئاااديتش رلاااار يَضواااتت و ز:ااادْ ا ُأ ااااا(
93
)
؟ لااار يَضوااااتت و ُأ
ا(
94
)
َةئد ووت(
95
)
ف ال(
96
)
ريضا ال اعلَ ا و ةسو هَئتل(
97
)
بن س تاع ال عال ماا ةعلا فا ال اف ُبل اا
ل اان انااا ز:ااادِ ااا ر ااك ُااد ووتَ تعاا رداِ ا ع اات لتيَصت ااح ا عنااى لَاانت َ تاان ذلااك رن صاااو تن ااال
ماااا رن ةاعااالَ ال عااالت ا ااالك و(
98
) اااللك(
99
) لااايس سئاااديتاما لل ااال بااال
للمعناااى بااااء ال اااال
.اس اُا 72
ُن(
100
)
حَ مَ لواا َ عغاا ا تةاا ا وَةَعواا َ بهئااديتش ُيااا لاار يَضااتت و ز:ااد ُيااا لاار يَضااتت و(
101
)
ُأ ااا
ا ااالا فااا وَ اااتوحت ا ااان تفت(
102
) لااايس بءااا فء؛ ن تعناااى ا جملاااق(
103
)
[ ا لااا غٍاااي تعناااى ا جملاااق2
] 72
ُن(
100
)
حَ مَ لواا َ عغاا ا تةاا ا وَةَعواا َ بهئااديتش ُيااا لاار يَضااتت و ز:ااد ُيااا لاار يَضااتت و(
101
)
ُأ ااا
ا ااالا فااا وَ اااتوحت ا ااان تفت(
102
) لااايس بءااا فء؛ ن تعناااى ا جملاااق(
103
)
[ ا لااا غٍاااي تعناااى ا جملاااق2
] الثا يااق(
104
)
؛ ُذ تعنااى ا جملااق ا لاا-ع غاا زعماا-
ش رن ز:اادا لاار يَضااتت و سَ اا ة تعنااى ا جملااق الثا يااقش
ر لر يَضتت و ُأ سَ ة. "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ؟ ةأغناك ذلك عن سئديت ا ع ه البه ق
ةااااإن احااااتن تسر(
112
)
َسئااااديت ال عاااال(
113
) ت ع لَاااا ة تاااان حيااااس ُن
ا عاااا ه لاااايس كااااا جنء تاااان
ال عال ةاللك أز للم انف(
114
) ريضاا رأ سااتى رن الهئاديت(
115
) عناد اللك بدالة ما(
116
)
ُأ ر اا
لاااار يةئَ ااااد ت و ح ااااتْا ةيَ و سة اااادة ال ااااال ة بعااااد سئااااديت(
117
)
ُيااااا ؛ ُذ صاااااو يْااااا تدضْااااا ةدَ َاااالَ الهخااااالةفة
بٍن(
118
)
.بمل ف اله سٍي ذلك خطأ اضح ما بي ن ا
ةاااااااإن ُيااااااالش ُن(
119
) ا ع ااااااات فااااااا ا جملاااااااق ا ااااااالك وِ فااااااا با اااااااع ال اعااااااال
ة ياااااااف ح لوهَااااااا ة فااااااا
سئااديتك(
120
) ا ه ُلاا با ااع ا عاا ه؟ ُلاا ش ال اعاال ا عاا ه فاا ااال ا سااألق لااامة سَ مًّ ى(
121
)
احااد
.ةال ةتق ف ا عنى
ُ مااااا لااااار رةَُااااد ت وو(
122
)
رداِ ا ع ااااات داخلاااااق عغاااا ال اعااااال ااااا أ سةداااالف رداِ اأحاااااه ناء :بئااااا
ا ستثنى باس اق(
123
) . "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ال ا رن الهئديتش رَلَرو يَضواتت و(
105
)
ز:اد(
106
)
ُأ ُياا(
107
)
؟ ُياا لار يَضواتت و
ُأ ااا بداالف ال عاال ت ع لاا ا د اا و(
108
)
بئاااء ال اعاال مااا ُاادو ا ةيمااا سئااد حاالف ال عاال
ةا عل ا د و(
109
)
بئاء ا ع ه(
110
). ُن(
111
)
َ وت و َ َُد ووتَ ش رضَتَ َ ز:د سَ ة خاص قْ؟ رغناك ذلاك عان سئاديت ا ع ات. ُن وت ا و
.َُد ووتَ ش رَأَ بَسَ ز:د ... ؟ ةأغناك ذلك عن سئديت ا ع ه البه ق
ةااااإن احااااتن تسر(
112
)
َسئااااديت ال عاااال(
113
) ت ع لَاااا ة تاااان حيااااس ُن
ا عاااا ه لاااايس كااااا جنء تاااان
ال عال ةاللك أز للم انف(
114
) ريضاا رأ سااتى رن الهئاديت(
115
) عناد اللك بدالة ما(
116
)
ُأ ر اا
لاااار يةئَ ااااد ت و ح ااااتْا ةيَ و سة اااادة ال ااااال ة بعااااد سئااااديت(
117
)
ُيااااا ؛ ُذ صاااااو يْااااا تدضْااااا ةدَ َاااالَ الهخااااالةفة
بٍن(
118
)
.بمل ف اله سٍي ذلك خطأ اضح ما بي ن ا الثا يااق(
104
)
؛ ُذ تعنااى ا جملااق ا لاا-ع غاا زعماا-
ش رن ز:اادا لاار يَضااتت و سَ اا ة تعنااى ا جملااق الثا يااقش
ر لر يَضتت و ُأ سَ ة. ال ا رن الهئديتش رَلَرو يَضواتت و(
105
)
ز:اد(
106
)
ُأ ُياا(
107
)
؟ ُياا لار يَضواتت و
ُأ ااا بداالف ال عاال ت ع لاا ا د اا و(
108
)
بئاااء ال اعاال مااا ُاادو ا ةيمااا سئااد حاالف ال عاال
ةا عل ا د و(
109
)
بئاء ا ع ه(
110
). ُن(
111
)
َ وت و َ َُد ووتَ ش رضَتَ َ ز:د سَ ة خاص قْ؟ رغناك ذلاك عان سئاديت ا ع ات. ُن وت ا و
.َُد ووتَ ش رَأَ بَسَ ز:د ... "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك املسألة الخامسة
ش رن يك ن ال اغل ضمٍيا تن ال حَ بَبتيًّا(
124
)
ل ل(
125
)
ص وسانش
ُُّحاادااماش رن يكاا ن الس اابٍَّت ف(
126
) ااا ا ن اا الضاامٍية ااا ا تةاا ا داا ش رز:ااد(
127
) لاا ر يَضوااتت و
رخااا ُأ ااا ؟ لااك فاا ااال ا سااألق ُذا لاار سَدو متاالو عغاا اأبهااداء رن سَدو متاالَ عغاا(
128
)
َ ر ت مااا وت اا و(
129
)
ةإن حَ مَ لو َ عغ السبٍّف ب بهئاديتش رلار ة تانو ز:ادْ ا ُأ اا ؟ لار يَضواتت و رخاا ُأ اا(
130
)
َ ةئاد ووت
َال عااااااااالَ ال اعااااااااال(
131
) تد ااااااااا وْ ا تة ااااااااافَخ تْا
عااااااااان ا عااااااااا ه ليعااااااااا د عليااااااااا ن ا د ااااااااا و وةسوبَهةااااااااا ة(
132
)
الهأخٍي(
133
). 73 ُن حَ مَ لوهَااا ة عغااا الضااامٍي وةعااا َ بهئاااديتش رلااار يَضواااتت و ز:اااد رخاااا(
134
)؟ لااار(
135
)
يَضواااتت و رخاااا ُأ
اااااا ا ااااالا َُاااااد وَ ا ااااان تفة ةيااااا سخاااااالةفة ا جملهاااااٍن فااااا ا عناااااى ماااااا تَ ااااات فااااا ا ساااااألق التابعاااااق [3 ]ر
َ َ ال اااا ا رن يةئَ ااااد وَ ش رَضَاااات(
136
) ز:ااااد خاص ااااقْ رخااااا ... ؟(
137
)
ِ. ُ مااااا ُاااادو ا "خاص ااااقْ" لتتَسة ااااد تَ سَ ااااد ردا
ا ع ت ال ف(
138
) ذة ت تَتو ف بمل
ق اله سٍي؛ ُذ لار يةم ان(
139
) سئاديت رداِ اأحاه ناء تدل ةاق بئااء(
140
)
ا ستثنى(
141
) . ُن حَ مَ لوهَااا ة عغااا الضااامٍي وةعااا َ بهئاااديتش رلااار يَضواااتت و ز:اااد رخاااا(
134
)؟ لااار(
135
)
يَضواااتت و رخاااا ُأ
اااااا ا ااااالا َُاااااد وَ ا ااااان تفة ةيااااا سخاااااالةفة ا جملهاااااٍن فااااا ا عناااااى ماااااا تَ ااااات فااااا ا ساااااألق التابعاااااق [3 ]ر
َ َ ال اااا ا رن يةئَ ااااد وَ ش رَضَاااات(
136
) ز:ااااد خاص ااااقْ رخااااا ... ؟(
137
)
ِ. ُ مااااا ُاااادو ا "خاص ااااقْ" لتتَسة ااااد تَ سَ ااااد ردا
ا ع ت ال ف(
138
) ذة ت تَتو ف بمل
ق اله سٍي؛ ُذ لار يةم ان(
139
) سئاديت رداِ اأحاه ناء تدل ةاق بئااء(
140
)
ا ستثنى(
141
) . "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ال وِ الثا يقش رن سك ن بالع س د ش رز:د(
142
) لر يَضوتت و رخ ُأ ُيا ؟ لاك(
143
)
فا اال
ريضااا ُذا لاار سدماال عغاا اأبهااداء رن سدماال عغاا ر ت(
144
) ال اااغتلٍَونت وت اا و َ ةااإن حَ مَ لواا َ عغاا الساابٍّف ت
َ وَةَعواا(
145
)
َبهئااديتش رَرةات ااٍن(
146
)
ز:ااد لاار يَضوااتت و رخاا ُأ ُيااا(
147
) ا االا(
148
)
َُااد وَ بعضاا ر ا َ و لَاا رن
َ يةئَ اااااد وَ ش رَضةاااااتت(
149
) ز:اااااد ... ؟ ُن حَ مَ لوااااا َ عغااااا الضااااامٍي اااااب َ بهئاااااديت(
150
)ش رَضَاااااتَ َ ز:ااااادْ ا(
151
)
ْخاص اااااق
رخ ؟ لر يَضوتت و رخ. ُأ ُيا وأما الخمسة الثانية: فإحداها(
152
)
ش رن يك ن ال اغل ضمٍيا ته ال تتة عا تاع حابٍّف(
153
)
دااا ش رز:اااد(
154
) ضَاااتَ َ رخاااا ؟ ةيجاااع(
155
)
ُذا رودت ا عمااال عغااا ال عااال د ن اأبهاااداء رن سدمااال عغااا
الضااامٍي ا ه ااال أ غٍااااي ةحيةاااع اأحااار السااااابا بهئاااديتش رَضَااااتَ َ ز:اااد(
156
)
رخاااا ضَااااتَ َ رخاااا ؟ ُ مااااا
اتهناع ا عماالة عغاا الساابٍّف ت ااك ناا سئاا هش رَز:اادْ ا(
157
)
َرَاَ ااان(
158
)
َ... ؟ :ةاافَد ت ذلااك ُلاا رن تةعَااد ت َ ةعاال
ا ضمت ا ه ل ُل ظاات(
159
) ا تمهنع ف بميع رب ا العتبيق(
160
) . 74
:الثانيااة
رن يكاا ن(
161
) ال اااغل(
162
) ضاامٍيا ته ااال(
163
)
تتة عااا كااال ف ُبل ااا ل اان أ تااع حاابٍّف
بااال تاااع ضااامٍي تن ااال دااا ش رز:اااد(
164
)
لااار يَضواااتت و ُأ ُياااا(
165
)
؟ ةيهعاااٍ نة(
166
)
ةً اااا ريضاااا ا عمااالة عغااا
الضمٍي ا ه ل ا الضمٍي ا تة ا ةحيةع "ز:دا" ما وةعه(
167
)
فا ال اف ُبل اا ذلاك بهئاديتش رلار
يَضوااتت و ز:ااد ُأ ُيااا(
168
)
؟ لاار يَضوااتت و ُأ ُيااا(
169
)
َ . علااق ا نااع ةً ااا كااال ف ُبل ااا؛ ُذ لاا َ َاابو(
170
)
لكااان
َالهئديتش رز:دْ ا رَاَ ان(
171
)... ر ش رز:دْ ا(
172
)
. "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك :الثالثااة رن يكاا ن ال اااغ
ل ضاامٍيا ته ااال(
173
)
تن اا با تااع ضاامٍي تن اال ةااال يخلاا ُتااا رن
[3 ] يك ن ال عل تن با (ظَن ) ر أ ةإن(
174
) لر ي ن(
175
)
تن با(
176
) (ظَن ) دا ش رز:اد(
177
)
لار
يَضوااااتتبو ة ُأ ااااا(
178
)
ةإ اااا يجااااع عليااااك ُذا رودت ا عماااال عغاااا ال عاااال د ن اأبهااااداء رن سدماااال عغاااا
الضااااااامٍي
ا ه ااااااال ماااااااا باااااااع فااااااا ا ساااااااألهٍن الساااااااابئهٍن ا عمااااااال عغااااااا الضااااااامٍي(
179
) ا ه ااااااال(
180
)
ةدينئااااال(
181
) سَنو ت اااااعة اأحااااارَ ؛ ن(
182
) الضااااامٍي(
183
) ا ه ااااال اناااااا(
184
)
تن ااااا اااااا ال ااااااء ةهئااااا هش
رز:دْ ا(
185
) لر يَضوتتبو ة ُأ ا ؟ الهئديتش رلر(
186
) يَضوتت و ز:دْ ا(
187
)
ُأ ا ؟ لر يَضوتت بو ة ُأ ا(
188
) . :مهنااااااع وةعاااااا با عماااااال(
189
)
عغاااااا "ااااااا "؛ ن الهئااااااديت حينئاااااال ش رَضَااااااتَبَ ة(
190
)
ز:ااااااد خاص ااااااقْ؟ لاااااار
يَضواااتتبو ة(
191
) ُأ اااا . ُ ماااا ُااادو ا "خاص اااق" لهعااالُّ و سئاااديت رداِ ا ع ااات
ُ ماااا بطااال اااالا الهئاااديت ااا
يةاافَد ت ُلاا تعااد ت ةعاال ال اااات ُلاا ضاامٍي ااا أ يجاا ز(
192
)
ُ مااا صَ ااح سئاادير الضاامٍي عغاا تة َ س ت ااتت ت
ل ْا
؛
. تةفَخ ت عن سئديتْا :مهنااااااع وةعاااااا با عماااااال(
189
)
عغاااااا "ااااااا "؛ ن الهئااااااديت حينئاااااال ش رَضَااااااتَبَ ة(
190
)
ز:ااااااد خاص ااااااقْ؟ لاااااار
يَضواااتتبو ة(
191
) ُأ اااا . ُ ماااا ُااادو ا "خاص اااق" لهعااالُّ و سئاااديت رداِ ا ع ااات
ُ ماااا بطااال اااالا الهئاااديت ااا
يةاافَد ت ُلاا تعااد ت ةعاال ال اااات ُلاا ضاامٍي ااا أ يجاا ز(
192
)
ُ مااا صَ ااح سئاادير الضاامٍي عغاا تة َ س ت ااتت ت
ل ْا
؛
. تةفَخ ت عن سئديتْا ل ْا
؛
. تةفَخ ت عن سئديتْا ُن كااان تااان بااا (ظَااان ) باااز(
193
)
لاااك ا عمااال عغاا ر ماااا واا(
194
) دااا ش رز:ااد(
195
)
لااار يَ َن ااا ة
ُاامااا ُأ ااا(
196
). "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ةااإن
َ حَ مَ لواا َ عغاا ا ه اال َ َاابو(
197
)
الهئااديتش رلاار يَ ةاان(
198
)
ز:اادْ ا ُاامْ ااا ُأ ااا ؟
لااار يَ ةن ااا ة ُاامْ اااا ُأ اااا(
199
). ُن(
200
) حملااا عغااا ا ن ااال(
201
)
وَةَعوااا َ الهئاااديتش رَظَن ااا ة(
202
)
ز:اااد(
203
)
خاص ااقْ ُاامْ ااا... "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك خاص قْ رَاَ انَ لر يَضوتت و ُأ ُيا 74
:الثانيااة
رن يكاا ن(
161
) ال اااغل(
162
) ضاامٍيا ته ااال(
163
)
تتة عااا كااال ف ُبل ااا ل اان أ تااع حاابٍّف
بااال تاااع ضااامٍي تن ااال دااا ش رز:اااد(
164
)
لااار يَضواااتت و ُأ ُياااا(
165
)
؟ ةيهعاااٍ نة(
166
)
ةً اااا ريضاااا ا عمااالة عغااا
الضمٍي ا ه ل ا الضمٍي ا تة ا ةحيةع "ز:دا" ما وةعه(
167
)
فا ال اف ُبل اا ذلاك بهئاديتش رلار
يَضوااتت و ز:ااد ُأ ُيااا(
168
)
؟ لاار يَضوااتت و ُأ ُيااا(
169
)
َ . علااق ا نااع ةً ااا كااال ف ُبل ااا؛ ُذ لاا َ َاابو(
170
)
لكااان
َالهئديتش رز:دْ ا رَاَ ان(
171
)... ر ش رز:دْ ا(
172
)
. خاص قْ رَاَ انَ لر يَضوتت و ُأ ُيا 74 :الثالثااة رن يكاا ن ال اااغ
ل ضاامٍيا ته ااال(
173
)
تن اا با تااع ضاامٍي تن اال ةااال يخلاا ُتااا رن
[3 ] يك ن ال عل تن با (ظَن ) ر أ ةإن(
174
) لر ي ن(
175
)
تن با(
176
) (ظَن ) دا ش رز:اد(
177
)
لار
يَضوااااتتبو ة ُأ ااااا(
178
)
ةإ اااا يجااااع عليااااك ُذا رودت ا عماااال عغاااا ال عاااال د ن اأبهااااداء رن سدماااال عغاااا
الضااااااامٍي
ا ه ااااااال ماااااااا باااااااع فااااااا ا ساااااااألهٍن الساااااااابئهٍن ا عمااااااال عغااااااا الضااااااامٍي(
179
) ا ه ااااااال(
180
)
ةدينئااااال(
181
) سَنو ت اااااعة اأحااااارَ ؛ ن(
182
) الضااااامٍي(
183
) ا ه ااااال اناااااا(
184
)
تن ااااا اااااا ال ااااااء ةهئااااا هش
رز:دْ ا(
185
) لر يَضوتتبو ة ُأ ا ؟ الهئديتش رلر(
186
) يَضوتت و ز:دْ ا(
187
)
ُأ ا ؟ لر يَضوتت بو ة ُأ ا(
188
) . "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك 75 :املسألة الخامسة
رن يك ن ال اغل ضمٍيين ته لٍن أ يهأت(
218
)
) ذلك ُأ ف باا (ظَان
دااااا ش رز:ااااااد ظَن ااااا ة(
219
) ُاامْ ااااااا(
220
). :جاااااع ةً ااااااا(
221
)
"ا عمااااال عغاااااا ا تةااااا ا. رتااااااا دااااا ش "رز:ااااااد ضَااااااتَبَ ة؟
ةممهنعاااااااااق(
222
)
[ َ وَةعوااااااااا َ ر َ َااااااااابو4 ر]؛ ااااااااااك(
223
)
فااااااااا الااااااااا ب ٍن بميعااااااااااا بمعوااااااااا َ باااااااااٍن ال اعاااااااااال
ا عاااا ه(
224
)ش ضاااامٍيين ته اااالٍن ساااامًّ ى(
225
)
َ احااااد فاااا غٍااااي بااااا (ظاااان ةَئَ ااااد(
226
)
َ عَاااادت(
227
))(
228
)
ذلك تمهنع(
229
). :املسألة الخامسة
رن يك ن ال اغل ضمٍيين ته لٍن أ يهأت(
218
)
) ذلك ُأ ف باا (ظَان
دااااا ش رز:ااااااد ظَن ااااا ة(
219
) ُاامْ ااااااا(
220
). :جاااااع ةً ااااااا(
221
)
"ا عمااااال عغاااااا ا تةااااا ا. رتااااااا دااااا ش "رز:ااااااد ضَااااااتَبَ ة؟
ةممهنعاااااااااق(
222
)
[ َ وَةعوااااااااا َ ر َ َااااااااابو4 ر]؛ ااااااااااك(
223
)
فااااااااا الااااااااا ب ٍن بميعااااااااااا بمعوااااااااا َ باااااااااٍن ال اعاااااااااال
ا عاااا ه(
224
)ش ضاااامٍيين ته اااالٍن ساااامًّ ى(
225
)
َ احااااد فاااا غٍااااي بااااا (ظاااان ةَئَ ااااد(
226
)
َ عَاااادت(
227
))(
228
)
ذلك تمهنع(
229
). ُااد اس ااح الئاا ه فاا ااال ا ساااال الع اات بمااا لاار سة ضَااعو(
230
)
علياا يَااد(
231
)
ُباال ذلااك ااال
ا ساال ر ل بأن(
232
) سةلَئ عَ با ساال العَ و ات ا و ةهوعتبَاق للع ات أ ا سااال ال اف ر ودااا(
233
) ربا اناو(
234
)
ِالبغداد ُّ ا لئ ع بتمَ لتكت النُّدا(
235
)
. ا عمد هلل حد سَم و(
236
). ُااد اس ااح الئاا ه فاا ااال ا ساااال الع اات بمااا لاار سة ضَااعو(
230
)
علياا يَااد(
231
)
ُباال ذلااك ااال
ا ساال ر ل بأن(
232
) سةلَئ عَ با ساال العَ و ات ا و ةهوعتبَاق للع ات أ ا سااال ال اف ر ودااا(
233
) ربا اناو(
234
)
ِالبغداد ُّ ا لئ ع بتمَ لتكت النُّدا(
235
)
. ا عمد هلل حد سَم و(
236
). الهوامش و:اإلحاالت "ص وِ ل حق الغالف تن نسخق ت هبق الك جتس "ك ؟ ةاااا"خاص ق" رغناا عاان ا ع اات سئااديتاا ُلاا با ااع ال اعاال ت يااد رن(
204
)
ا ع اات
ف با ب
صَ ح ع دة الضمٍي ا ئد(
205
) عغ "ز:د" ا فخ ت ل ا
؛
تة ئَ د(
206
)
.ْ ي ق :املساااألة الرابعاااة
رن يكااا ن ال ااااغل ضااامٍيا ته اااال تن ااا با تاااع حااابٍّف(
207
)
ةيااا اله ااايل
الساباش ُن كان ال عل تن(
208
)
غٍي با(
209
) (ظَان ) باع ُذا رودت ا عمال(
210
) عغا ال عال رن س دمال
عغ الضمٍي(
211
) ا ه ل د ش رز:د(
212
)
لر يضتبو ة رخ ؟ :املساااألة الرابعاااة
رن يكااا ن ال ااااغل ضااامٍيا ته اااال تن ااا با تاااع حااابٍّف(
207
)
ةيااا اله ااايل
الساباش ُن كان ال عل تن(
208
)
غٍي با(
209
) (ظَان ) باع ُذا رودت ا عمال(
210
) عغا ال عال رن س دمال
عغ الضمٍي(
211
) ا ه ل د ش رز:د(
212
)
لر يضتبو ة رخ ؟ :املساااألة الرابعاااة
رن يكااا ن ال ااااغل ضااامٍيا ته اااال تن ااا با تاااع حااابٍّف(
207
)
ةيااا اله ااايل
الساباش ُن كان ال عل تن(
208
)
غٍي با(
209
) (ظَان ) باع ُذا رودت ا عمال(
210
) عغا ال عال رن س دمال
عغ الضمٍي(
211
) ا ه ل د ش رز:د(
212
)
لر يضتبو ة رخ ؟
ُن(
213
)
كااااان تاااان بااااا (ظَاااان ) حملهاااا(
214
)
عغاااا ر مااااا واااا داااا ش رز:ااااد(
215
)
لاااار يَ ةن اااا ة ُاامااااا 75
:املساااألة الرابعاااة
رن يكااا ن ال ااااغل ضااامٍيا ته اااال تن ااا با تاااع حااابٍّف(
207
)
ةيااا اله ااايل
الساباش ُن كان ال عل تن(
208
)
غٍي با(
209
) (ظَان ) باع ُذا رودت ا عمال(
210
) عغا ال عال رن س دمال
عغ الضمٍي(
211
) ا ه ل د ش رز:د(
212
)
لر يضتبو ة رخ ؟
ُن(
213
)
كااااان تاااان بااااا (ظَاااان ) حملهاااا(
214
)
عغاااا ر مااااا واااا داااا ش رز:ااااد(
215
)
لاااار يَ ةن اااا ة ُاامااااا
رخ ؟ ُد سبٍن ي ي قة(
216
) الهئديت(
217
). الهوامش و:اإلحاالت تن ا تابع ا عديثق ال ف ستبم أبن ا ا ش عغ ة د
يل ابن
ا ا ا او ش آثاو تلاب الند عمادِ وف ن ا هبات باتعق ا لك حع د الت:اض
د.ط
1404
ه :حف عبد التحمن الضبع
ابن ا ا رثت ف الند العتِي
داو ا عديس ت ت
ط1
1998
ع ا
و الدين ابن ا ا ا او ش حياس تنهج الند ال ت ق العا يق لل ها
ِالئاات ط1
1989
. (2)
ابن ا تبيد ا ج ات ا نضد
ش 1/
77
. (3)
ابن حجت الدُّ وَو الكاتنق
ش 2/
415
. (4)
تدمد بن عغ اليمنف
ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق بٍي ت
د.ط د.ت
ش 1/
401
. (3)
ابن حجت الدُّ وَو الكاتنق
ش 2/
415
. (4)
تدمد بن عغ اليمنف
ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق بٍي ت
د.ط د.ت
ش 1/
401
. (4)
تدمد بن عغ اليمنف
ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق بٍي ت
د.ط د.ت
ش 1/
401
. (4)
تدمد بن عغ اليمنف
ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق بٍي ت
د.ط د.ت
ش 1/
401
. (5)
ْذ ت عغ ة دِ يل ثٍي" ا تن تفل ات ابن ا ا ف هاب "ابن ا ا ا او ش آثاو تلاب الند ش
13
189
215
287
325
353
. (5)
ْذ ت عغ ة دِ يل ثٍي" ا تن تفل ات ابن ا ا ف هاب "ابن ا ا ا او ش آثاو تلاب الند ش
13
189
215
287
325
353
. (6)
سن ت ستبمه ف
ش ابن حجت رحمد بن عغ العسئالني ُ باء الغمت بأبناء العمت سدئيا ش حسن حبء ف
ن ت جنق ُحياء الحياث اإلحالتي ف ا جلس ا عغ لل ئ ن اإلحالتيق ت ت
د.ط
1389هش 1/
540
ِ السي طي بغيق ال عا
ش 1/
148 ابن العماد ا عنبغ ولوات اللاع
ش 8/
616
. ي
و
ن
ابن ا وتبويَد ا ج
ات ا نضد
ش 1/
160 :ن ت
ش
ِالسي طي بغيق ال عا
ش 2/
390
. الهوامش و:اإلحاالت 76
(1)
ي
ن ت ستبمه رخباو ةيما ي
أتي تن ت ن ات
ةيما ذ ت تدئئ اا تن تتابعش صالح الدين خليل بن
ريبك ال د رعيان الع ت رع ان الن ت سدئيا ش عغ رب ز:د زتالا داو ال ت
بٍي ت -
دت ا
ط1
1418شه 3/5 ابن واةع سئي الدين تدمد بن هجتس السالتي ال ةيات سدئيا
ش
صا ح
ت د عباس ِ او ع اد تعت ف تفحسق التحالق بٍي ت
ط1
1402هش 2/
234
ابن حجت رحمد
بن عغ العسئالني الدُّ وَو الكاتنق ف رعيان ا ااق الثاتنق سدئيا
ش
تدمد حيد باد ا عا تطبعق
ا دني الئااتِ ط2 د.تش 2/
415
ابن تغت بتد ي حف بن تغت بتد ا عن ي ا ه ل ال اف
ا سه ف بعد ال اف سدئيا ش تدمد تدمد رتٍن ال يئق ا ت:ق العاتق لل هاِالئاات
د.ط د.تش
7/
131
ابن ت لح ُبتااير بن تدمد بن عبد هللا ا ئ د ا وود ف ذ ت رصعا اإلتا رحمد
سدئيا
ش عبد
التحمن بن حليمان العثيمٍن ت هبق التود الت:اض
ط1
1410هش 2/
66
ابن ا وتبويَد
ي حف بن حسن ال الح ا عنبغ ا ج ات ا
نضد ف طبئات تهأخت رصعا رحمد سدئيا
ش
عبد
التحمن بن حليمان العثيمٍن ت هبق العبيكان الت:اض
ط1
1421هش 1/
77
باله الدين عبد
التحمن بن رِي ب ت السي ط
ي
بغيق ال عاِ ف طبئات اللغ :ٍن النداِ سدئيا
ش
تدمد رب ال ضل
ُبتااير ا هبق الع ت:ق صيدا لبنان .د
ط د.تش 2/
68
ابن العماد ا عنبغ عبد الح بن رحمد
بن تدمد ولوات اللاع ف رخباو تن ذاع سدئيا
ش
تدم د ا و اؤ ط داو ابن ثٍي دت ا-
بٍي ت
ط1
1406هش 6/
191
ابن حميد تدمد بن عبد هللا النجد السعع ال ابلق عغ ضتااح
ا عنابلق سدئيا
ش ب ت بن عبد هللا رب ز:د
عبد التحمن بن حليمان العثيمٍن تفحسق التحالق 76 بٍي ت
ط1
1416هش 2/
662
ِ. الهوامش و:اإلحاالت (
13
)
سن ت ستبمه ف
ش تدمد بن عبد التحمن بن تدمد
السخا الض ء الالتع ال الئتن الهاحع
تن وات داو ت هبق ا عياِ بٍي ت د.ط د.ت
ش 6/
113
ِ السي طي بغيق ال عا
ش 2/
222
. (
13
)
سن ت ستبمه ف
ش تدمد بن عبد التحمن بن تدمد
السخا الض ء الالتع ال الئتن الهاحع
تن وات داو ت هبق ا عياِ بٍي ت د.ط د.ت
ش 6/
113
ِ السي طي بغيق ال عا
ش 2/
222
. (
14
)
عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق خطيق
تد ظق ف ا هبق السليما يق بإصطنب ه
ست يا سد وُرش1327
1
. / (
14
)
عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق خطيق
تد ظق ف ا هبق السليما يق بإصطنب ه
ست يا سد وُرش1327
1
. / (
14
)
عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق خطيق
تد ظق ف ا هبق السليما يق بإصطنب ه
ست يا سد وُرش1327
1
. / (
15
)
ابن ا ا ا او
تغنف اللبيع نسخق خطيق تد ظق ف ا هبق السليما يق الئديمق بإصطنب ه
ست يا سد وُرش696
ه1
.ر (
15
)
ابن ا ا ا او
تغنف اللبيع نسخق خطيق تد ظق ف ا هبق السليما يق الئديمق بإصطنب ه
ست يا سد وُرش696
ه1
.ر (
16
)
ابن ا ا ا او ر ضح ا سالك نسخ ق خطيق تد ظق ف ت هبق عاطف رةند ف ُصطنب ه
ست يا سد وُرش2442
. (
17
)
رةدت الا تن البدس الل ي هب اآلن ش. بابت بن عبد هللا الستيع عن ت ن ات ابن ا ا ا او
(
18
)
.ين ت ص و النسخهٍن ف ن ايق ال الدواحق (
19
)
ابن ا ا ا او تغنف اللبيع ه1 ر نسخق السليما يق الئدي
ش مق
696
. (
21
)
ابن َُدت يد حاويق ابن َُدت يد عغ ر ضح ا سالك
ش 1
. الهوامش و:اإلحاالت سن ت ستبمه رخباو ف ا ن ات اآلسيق
ةيما ذ ت تدئئ اا تن تتابعش ابن عبد ا لك تدمد بن
تدمد بن عبد ا لك ا او ا تا ء ف الليل اله ملق ل هاِي ا ص ه ال لق سدئيا
ش
ُحسان
عباس تدمد بن وت: ق ِ او ع اد تعت ف داو الغت اإلحالتي س نس
ط1
2012
ش 3/
348
رحمد بن رحمد بن عبد هللا بن تدمد
الغتبويينف عن ان الدوايق ةيمن عةتتفَ تن العلماء ف ا ااق
السابعق ببجايق سدئيا
ش
عاده ض تن وات داو اآلةاق ا جديدِ بٍي ت
ط2
1979
ش317
عبد
الباق بن عبد ا جيد اليماني ُواوِ الهعيٍن ف ستابر النداِ اللغ :ٍن سدئيا
ش
عبد ا جيد
ديا تت ن ا لك ةي ل للبد ث الدواحات اإلحالتيق
ط1
1406ه ش236
تدمد بن رحمد بن
عثمان بن ُايماز
اللاٍّف ساو:خ اإلحال َ َ ةيات ا ااٍي ا عال
سدئيا ش ب او ع اد تعت ف داو
الغت اإلحالتي بٍي ت
ط1
2003
ش
15
/
172
ابن وا ت تدمد بن وا ت بن رحمد ة ات ال ةيات
سدئيا
ش
ُحسان عباس داو صادو بٍي ت
ط1
1974
ش 3/
109
صالح الدين خليل بن ريبك
ال د ال اف بال ةيات سدئيا
ش رحمد ا اؤ ط
ستكي ت ط داو ُحياء الحياث بٍي ت1420شه
22
/
165 ال ٍي زآباد البلغق ف ستابر رامق الند اللغق سدئيا
ش
تدمد ا ت تطبعق
: ال ي ل الك
ط1
1407ه ش160
ِ السي طي بغيق ال عا
ش 2/
210
ابن العماد ا عنبغ 77 ولوات اللاع
ش 7/
575
تدمد تد ظ ستابر ا فل ٍن اله نسيٍن داو الغت اإلحالتي بٍي ت
ط2
1994
ش 3/
391
. ولوات اللاع
ش 7/
575
تدمد تد ظ ستابر ا فل ٍن اله نسيٍن داو الغت اإلحالتي بٍي ت
ط2
1994
ش 3/
391
. (
10
)
ال يخ خالد بن عبد هللا ا جتبا
ا زات
اله ت:ح بمضم ن اله ضيح ف الند ن ت داو ال هع
العلميق بٍي ت
ش ط1
1421هش 1/5 . (
10
)
ال يخ خالد بن عبد هللا ا جتبا
ا زات
اله ت:ح بمضم ن اله ضيح ف الند ن ت داو ال هع
العلميق بٍي ت
ش ط1
1421هش 1/5 . (
12
)
ابن العماد
ا عنبغ ولوات اللاع
ش 8/
330
. الهوامش و:اإلحاالت التابعق تن تساال ا جم عق الثا يق (
29
)
.سن ت ا سألق ا ل الثا يق الثالثق تن تساال ا جم عق الثا يق (
29
)
.سن ت ا سألق ا ل الثا يق الثالثق تن تساال ا جم عق الثا يق (
29
)
.سن ت ا سألق ا ل الثا يق الثالثق تن تساال ا جم عق الثا يق (
30
)
.سن ت ا سألق ا خاتسق تن تساال ا جم عق الثا يق (
30
)
.سن ت ا سألق ا خاتسق تن تساال ا جم عق الثا يق (
30
)
.سن ت ا سألق ا خاتسق تن تساال ا جم عق الثا يق (
31
)
ين ت
ش ب اد ي حف
العتبا ظااتِ اأوهغاه ف العتبيق وحالق تابسهٍي ا جاتعق ا ود يق كليق
الدواحات العليا1412ه ال ل الثانيش ص وِ ال ااتِ ف ع و اأحهجاج ش42
تا بعداا سن ت
رتثلق اأوهغاه ف
ش
: عضيمق ة اوس ها حيب ش165
-
169
. (
31
)
ين ت
ش ب اد ي حف
العتبا ظااتِ اأوهغاه ف العتبيق وحالق تابسهٍي ا جاتعق ا ود يق كليق
الدواحات العليا1412ه ال ل الثانيش ص وِ ال ااتِ ف ع و اأحهجاج ش42
تا بعداا سن ت
رتثلق اأوهغاه ف
ش
: عضيمق ة اوس ها حيب ش165
-
169
. (
31
)
ين ت
ش ب اد ي حف
العتبا ظااتِ اأوهغاه ف العتبيق وحالق تابسهٍي ا جاتعق ا ود يق كليق
الدواحات العليا1412ه ال ل الثانيش ص وِ ال ااتِ ف ع و اأحهجاج ش42
تا بعداا سن ت
رتثلق اأوهغاه ف
ش
: عضيمق ة اوس ها حيب ش165
-
169
. (
32
)
ال يخ تدمد بن ت ط بن حسن الدتياطي ا خضت حاويق ا خضت عغ وتح ابن عئيل ل يق
ابن تالك تطبعق ت ط الباِي ا علٍّف
الئااتِ د.ط
1359هش 1/3. (
32
)
ال يخ تدمد بن ت ط بن حسن الدتياطي ا خضت حاويق ا خضت عغ وتح ابن عئيل ل يق
ابن تالك تطبعق ت ط الباِي ا علٍّف
الئااتِ د.ط
1359هش 1/3. (
33
)
ف " " "ك"ش بسر هللا التحمن التحير ب نسهعٍن ُاه ال يخ.... ف "د"ش بسر هللا التحمن
....التحير ُاه ال يخ (
33
)
ف " " "ك"ش بسر هللا التحمن التحير ب نسهعٍن ُاه ال يخ.... الهوامش و:اإلحاالت /
(
22
)
ين ت
ش
ابن ا ا ا او تسألق ا ع مق ف سل ٍي ُت:ع ف ُ ل تعال ش (ُن وحمق هللا ُت:ع تن
ا دسنٍن) سدئيا د. عبد ال هاح ا عم ز ط
باعق ن ت داو عم او عم ان ط1
1405ه ش30
ابن ا ا
ا او وحالق ف س بي الن ع ف ُعتا "ةضالْ لغقْ خالةْا ريضْا الر بتًّا" سدئيا
ش
حسن
ت س ى ال اعت ن ت داو ا وُر عم ان ط1
1404ه ش8
ابن ا ا ا او ا ساال الس ت:ق ف
الند
سدئيا
ش
عغ حسٍن الب ا داو طيبق للن ت اله زيع الت:اض د.ط د.تش9 . 78 (
25
)
ين ت
ش
ابن ع و ا ئت ت سدئيا
ش
رحمد ا ج او عبد هللا ا جب و تطبعق العاني بغداد
ط1
1391هش 1/
91
. (
26
)
ين ت
ش
ابن ع و ا ئت ت
ش 1/
91
ابن ع و تة ثةل ا ئت ت سدئيا صالح حعد ا ليطي داو اآلةاق
ِالعتبيق الئاات
ط1
1427ه ش149
155
. (
27
)
ل لا ُاه رب حيان ا دلس ف ف الهلييل اله ميل ف وتح ها التس يل سدئيا ش حسن اندا
داو الئلر داو ن ز ُوبيليا ط1
1419هش 6/
293
تا بعداا عن با اأوهغاهش ( اأوهغاه للك
.)با تة لَ ا بند ُاه ابن تَ ضَاء الئتطٍّف رحمد بن عبد التحمن بن تَ ضَاء اللخمف الئتطٍّف ف
هاب التد عغ النداِ سدئيا ش ِتدمد ُبتااير البنا داو اأعه ا الئاات
ط1
1399ه ش103
. سن ت رتثلق اأوهغاه ف
ش
ِعضيمق ة اوس ها حيب : تطبعق السعادِ الئاات
ط1
1395ه ش
165
-
169
. (
28
)
سن ت ا سألق ا ل الثا يق التابعق ا خاتسق تن تساال ا جم عق ا ل ا سألق ا ل الثالثق
. التابعق تن تساال ا جم عق الثا يق (
28
)
سن ت ا سألق ا ل الثا يق التابعق ا خاتسق تن تساال ا جم عق ا ل ا سألق ا ل الثالثق
. التابعق تن تساال ا جم عق الثا يق (
28
)
سن ت ا سألق ا ل الثا يق التابعق ا خاتسق تن تساال ا جم عق ا ل ا سألق ا ل الثالثق
. الهوامش و:اإلحاالت 80
ل ضمٍيان ته الن حملهَ ة عغ ا تة ا ته ما ُأ رن ذلك أ يك ن ُأ ف با(
ظَنَنو ة)
ف(
ةَئَ دو تة
عَدت تو ة"). (
39
)
.ف "ر"ش ةإن ا
(
40
)
ف "د"ش.ُسمٍن
(
41
)
.ف "ر" "د"ش ةخمسق
(
42
)
."ُ ل ش "الضمٍي" حاُط تن " " "ك
(
43
)
."ُ ل ش "ر ثي حببيًّا كان ر ضمٍيا ته ال كان الضمٍي ر " حاُط تن "د
(
44
)
. ف "د"ش ز:د ضتبه
(
45
)
.ف " "ش حدا
(
46
)
"ف " " "د. "ك"ش ز:د ضتب رخا
(
47
)
.ف "ر"ش يجعل
(
48
)
.""بميعا" حاُطق تن "ر
(
49
)
" " ف."ك"ش ُن
(
50
)
""ذلك" حاُطق تن "ر. (
51
)
ف "ر"ش ع. (
52
)
.ف "د"ش كان ذلك ال اغل
(
53
)
....ف " " "ك"ش تهدمل اأبهداءت ا عملَ .... ف "د"ش تدهمال لالبهداء ا عملت
(
54
)
ف "ك"ش عغ ي عل. :ن ت ف ا سألق
ش
تدمد بن عبد هللا الطائي ا جياني ابن تالك تس يل ال ااد س ميل
ا ئاصد سدئيا
ش تد مد كاتل بتكات داو ال ها العتِي للطباعق الن ت
بٍي ت
1387ه ش82
رب حيان
ا دلس ف الهلييل اله ميل
ش 6/
348
ُبتااير بن ت س ى ال اطٍّف ا ئاصد ال اةيق ف وتح ا خالصق
الكاةيق سدئيا
ش عبد التحمن العثيمٍن زتالا تع د البد ث العلميق ُحياء الحياث اإلحالتي بجاتعق ر
الئتى ت ق ا تتق
ط1
1428هش 3/
75
. (
55
)
.ف "ر"ش ف ا ه
(
56
)
. ف "د"ش ضتب
(
57
)
يحيج ح التةع باأبهداء عغ الن ع ف ا ثالٍن ا لٍَونت تن ا سألهٍن اماش ز:دْ ا ضتبهة ة ز:دْ ا ضتب ة
ر خا ؛ ن التةع أ سئديت ةي رتا الن ع ةيدهاج ُل سئديتت ةعل اصع عد الهئديت ر ل
؛
رخفُّ كةلو َ ق ن ال ال تع يك ن بملق احدِ للك ليس ف ال ال تا يئه ف سئديت ةعل؛ ن (
48
)
.""بميعا" حاُطق تن "ر (
50
)
""ذلك" حاُطق تن "ر. (
52
)
.ف "د"ش كان ذلك ال اغل
(
53
)
....ف " " "ك"ش تهدمل اأبهداءت ا عملَ .... الهوامش و:اإلحاالت ف "د"ش بسر هللا التحمن
....التحير ُاه ال يخ (
34
)
" " ف "ك"ش ُاه
ال يخ اإلت ا العالتق بماه الدين بن ا ا-
وحم هللا تعال- ف "د"ش
ُاه ال يخ
بماه الدين بن ا ا-
وحم هللا تعال-. (
34
)
" " ف "ك"ش ُاه
ال يخ اإلت ا العالتق بماه الدين بن ا ا-
وحم هللا تعال- ف "د"ش
ُاه ال يخ
بماه الدين بن ا ا-
وحم هللا تعال-. 79
(
37
)
.ف " " "ك"ش آخت
(
38
)
ُاه ابن ع و ف ا ئت
ش 1/
91
ش" اأحر الامة و هَغَلة عن ف الا البا ُن كان ل ضمٍي اح د ر
حبٍّفٌّ احد حملهَ ة علي ُن كان ل حببان ر ضمٍيان تن الن ر ضمٍي تن ل حبٍّفٌّ حملهَ ة عغ
ر ما و ُن كان ل ضمٍي ته ل تتة ا تع حبٍّف ر ضمٍي تن ل حملهَ ة عغ الضمٍي ا ه ل أ 79 غٍي ُن كان ل ضمٍي ته ل تن تع ضمٍي تن ل ر حبٍّف حم لهَ ة عغ ر ما و ف با
ظنن ف(
ةئدت عدت) ف غٍي ذلك تن ا ب ا أ يج ز حملة ة ُأ عغ الضمٍي ا ه ل
ُن كان
ل ضمٍيان ته الن حملهَ ة عغ ا تة ا ته ما ُأ رن ذلك أ يك ن ُأ ف با(
ظَنَنو ة)
ف(
ةَئَ دو تة
عَدت تو ة"). غٍي ُن كان ل ضمٍي ته ل تن تع ضمٍي تن ل ر حبٍّف حم لهَ ة عغ ر ما و ف با
ظنن ف(
ةئدت عدت) ف غٍي ذلك تن ا ب ا أ يج ز حملة ة ُأ عغ الضمٍي ا ه ل
ُن كان
ل ضمٍيان ته الن حملهَ ة عغ ا تة ا ته ما ُأ رن ذلك أ يك ن ُأ ف با(
ظَنَنو ة)
ف(
ةَئَ دو تة
عَدت تو ة"). غٍي ُن كان ل ضمٍي ته ل تن تع ضمٍي تن ل ر حبٍّف حم لهَ ة عغ ر ما و ف با
ظنن ف(
ةئدت عدت) ف غٍي ذلك تن ا ب ا أ يج ز حملة ة ُأ عغ الضمٍي ا ه ل
ُن كان
ل ضمٍيان ته الن حملهَ ة عغ ا تة ا ته ما ُأ رن ذلك أ يك ن ُأ ف با(
ظَنَنو ة)
ف(
ةَئَ دو تة
عَدت تو ة"). الهوامش و:اإلحاالت ال عل ُد وة غتلَ بن ع الضمٍي. ين ت
ش حيب : ال ها سدئيا
ش عبد السال تد مد ااو ن ت هبق
ِا خا ج الئاات
ط3
1408هش 1/
81
ابن ال و اق تدمد بن عبد هللا بن العباس علل الند
سدئيا
ش
تدم د باحر تدمد الدو يش ت هبق التود الت:اض
ط1
1420ه ش311
عغ بن تدمد
بن عغ ا عضتتي اإلوبيغ
ابن خت
ف
وتح بمل النباج سدئيا ش حل ى تدمد عت باتعق ر
الئتى
د.ط
1419هش 1/
403 عثمان بن عمت ابن ا عابع اإليضاح ف وتح ا ل سدئيا
ش
ت س ى
بنا العليغ تطبعق العاني بغداد
د.ط
1982هش 1/
312
ا عسن بن ُاحر بن عبد هللا
ا تاد
س ضيح ا ئاصد ا سالك ب تح رل يق ابن تالك سدئيا
ش
عبد التحمن عغ حليمان داو ال ت
العتِي دت ا
ط1
1428هش 2/
618 عبد هللا بن عبد التحمن ابن عئيل ا ساعد عغ تس يل
ال ااد سدئيا ش
تدمد كاتل بتكات ن ت باتعق ر الئتى طباعق داو ال ت دت ا داو ا دني
ِبد
ط1
1400
-
1405هش 1/
422
ال اطٍّف
ا ئاصد ال اةيق
ش 3/
105
. (
58
)
" " ف . "ك"ش ُا (
59
)
.ف "د"ش الثالثق (
60
)
. ف "ر"ش ر ن (
60
)
. ف "ر"ش ر ن (
61
)
. ف "ر" "د"ش الالبس ز:د ُا رب . ف "ك"ش رأبس ُا ز:د رب
:حيجح التةع با عمل عغ ال اغل ف ا ثاه الثاني تن ا سألهٍن اما ُ ل ش "رز:د ُا ؟ رز:د ُا رب ؟"؛ ن
ا َ و لَ ف امنِ اأحه ا رن
يَلتًَ َا ةعل ؛ ن اأحه ا يئع عغ ا حداث. الهوامش و:اإلحاالت ف "د"ش تدهمال لالبهداء ا عملت
(
54
)
ف "ك"ش عغ ي عل. :ن ت ف ا سألق
ش
تدمد بن عبد هللا الطائي ا جياني ابن تالك تس يل ال ااد س ميل
ا ئاصد سدئيا
ش تد مد كاتل بتكات داو ال ها العتِي للطباعق الن ت
بٍي ت
1387ه ش82
رب حيان
ا دلس ف الهلييل اله ميل
ش 6/
348
ُبتااير بن ت س ى ال اطٍّف ا ئاصد ال اةيق ف وتح ا خالصق
الكاةيق سدئيا
ش عبد التحمن العثيمٍن زتالا تع د البد ث العلميق ُحياء الحياث اإلحالتي بجاتعق ر
الئتى ت ق ا تتق
ط1
1428هش 3/
75
. 80
(
55
)
.ف "ر"ش ف ا ه
(
56
)
. ف "د"ش ضتب
(
57
)
يحيج ح التةع باأبهداء عغ الن ع ف ا ثالٍن ا لٍَونت تن ا سألهٍن اماش ز:دْ ا ضتبهة ة ز:دْ ا ضتب ة
ر خا ؛ ن التةع أ سئديت ةي رتا الن ع ةيدهاج ُل سئديتت ةعل اصع عد الهئديت ر ل
؛
رخفُّ كةلو َ ق ن ال ال تع يك ن بملق احدِ للك ليس ف ال ال تا يئه ف سئديت ةعل؛ ن 80 ال عل ُد وة غتلَ بن ع الضمٍي. ين ت
ش حيب : ال ها سدئيا
ش عبد السال تد مد ااو ن ت هبق
ِا خا ج الئاات
ط3
1408هش 1/
81
ابن ال و اق تدمد بن عبد هللا بن العباس علل الند
سدئيا
ش
تدم د باحر تدمد الدو يش ت هبق التود الت:اض
ط1
1420ه ش311
عغ بن تدمد
بن عغ ا عضتتي اإلوبيغ
ابن خت
ف
وتح بمل النباج سدئيا ش حل ى تدمد عت باتعق ر
الئتى
د.ط
1419هش 1/
403 عثمان بن عمت ابن ا عابع اإليضاح ف وتح ا ل سدئيا
ش
ت س ى
بنا العليغ تطبعق العاني بغداد
د.ط
1982هش 1/
312
ا عسن بن ُاحر بن عبد هللا
ا تاد
س ضيح ا ئاصد ا سالك ب تح رل يق ابن تالك سدئيا
ش
عبد التحمن عغ حليمان داو ال ت
العتِي دت ا
ط1
1428هش 2/
618 عبد هللا بن عبد التحمن ابن عئيل ا ساعد عغ تس يل
ال ااد سدئيا ش
تدمد كاتل بتكات ن ت باتعق ر الئتى طباعق داو ال ت دت ا داو ا دني
ِبد
ط1
1400
-
1405هش 1/
422
ال اطٍّف
ا ئاصد ال اةيق
ش 3/
105
. الهوامش و:اإلحاالت ين ت
ش
حيب : ال ها
ش 1/
102
ابن
ال و اق علل الند ش312
ِ ابن يعيش وتح ا ل ت هبق ا هنٍّف الئاات
د.ط د.ت
ش 1/
81
ابن تالك تدمد
بن عبد هللا الطائي ا جياني وتح التس يل سدئيا
ش
عبد التحمن السيد تدمد بد ا خه ن هجت للطباعق
الن ت اله زيع اإلعالن ِالئاات
ط1
1410هش 2/
141 رب حيان ا دلس ف الهلييل اله ميل
ش 6/
295
318
350 ا عسن بن ُاحر بن عبد هللا ا تاد ا جنى الداني ف حت ف ا عاني سدئيا
ش
ِ ةخت الدين ُبا
تدمد دير ةاضل داو ال هع العلميق بٍي ت
ط1
1413ه ش343
ال اطٍّف
ا ئاصد ال اةيق
ش 3/
75
. (
62
)
" " ف
."ك"ش ز:د (
64
)
ةلك ا خياو ُن و ب اأحر ا غ ه عن ُن و وةعه ذلك حمال عغ السبٍّف ت :ج ز ةي
.التةع عغ اأبهداء تع عد ا عمل عغ السبٍّ ت ف ا عمل عغ السبٍّف ت ف ال ا سألق ا سألهٍن الل هٍن
سلً ا ر ل تن ا عمل عغ اأبهداء؛ ل ب د امنِ اأحه ا ال ف سطلع ال عل. ين ت
ش
ابن ع و تة ثةلة
ا ئت
ش
155
ابن رِي التبيع عبيد هللا بن رحمد ا وبيغ السب ف البسيط ف وتح بمل النباج
سدئيا
ش عياد بن
عيد الثبي ف داو الغت اإلحالتي بٍي ت
ط1
1407هش 2/
659
ابن النداس تدمد 81 بن ُبتااير ا علٍّف الهعليئق عغ ا ئت- وتح ا ئت ا سم ى الهعليئق سدئيا
ش
خٍي عبد التاض ف
ِعبد اللطيف ت هبق داو النتان للن ت اله زيع ا دينق ا ن و
ط1
1426هش 1/
392
رب حيان
ا دلس ف الهلييل اله ميل
ش 6/
139 ا تاد ا جنى الداني ش343
. آ ُذا حة متلَ عغ ال اغل ةإ يهعٍ ن حمل عغ رحداما؛ أحهدالق رن يةدمل علً ما تعْا؛ أخهالة ما ف اإلعتا
أ
الله
ر بن ُبتااير ا علٍّف الهعليئق عغ ا ئت- وتح ا ئت ا سم ى الهعليئق سدئيا
ش
خٍي عبد التاض ف
ِعبد اللطيف ت هبق داو النتان للن ت اله زيع ا دينق ا ن و
ط1
1426هش 1/
392
رب حيان
ا دلس ف الهلييل اله ميل
ش6/
139 ا تاد ا جنى الداني
ش343
. (
86
)
.ف " "ش ن ا ل
(
87
)
يجت
الضمٍي ا ن ل أ ال احهئالل تجتى السبٍّف ت ف بميع تساال الا البا . ين ت
ش
رب
العالء رحمد بن عبد هللا بن حليمان الهن خ
ا عت
وحالق ا الا ق سدئيا
ش
عبد العن:ن ا يمنف داو ال هع
العلميق بٍي ت ط1
1424ه ش223 ال او ُاحر بن عغ بن تدمد البطلي س ف
: وتح ها حيب
سدئيا
ش
عن:نِ اللبياني
رطت حق د ه وا باتعق طيبق
ِبا دينق ا ن و1434ه ش407
ابن ع و
وتح ا جمل
ش 1/
373
اظت ا جيش تدمد بن ي حف بن رحمد سم يد الئ اعد ب تح تس يل
ال ااد سدئيا
ش
عغ تدمد ِةاخت زتالا داو السال للطباعق الن ت اله زيع الحيبمق الئاات
ط1
1428هش 4/
1711
1716
. (
88
)
.ف " " "ك"ش ةا ن الت
(
89
)
.ف "ر"ش ةا ن الن كال ااتان
(
90
)
.ف "د"ش السببٍن
(
91
)
.ف "ظ"ش ُد ت ى عغ ر ك
(
92
)
. ذلك ف ا سألق الثالثق
(
93
)
. ف "ر"ش ُأ ب (
92
)
. ذلك ف ا سألق الثالثق (
92
)
. ذلك ف ا سألق الثالثق (
93
)
. ف "ر"ش ُأ ب ُذا دخل امنِ اأحه ا عغ الناف ةا ثي رن يك ن اأحه ا دض الهئت:ت :هد ه الن ي ُل ُثبات
ئ ل تعال ش {رَلَرو نَ و تَحو لَكَ صَدو وَكَ } ُد يبئ ف الئليل اأحه ا ة عغ حئيئه ا تتاد ا فلف انا. حبع
اُه او ا فلف عغ الا ا حل- ُن كان ُليال- رن امنِ اأحه ا ةي لطلع اله ديا تعل ر أ
َيةسهخد تن رد ات اأحه ا لطلع اله ديا ُأ ال منِ "ال" ُأ رن "ال" أ يةسو هَ و َرة ب ا ف الن ي ةهعٍ ن
احهخدا ال منِ. الهوامش و:اإلحاالت ُذا حة متلَ عغ ال اغل ةإ يهعٍ ن حمل عغ رحداما؛ أحهدالق رن يةدمل علً ما تعْا؛ أخهالة ما ف اإلعتا
ةأحداما تتة ا اآلخت تن . ين ت
ش
رب حعيد ا عسن بن عبد هللا بن ا
تزبان
السٍياف وتح ال ها
سدئيا
ش
رحمد حسن ت دل عغ حيد عغ داو ال هع العلميق بٍي ت
ط1
2008
ش 1/
415
. (
67
)
" " ف ِ"ك"ش تع ا ع ه. ف "د"ش تع ا عل. الكلمهان حاُطهان تن "ر". ُد عل ل ا فلف ف ال ئت
الهاليق لهئديت ال اع .ل (
67
)
" " ف ِ"ك"ش تع ا ع ه. ف "د"ش تع ا عل. الكلمهان حاُطهان تن "ر". ُد عل ل ا فلف ف ال ئت
الهاليق لهئديت ال اع .ل (
70
)
ُ ل ش " :ةئَ د وة ُذا ب ش رَرَاان ز:دا رخ ضَتَ َ رخ غالتَ ة ؟" حاُط تن " " "د" "ك". ودت ف
"ر"ش
..... ِعلو ُذا كان ب ش رراان
(
)اه
" " "ك"
اه
"ر" ا
"ر"ش
..... ِعلو ُذا كان ب ش رراان
(
71
)
.ف "ر"ش ف تثاه. ف " " "ك"ش تمن تثاه
(
72
)
تن انا سبدر ن ."سخق ت هبق ا حد ُد وتنت ل ا با عتف "ظ
(
73
)
الهئديتش ضَتَبو ة ز:دْ ا ضَتَبوهة ة. ين ت
ش
حيب : ال ها
ش 1/
81 رب البيكات عبد
التحمن
بن تدمد
ابن
ا باو
اإل اف ف تساال ا خالف بٍن الند :ٍن الب ت:ٍن الك ةيٍن سدئيا
ش
تدمد تديف الدين عبد
ا عميد ا هبق الع ت:ق بٍي ت
ط1
1407هش 1/
82 ابن يعيش وتح ا ل
ش 2/
30
ال اطٍّف
ا ئاصد ال اةيق
ش 3/
68
. 82
(
74
)
.ف "ظ"ش ا ئدو
(
75
)
.ِف "د"ش للضت و
(
76
)
. ف "ر"ش ز:د رضتبه
(
77
)
.ف " " "د" "ظ" "ك"ش ةعل تضمت
(
78
)
.ف " " "د" "ظ" "ك"ش ح ل
(
79
)
.ف "ر"ش ا رل ا
(
80
)
. "تن" حاُطق تن "ر" ف الا ا ضع الل يلي
(
81
)
.ف "د"ش التابعق التابعق
(
82
)
. ف " " "ك"ش رز:د ُيا 82 (
83
)
.ف " " "ك"ش ُ ما ُد بنا للم ع ه. الهوامش و:اإلحاالت ف "د"ش ُ ما ُدتنا ا ع ه
(
84
)
.ف "ر" " " "ك"ش ف الا
(
85
)
. ف "ظ"ش عغ ر و
(
86
)
.ف " "ش ن ا ل
(
87
)
يجت
الضمٍي ا ن ل أ ال احهئالل تجتى السبٍّف ت ف بميع تساال الا البا . ين ت
ش
رب
العالء رحمد بن عبد هللا بن حليمان الهن خ
ا عت
وحالق ا الا ق سدئيا
ش
عبد العن:ن ا يمنف داو ال هع
العلميق بٍي ت ط1
1424ه ش223 ال او ُاحر بن عغ بن تدمد البطلي س ف
: وتح ها حيب
سدئيا
ش
عن:نِ اللبياني
رطت حق د ه وا باتعق طيبق
ِبا دينق ا ن و1434ه ش407
ابن ع و
وتح ا جمل
ش 1/
373
اظت ا جيش تدمد بن ي حف بن رحمد سم يد الئ اعد ب تح تس يل
ال ااد سدئيا
ش
عغ تدمد ِةاخت زتالا داو السال للطباعق الن ت اله زيع الحيبمق الئاات
ط1
1428هش 4/
1711
1716
. ين ت
ش
السٍياف وتح ال ها
ش 3/
416
جر الدين تدمد بن ا عسن التض ف ا ححياباذ وتح
التض ف عغ الكا
ةيق سدئيا
ش
ي حف حسن عمت تطابع ال ت ق بٍي ت ن ت باتعق بنغاز ليبيا د.ط د.تش
4/
447
رب حيان ا دلس ف تةدَ م د بن ية حة ف اوت اف الضت تن لسان العت سدئيا
ش
وبع عثمان تدمد
ِت هبق ا خا ج الئاات
ط1
1418هش 4/
1861
ابن ا ا ا او عبد هللا بن ي حف ا عنبغ تغنف
اللبيع عن هع ا عاو:ع سدئيا
ش
تازن
ا باوك تدمد عغ ح
مد هللا داو ال ت دت ا ط6
1985
ش21
457 اظت ا جيش سم يد الئ اعد
ش 9/
4320
4477
عبد التحمن بن رِي ب ت بن تدمدالسي طي امع
ال اتع ف وتح بمع ا ج اتع سدئيا ش : عبد العاه حالر ت ت ن ت داو البد ث العلميق الك
د.ط
1394
-
1400هش 4/
312
. 83 8
(
94
)
حة لت فَ ضمٍية الن ع ا ن ل ن ال عل تسل ط عغ اأحر ال اات. ين ت التض ف وتح التض ف عغ
الكاةيق
ش 1/
474
. (
95
)
.ِف "د"ش ةئدو
(
96
)
.ف "ر"ش ف الا
(
97
)
"ف "ر" "د."ظ"ش ا هغل
(
98
)
.ف "ر"ش ةاعل ا لك و. ف " " "ك"ش ةاعل ا ع ه ا لك و
(
99
)
ر ش ا لك و ف ا سألق الثالثق ال ف ُبل ال ت ثال اش رز:د ضَتَ َ رخ غالتَ ة َ؟ الهئديت ةً اش ر
رَاان
ز:دْ ا رخ ضَتَ َ رخ غالتَ ة؟ سخهل ان ف رن ال اعل ا ئد و ف ا سألق الثا لثق احر ظاات ف التابعق
. ضمٍي تن ل الا اأخهالف أ رثت ل ؛ ن الضمٍي ا ن ل كالسبٍّف ف بميع تساال الا البا
ين ت
ش ابن ع و وتح ا جمل
ش 1/
373
. (
100
)
.ف " " "ك"ش ةإن
(
101
)
."ُ ل ش "لر يَضوتت و " حاُطق تن "ر
(
102
) ين ت
ش
ابن ع و عغ بن تفتن بن تة دَ م د ا و عَ ضوتَتت ي اإلوبيغ وتح ا ئت ت نسخق خطيق
تد ظق ف ا خنا ق العاتق بالتباط سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
103
)
.ف "ك"ش ن ا جملق
(
104
)
.ف "ك"ش غٍي تعنى الثا يق
(
105
)
ف " " "ك"ش رن الهئديت ف ش . لر يضت . ف "د" "ظ"ش رن الهئديتش لر يضت
(
106
)
."ف كل النسخ تا عدا " "ش "ز:دْ ا
(
107
)
.ف كل النسخ تا عدا "ر"ش "ُأ ا ". حيه ت حة تن ال ا فلف بعد ُليل عد ص ا ال اأخهالةات
(
108
)
.ف "ر"ش ت ع ل ا د و
(
109
)
بملق " بئاء ال اعل ما ُدو ا ةيما سئد حلف ال عل ةاعل
."ا د و" حاُطق تن "ظ
(
110
)
ذلك ف ال ا سألق حينما حملو َ اأحرَ ا غ ه عن عغ الضمٍي ا ن ةن بوهَ ة تثال اش رز:دْ ا
. ُيا لر يَضو تت و ُأ ا . الهئديتش رَلَرو يَضوتت و ز:دْ ا ُأ ا لر يَضوتت و ُأ ا
(
111
)
.ف "ر"ش ةإن
(
112
) ف "ر"ش احهلنتهر. ف " "ش حتن تسر. (
113
) ف " " "ك"ش "سئد "يت ف ا ل ال عل". ف "د.""ظ"ش "سئديت ا ل ال عل
(
114
)
.ف "ظ"ش أز ا نف
(
115
)
.ف " "ش رأ استى رن الهئديت. ف "د"ش ُأ رن الهئديت. ف "ظ"ش رأ ستى رن الئديت 84
(
94
)
حة لت فَ ضمٍية الن ع ا ن ل ن ال عل تسل ط عغ اأحر ال اات. ين ت التض ف وتح التض ف عغ
الكاةيق
ش 1/
474
. (
95
)
.ِف "د"ش ةئدو
(
96
)
.ف "ر"ش ف الا
(
97
)
"ف "ر" "د."ظ"ش ا هغل
(
98
)
.ف "ر"ش ةاعل ا لك و. ف " " "ك"ش ةاعل ا ع ه ا لك و
(
99
)
ر ش ا لك و ف ا سألق الثالثق ال ف ُبل ال ت ثال اش رز:د ضَتَ َ رخ غالتَ ة َ؟ الهئديت ةً اش ر
رَاان
ز:دْ ا رخ ضَتَ َ رخ غالتَ ة؟ سخهل ان ف رن ال اعل ا ئد و ف ا سألق الثا لثق احر ظاات ف التابعق
. ضمٍي تن ل الا اأخهالف أ رثت ل ؛ ن الضمٍي ا ن ل كالسبٍّف ف بميع تساال الا البا
ين ت
ش ابن ع و وتح ا جمل
ش 1/
373
. (
100
)
.ف " " "ك"ش ةإن
(
101
)
."ُ ل ش "لر يَضوتت و " حاُطق تن "ر
(
102
) ين ت
ش
ابن ع و عغ بن تفتن بن تة دَ م د ا و عَ ضوتَتت ي اإلوبيغ وتح ا ئت ت نسخق خطيق
تد ظق ف ا خنا ق العاتق بالتباط سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
103
)
.ف "ك"ش ن ا جملق
(
104
)
.ف "ك"ش غٍي تعنى الثا يق
(
105
)
ف " " "ك"ش رن الهئديت ف ش . لر يضت . ف "د" "ظ"ش رن الهئديتش لر يضت
(
106
)
."ف كل النسخ تا عدا " "ش "ز:دْ ا
(
107
)
.ف كل النسخ تا عدا "ر"ش "ُأ ا ". حيه ت حة تن ال ا فلف بعد ُليل عد ص ا ال اأخهالةات
(
108
)
.ف "ر"ش ت ع ل ا د و
(
109
)
بملق " بئاء ال اعل ما ُدو ا ةيما سئد حلف ال عل ةاعل
."ا د و" حاُطق تن "ظ
(
110
)
ذلك ف ال ا سألق حينما حملو َ اأحرَ ا غ ه عن عغ الضمٍي ا ن ةن بوهَ ة تثال اش رز:دْ ا
. ُيا لر يَضو تت و ُأ ا . الهئديتش رَلَرو يَضوتت و ز:دْ ا ُأ ا لر يَضوتت و ُأ ا
(
111
)
.ف "ر"ش ةإن
(
112
) ف "ر"ش احهلنتهر. ف " "ش حتن تسر. (
113
) ف " " "ك"ش "سئد "يت ف ا ل ال عل". "ظ"ش ةإن
(
120
) ف "ر"ش ف با ع ا " " ل اعل ةيك ن ف سئديت. ف."ك"ش ف با ع سئديتك. ف "د"ش ف با ع سئدو:ك
(
121
)
.ف "ر"ش ا سمى احد. ف " " "ك"ش لسمى احد
(
122
)
.ف "ر"ش ُ ما ُدو
(
123
)
َُذا حة مل اأحر ا غ ه عن عغ ال اغل ال اُع بعد "ُأ" ما ف د ش رز:دْ ا ُيا لر ي ضوتت و ُأ ا
بع رن يك ن ال عل ا ئد و تة ثوبَهْا؛ ن اأحر ا غ ه عن يئع تن ال عل ا ئد و ت ُع ال اغل تا
بعد "ُأ" تثب أ غٍي؛ ن اأحه ناء ا ت غ أ يك ن ُأ بعد غٍي ا بع اأحر ا غ ه عن لر سئع
ُبل "ُأ" ةهنئة ضَ َ و يَ ال علت ا ئد وت
ما ئض "ُأ" ال اُعق ُبل ال اغل َ و يَ ال علت ا س ت تت؛
ة بع ُضماو ال عل ا و ةثوبَ ت لي اةا ف ا عنى ال علَ ا س ت تَ ا ن ي ا نئ ضَ ية ة با"ُأ" ةالهئديت ف
ا ثاه ا لك وش رُيا ضَت َ ز:د ؟ ُيا لر يَضو تت و ُأ ا ؛ للا لر يصح ُ اة ا ع تت ا عهمتدت عغ الن ي
اأحه ناء ف ا جملق ا ئد وِ لعد ب د "ُأ" ُبل اأحر ا غ ه عن . ين ت
ش
التض ف وتح التض ف
عغ الكاةيق
ش 1/
474 ابن ا ا ا او تغنف اللبيع ش837
. (
124
)
.ف "ر"ش حَ بَبْا (
119
)
"ف "ر
. "ظ"ش ةإن
(
120
) ف "ر"ش ف با ع ا " " ل اعل ةيك ن ف سئديت. ف."ك"ش ف با ع سئديتك. ف "د"ش ف با ع سئدو:ك
(
121
)
.ف "ر"ش ا سمى احد. ف "د.""ظ"ش "سئديت ا ل ال عل
(
114
)
.ف "ظ"ش أز ا نف
(
115
)
.ف " "ش رأ استى رن الهئديت. ف "د"ش ُأ رن الهئديت. ف "ظ"ش رأ ستى رن الئديت (
97
)
"ف "ر" "د."ظ"ش ا هغل
(
98
)
.ف "ر"ش ةاعل ا لك و. ف " " "ك"ش ةاعل ا ع ه ا لك و
(
99
)
ر ش ا لك و ف ا سألق الثالثق ال ف ُبل ال ت ثال اش رز:د ضَتَ َ رخ غالتَ ة َ؟ الهئديت ةً اش ر
رَاان
ز:دْ ا رخ ضَتَ َ رخ غالتَ ة؟ سخهل ان ف رن ال اعل ا ئد و ف ا سألق الثا لثق احر ظاات ف التابعق
. ضمٍي تن ل الا اأخهالف أ رثت ل ؛ ن الضمٍي ا ن ل كالسبٍّف ف بميع تساال الا البا
ين ت
ش ابن ع و وتح ا جمل
ش 1/
373
. (
)ن
ر ي تت
(
102
) ين ت
ش
ابن ع و عغ بن تفتن بن تة دَ م د ا و عَ ضوتَتت ي اإلوبيغ وتح ا ئت ت نسخق خطيق
تد ظق ف ا خنا ق العاتق بالتباط سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
103
)ف "ك"ش ن ا جملق (
102
) ين ت
ش
ابن ع و عغ بن تفتن بن تة دَ م د ا و عَ ضوتَتت ي اإلوبيغ وتح ا ئت ت نسخق خطيق
تد ظق ف ا خنا ق العاتق بالتباط سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
103
)
.ف "ك"ش ن ا جملق
(
104
)
.ف "ك"ش غٍي تعنى الثا يق
(
105
)
ف " " "ك"ش رن الهئديت ف ش . لر يضت . ف "د" "ظ"ش رن الهئديتش لر يضت (
115
)
.ف " "ش رأ استى رن الهئديت. ف "د"ش ُأ رن الهئديت. ف "ظ"ش رأ ستى رن الئديت 84 (
116
)
.ف " " "ك"ش علة ما
(
117
)
. ف " " "د" "ك"ش بعد سئديت
(
118
)
.ف "ر"ش تن
(
119
)
"ف "ر
. ف " " "ك"ش لسمى احد ُ
(
123
)
َُذا حة مل اأحر ا غ ه عن عغ ال اغل ال اُع بعد "ُأ" ما ف د ش رز:دْ ا ُيا لر ي ضوتت و ُأ ا
بع رن يك ن ال عل ا ئد و تة ثوبَهْا؛ ن اأحر ا غ ه عن يئع تن ال عل ا ئد و ت ُع ال اغل تا
بعد "ُأ" تثب أ غٍي؛ ن اأحه ناء ا ت غ أ يك ن ُأ بعد غٍي ا بع اأحر ا غ ه عن لر سئع
ُبل "ُأ" ةهنئة ضَ َ و يَ ال علت ا ئد وت
ما ئض "ُأ" ال اُعق ُبل ال اغل َ و يَ ال علت ا س ت تت؛
ة بع ُضماو ال عل ا و ةثوبَ ت لي اةا ف ا عنى ال علَ ا س ت تَ ا ن ي ا نئ ضَ ية ة با"ُأ" ةالهئديت ف
ا ثاه ا لك وش رُيا ضَت َ ز:د ؟ ُيا لر يَضو تت و ُأ ا ؛ للا لر يصح ُ اة ا ع تت ا عهمتدت عغ الن ي
اأحه ناء ف ا جملق ا ئد وِ لعد ب د "ُأ" ُبل اأحر ا غ ه عن . ين ت
ش
التض ف وتح التض ف
عغ الكاةيق
ش 1/
474 ابن ا ا ا او تغنف اللبيع ش837
. (
124
)َ َ ْا
ف "ر" (
126
)
.ف "ر"ش ا سبٍّف. ف "ظ"ش ا ستثنى (
128
)
."بملقش "اأبهداء رن سدمل عغ " حاُطق تن "ظ
(
129
)
. ذلك ن الضمٍي ا ن ل يجت تجتى ال اات السبٍّف ت ف بميع تساال با اأوهغاه ما حبا
ين ت
ش ابن ع و وتح ا جمل
ش 1/
373 ابن رِي التبيع البسيط
ش 2/
659
. (
130
)
تن ُ ل ش " لك ف ال ا سألق" ُل ُ ل ش "رخا ُأ ا " حاُط تن "ر". ود ا ثاه ف بئيق النسخ
..." ا لاش لر يَضوتت و ُأ ا رخا ُأ ا ". "ك"ش ما وةع
(
168
)
. ف " "ش لر يضت ز:دا ُأ يا . ف "ك"ش لر يضت ز:دا ُأ ُيا . ف "د"ش لر يضت ز:د ُأ ُيا
(
169
)
ُ ُ ل ش "لر يضت ُأ."يا " حاُط تن "ر
(
170
)
.ف "ر" "ظ"ش ة ع
(
171
)
.ف "د"ش رز:دا اان
(
172
)
.ف " " "ك"ش رز:د
(
173
)
."ُ ل ش "ته ال" تطم حق ف "ر
(
174
)
.ف "ظ"ش ُن
(
175
)
."ُ ل ش "لر ي ن" تطم حق ف "ر
(
176
)
. ف "ر"ش تن باب
(
177
)
.ف " " "ك"ش ز:د
(
178
)
.ف "ر"ش "ي ". ا ح تن الناسخ
(
179
)
.ف " " "ك"ش ضمٍي
(
180
)
.ف "ر"ش ا ن ل
(
181
)
."ُ ل ش "ةدينئل" حاُط تن كل النسخ تا عدا "ر
(
182
)
.ف "د"ش ُذ (
160
)
يلن علي بَ عولة ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يك ن غٍي ا ع ه؛ ن رصل ال اعلت رن
يك ن تة فَث تتْا ا ع ه ب تة هَأَث تتْا رصل ا و ةفَث تت رن يةغايت ا هأث تت يلن علي بَ عولة ا ع هت ال ضلقت
.و نا ةل حلة َ ا ع ه ف ُ لكش "رز:دْ ا ضَتَ َ " لر سَصت ح ا جملق؛ لعد اأحهغناء عن ا ع ه ةً ا
ين ت
ش ابن الستاج رب ب ت تة دَ م د بن الس ت الوبَغودَ اد ا ص ه ف الند سدئيا
عبد ا عسٍن
ال هغ تفحسق التحالق بٍي ت
ط1
1405هش 2/
121
240
السٍياف وتح ال ها
ش 1/
423
425 ابن تالك تس يل ال ااد ش84 ابن رِي التبيع البسيط
ش 2/
660
التض ف وتح التض ف عغ
الكاةيق
ش 4/
169 اظت ا جيش سم يد الئ اعد
ش 4/
1710
1715
1750
. (
162
)
.ف "ر"ش ال اعل (
163
)
.ف "ر"ش تن ال (
164
) ف " " "ك"ش ز:د. ف "ظ"ش ر
.رز:د (
164
) ف " " "ك"ش ز:د. ف "ظ"ش ر
.رز:د (
165
)
.ف "د"ش ُأ ُيا (
166
)
.ف "ظ"ش ليهعٍن (
167
)
" " ف . "ك"ش ما وةع (
168
)
. ا ح تن النساخ؛ ُذ أ تعنى له تاو "ُأ ا " سئديم ا
.عغ ا ع ه
(
131
)
.ف "د"ش ا ال اعل
(
132
)
.ف "ر"ش بتنتي ق 85 (
133
) ين ت
ش ابن تالك تدمد بن عبد هللا الطائي ا جياني وتح الكاةيق ال اةيق سدئيا
ش
عبد ا نعر رحمد
ات:د تت ن البدس العلمف ُحياء الحياث اإلحالتي بكليق ال تيعق الدواحات اإلحالتيق
باتعق ر
الئتى
ت ق ا تتق
ط1
1402
ه ش2/
590 رب حيان ا دلس ف الهلييل اله ميل
ش 6/
287
. (
134
) "ر ."خا " حاُطق تن "ر (
141
) الا غٍي باان ما حبا. ين ت
ش ابن ا ا ا او تغنف اللبيع ش837
. (
142
)
.ف "ر"ش ز:د. ف " " "ك"ش ز:دا (
143
)
.ف "ر"ش ذلك (
146
)
.ف "د"ش بهئديتش رةاٍن (
148
)
.ف "ر"ش لا (
150
) ُ ل ش "رَضةتت َ ز:د ... ؟ ُن حَ مَ لو َ عغ الض ."مٍي ب بهئديت" حاُطق تن "ر 86 (
160
) يلن علي بَ عولة ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يك ن غٍي ا ع ه؛ ن رصل ال اع
يك ن تة فَث تتْا ا ع ه ب تة هَأَث تتْا رصل
ا و ةفَث تت رن يةغايت ا هأث تت يلن علي بَ عولة ا ع هت ال
و نا ةل حلة َ ا ع ه ف ُ لكش "رز:دْ ا ضَتَ َ " لر سَصت ح ا جملق؛ لعد اأحهغناء عن ا ع ه
ين ت
ش ابن الستاج رب ب ت تة دَ م د بن الس ت الوبَغودَ اد ا ص ه ف الند سدئيا عبد ا ع
ال هغ تفحسق التحالق بٍي ت
ط1
1405هش 2/
121
240
السٍياف وتح ال ها
ش 1/
425 ابن تالك تس يل ال ااد ش84 ابن رِي التبيع البسيط
ش 2/
660 التض ف وتح التض ف
الكاةيق
ش 4/
169 اظت ا جيش سم يد الئ اعد
ش 4/
1710
1715
1750
. (
161
)
.ف "ظ"ش رن ي من
(
162
)
.ف "ر"ش ال اعل
(
163
)
.ف "ر"ش تن ال
(
164
) ف " " "ك"ش ز:د. ف "ظ"ش ر
.رز:د
(
165
)
.ف "د"ش ُأ ُيا
(
166
)
.ف "ظ"ش ليهعٍن
(
167
)
" " ف . ف " "ش لر يضت ز:دا ُأ يا . ف "ك"ش لر يضت ز:دا ُأ ُيا . ف "د"ش لر يضت ز:د ُأ ُيا
(
169
)
ُ ُ ل ش "لر يضت ُأ."يا " حاُط تن "ر (
168
)
. ف " "ش لر يضت ز:دا ُأ يا . ف "ك"ش لر يضت ز:دا ُأ ُيا . ف "د"ش لر يضت ز:د ُأ ُيا
(
169
)
ُ ُ ل ش "لر يضت ُأ."يا " حاُط تن "ر (
170
)
.ف "ر" "ظ"ش ة ع (
171
)
.ف "د"ش رز:دا اان (
172
)
.ف " " "ك"ش رز:د (
173
)
."ُ ل ش "ته ال" تطم حق ف "ر 87 (
183
)
."ُ ل ش "الضمٍي" حاُط تن "ظ
(
184
)
.ف " " "ك"ش ن اأحر انا
(
185
)
.ف كل النسخ تا عدا "ر"ش رز:د
(
186
)
.ف " " "ك"ش لر
(
187
)
.ف "د"ش ز:د
(
188
)
ين ت السٍياف وتح ال ها
ش 1/
423
425 ا عت وحالق ا الا ق ش222
اظت ا جيش سم يد
الئ اعد
ش 4/
1711
. (
189
)
.ف "د"ش با دل
(
190
)
. َ َف "د"ش رَضَت
(
191
)
. ف "ظ"ش لر يضت
(
192
)
يلن علي بَ عولة ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يك ن غٍي ا ع ه؛ ن رصل ال اعلت رن
يك ن تة فَث تتْا ا ع ه ب تهأث تتْا رصل ا فث تت رن يةغايتتَ ا هأث ت
ت. ين ت
ش
السٍياف وتح ال ها
ش 1/
425
ابن ع و وتح ا جمل
ش 1/
372 ابن رِي التبيع البسيط
ش 2/
659
التض ف وتح التض ف عغ
الكاةيق
ش 1/
475 ابن ا ا ا او تغنف اللبيع
ش
294
. (
193
)
.ف "ر"ش صاو
(
194
) ين ت
ش ابن ع و وتح ا جمل
ش 1/
375 ابن رِي التبيع البسيط
ش 2/
659
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
356
357 اظت ا جيش سم يد الئ اعد
ش 4/
1711
. (
195
)
.ف " " "ك"ش رز:دا
(
196
) لر يلن ف ال ا سألق بَ عولة ا ع هت ةاعال ف ا عنى
؛
ن ا ع ه ا ه تن ت ع ل "ظن " رخ ات ا
ت ع ه ف الل مل أ ف ا عئيئق ا ع ه ف ا عئيئق ُ ما ا تضم ن ا جملق؛ بدليل ر ك ل ُل ش
"ظنن ة ز:دْ ا تساةتا" لر يئع ال كُّ ف "ز:د" الل ا ا ع ه ا ه ُ ما ُع ال كُّ ف ح ت ةجاز
اس اُ ما ل ا؛ ن ما ليسا ف ا عئيئق ةاعال ت ع أ ب . ين ت
ش
السٍياف وتح ال ها
ش 1/
424
ابن
ال و اق علل ال ند ش286 ابن ع و وتح ا جمل
ش 1/
372 التض ف وتح التض ف عغ الكاةيق
ش
4/
170
. (
197
)
. ف "ر"ش ة ل
(
198
)
.ف "ر"ش رلر ي ن
(
199
)
تن ُ ل ش "ةإن حملو َ عغ ا ه ل بو َ ..." ُل ُ ل ش "...لر يَ ةن ة ُاامْ ا ُأ ا"
."حاُط تن "ظ
(
200
)
.ف "د"ش ةإن (
188
)
ين ت السٍياف وتح ال ها
ش 1/
423
425 ا عت وحالق ا الا ق ش222
اظت ا جيش سم يد
الئ اعد
ش 4/
1711
. (
189
)
.ف "د"ش با دل (
192
)
يلن علي بَ عولة ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يك ن غٍي ا ع ه؛ ن رصل ال اعلت رن
يك ن تة فَث تتْا ا ع ه ب تهأث تتْا رصل ا فث تت رن يةغايتتَ ا هأث ت
ت. ين ت
ش
السٍياف وتح ال ها
ش 1/
425
ابن ع و وتح ا جمل
ش 1/
372 ابن رِي التبيع البسيط
ش 2/
659
التض ف وتح التض ف عغ
الكاةيق
ش 1/
475 ابن ا ا ا او تغنف اللبيع
ش
294
. (
194
) ين ت
ش ابن ع و وتح ا جمل
ش 1/
375 ابن رِي التبيع البسيط
ش 2/
659
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
356
357 اظت ا جيش سم يد الئ اعد
ش 4/
1711
. (
195
)
.ف " " "ك"ش رز:دا (
194
) ين ت
ش ابن ع و وتح ا جمل
ش 1/
375 ابن رِي التبيع البسيط
ش 2/
659
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
356
357 اظت ا جيش سم يد الئ اعد
ش 4/
1711
. (
195
)
.ف " " "ك"ش رز:دا (
196
) لر يلن ف ال ا سألق بَ عولة ا ع هت ةاعال ف ا عنى
؛
ن ا ع ه ا ه تن ت ع ل "ظن " رخ ات ا
ت ع ه ف الل مل أ ف ا عئيئق ا ع ه ف ا عئيئق ُ ما ا تضم ن ا جملق؛ بدليل ر ك ل ُل ش
"ظنن ة ز:دْ ا تساةتا" لر يئع ال كُّ ف "ز:د" الل ا ا ع ه ا ه ُ ما ُع ال كُّ ف ح ت ةجاز
اس اُ ما ل ا؛ ن ما ليسا ف ا عئيئق ةاعال ت ع أ ب . ين ت
ش
السٍياف وتح ال ها
ش 1/
424
ابن
ال و اق علل ال ند ش286 ابن ع و وتح ا جمل
ش 1/
372 التض ف وتح التض ف عغ الكاةيق
ش
4/
170
. 88
(
197
)
. ف "ر"ش ة ل
(
198
)
.ف "ر"ش رلر ي ن
(
199
)
تن ُ ل ش "ةإن حملو َ عغ ا ه ل بو َ ..." ُل ُ ل ش "...لر يَ ةن ة ُاامْ ا ُأ ا"
."حاُط تن "ظ
(
200
)
.ف "د"ش ةإن
(
201
)
.ف "ر"ش ا ه ل 88
(
197
)
. ف "ر"ش ة ل
(
198
)
.ف "ر"ش رلر ي ن
(
199
)
تن ُ ل ش "ةإن حملو َ عغ ا ه ل بو َ ..." ُل ُ ل ش "...لر يَ ةن ة ُاامْ ا ُأ ا"
."حاُط تن "ظ
(
200
)
.ف "د"ش ةإن
(
201
)
.ف "ر"ش ا ه ل 88 (
202
)
. ف "ر"ش رَظَن
(
203
)
."ف " " "ك"ش ز:دا. ه حاُطق تن "د
(
204
)
.ف " " "د" "ظ" "ك"ش س يدان
(
205
)
. ف "ر"ش ا هئد
(
206
)
. ف "ظ"ش سئد
(
207
)
ف كل النسخش "... ضمٍيا ته ال تتة عا تع حبٍّف". الهصعيح تن رتثلت ا ال ف حاُ ا ا فلف تن
َحة و مت َا؛ ن ال اغل ُذا كان ضمٍيا ته ال تتة عا تع حبٍّف َب عَ ف ا غ ه عن ا عملة عغ الضمٍي
ا ه ل أ غٍي د ن س يل ُد ذ ت ا فلف ال ا سألق بعل ا ا سألق ا ل تن ا ساال ا خمس ف
ا جم عق الثا يق. :ن ت
ش
: ال او وتح ها حيب ش406
ابن ع و ا ئت
ش 1/
91
ابن
النداس الهعليئق عغ ا ئت
ش 1/
393
. (
202
)
. ف "ر"ش رَظَن
(
203
)
."ف " " "ك"ش ز:دا. ه حاُطق تن "د
(
204
)
.ف " " "د" "ظ" "ك"ش س يدان
(
205
)
. ف "ر"ش ا هئد
(
206
)
. ف "ظ"ش سئد
(
207
)
ف كل النسخش "... ضمٍيا ته ال تتة عا تع حبٍّف". الهصعيح تن رتثلت ا ال ف حاُ ا ا فلف تن
َحة و مت َا؛ ن ال اغل ُذا كان ضمٍيا ته ال تتة عا تع حبٍّف َب عَ ف ا غ ه عن ا عملة عغ الضمٍي
ا ه ل أ غٍي د ن س يل ُد ذ ت ا فلف ال ا سألق بعل ا ا سألق ا ل تن ا ساال ا خمس ف
ا جم عق الثا يق. :ن ت
ش
: ال او وتح ها حيب ش406
ابن ع و ا ئت
ش 1/
91
ابن
النداس الهعليئق عغ ا ئت
ش 1/
393
. (
208
)
ف ."ك"ش ال عل تع تن
(
209
)
. ف "ر"ش تن با
(
210
)
.ف "ر"ش ل مل
(
211
)
."ُ ل ش "الضمٍي" حاُط تن "ر
(
212
)
" " ف."ك"ش ز:د
(
213
) ف "د"ش ةإن
(
214
)
. ف "ر"ش حمل
(
215
)
" " ف ."ك"ش ز:د
(
216
)
.ف "د"ش ي ق
(
217
)
َ الهئديت ف ا عالق ا ل ش رلر يَضو تت و ز:دْ ا رخ ؟ لر يَضوتتبو ة رخ . الهئديت ف ا عالق الثا يق ُن حَ مَ لو
َ عغ ا ه ل ةن بو َ ش رلر يَ ةن ز:دْ ا ُاامْا رخ ؟ لر يَ ةن ة ُاامْا رخ . (
228
)
باز تج ء ةاعل ت ع ه "عد ةئد" ضمٍيين ته لٍن تة ه دت دَ ا عنى احهعماه تجاز ٌّ ؛ ن
ال اعل ا ع ه ةً ما أ بةد رن يك ا ت ب دين ةإذا عَدت َ ال اعلة سَ ة صاو عادتْ ا تعد تْ ا ف آن
احد الا تداه؛ ن تن عَدت َ ويئا كان ت ب دْا؛ للا ة أصل اأحهعماهش عَدت تَ نف غٍي ب لا ين ه
اسداد ال اعل ا ع ه. ين ت
ش رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد
عغ النجاو رحمد ي حف جاتي عالر ال هع بٍي ت
ط3
1403هش 1/
334
2/
106
السٍياف
وتح ال ها
ش 1/
424
3/
130 ابن يعيش وتح ا ل
ش 7/
88 ابن ع و وتح ا جمل
ش
1/
372 رب حيان ا دلس ف الهلييل اله ميل
ش 6/
113
. (
229
)
اتهنع ا سألق ن سئديتاا حٍن َ وعت ا غ هت عن حمالْ عغ الضمٍي ا ن ا ش ضَتَ َ ز:دْ ا ا
ضَتَبَ ة سئديتاا حٍن وَةوعت ت حمالْ ع .غ الضمٍي ا سهحي ا تة اش ضَتَبَ ة ز:د ضَتَبَ ة ر ش ضَتَ َ ز:د سَ ة
ة ي ال ب ا ه تَعَد ى ةتعولة ا ضمتت ا ه ل ُل ظاات ا تمهنع ف بميع رب ا العتبيق ف ال ب
الثاني تَعَد ى ةتعولة ال اات ُل ضمٍي ا ه ل ا تمهنع ف غٍي با (ظَن )؛ ا يلن علي ف ال ب ٍن-
ما حبا-
تن بَ عولت ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يةغايت ا ع ه ف الل مل ا عنى ر ف الل مل
ةئط. ين ت
ش
حيب : ال ها
ش 2/
366
ابن الستاج ا ص ه
ش 2/
121
241 ابن يعيش وتح ا ل
ش
7/
88
ابن ع و
وتح ا جمل
ش 1/
372
التض ف وتح التض ف عغ الكاةيق
ش 4/
169
ابن النداس
الهعليئق عغ ا ئت
ش 1/
393
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
112
357
. (
229
)
اتهنع ا سألق ن سئديتاا حٍن َ وعت ا غ هت عن حمالْ عغ الضمٍي ا ن ا ش ضَتَ َ ز:دْ ا ا
ضَتَبَ ة سئديتاا حٍن وَةوعت ت حمالْ ع .غ الضمٍي ا سهحي ا تة اش ضَتَبَ ة ز:د ضَتَبَ ة ر ش ضَتَ َ ز:د سَ ة
ة ي ال ب ا ه تَعَد ى ةتعولة ا ضمتت ا ه ل ُل ظاات ا تمهنع ف بميع رب ا العتبيق ف ال ب
الثاني تَعَد ى ةتعولة ال اات ُل ضمٍي ا ه ل ا تمهنع ف غٍي با (ظَن )؛ ا يلن علي ف ال ب ٍن-
ما حبا-
تن بَ عولت ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يةغايت ا ع ه ف الل مل ا عنى ر ف الل مل
ةئط. ين ت
ش
حيب : ال ها
ش 2/
366
ابن الستاج ا ص ه
ش 2/
121
241 ابن يعيش وتح ا ل
ش
7/
88
ابن ع و
وتح ا جمل
ش 1/
372
التض ف وتح التض ف عغ الكاةيق
ش 4/
169
ابن النداس
الهعليئق عغ ا ئت
ش 1/
393
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
112
357
. (
230
)
.ف "د" "ك"ش ي ضح
(
231
)
. ُ ل ش "يد" حاُط تن "ر". ودت ف " " "ك"ش ب
(
232
)
. ف "ظ"ش با
(
233
) ف "د"ش ضع ا. (
234
)
.ف " "ش ناد
(
235
)
ا رب ناو ا عسن بن صاف البغداد ةلد ف بغداد ب ا ن أ كان تة هَ َ ن تنْا ف العل بَتَاَ ف الند
تَ َتَ ةي ح ى صاو ر ح رال طبئه لَئ عَ س تَ لتكَ النداِ. تن ت ن اس ش ا عا ف الند
ا ئه د ف اله ت:ف رحل ا عا ف تعليل الئتاءات الع ت ل ع ت تساال احو تَ و كَلَ َا ف ف "ظ"ش با
(
233
) ف "د"ش ضع ا. (
234
)
.ف " "ش ناد
(
235
)
ا رب ناو ا عسن بن صاف البغداد ةلد ف بغداد ب ا ن أ كان تة هَ َ ن تنْا ف العل بَتَاَ ف الند
تَ َتَ ةي ح ى صاو ر ح رال طبئه لَئ عَ س تَ لتكَ النداِ. تن ت ن اس ش ا عا ف الند
ا ئه د ف اله ت:ف رحل ا عا ف تعليل الئتاءات الع ت ل ع ت تساال احو تَ و كَلَ َا ف 90
(
223
)
.ف "د"ش د
(
224
)
."ُ ل ش " ا ع ه" حاُط تن "د
(
225
)
.ف "ك"ش لسبٍّف
(
226
)
. ف "ر"ش يعد
(
227
) ف "ك"ش ظن ُع. د ُد
(
228
)
باز تج ء ةاعل ت ع ه "عد ةئد" ضمٍيين ته لٍن تة ه دت دَ ا عنى احهعماه تجاز ٌّ ؛ ن
ال اعل ا ع ه ةً ما أ بةد رن يك ا ت ب دين ةإذا عَدت َ ال اعلة سَ ة صاو عادتْ ا تعد تْ ا ف آن
احد الا تداه؛ ن تن عَدت َ ويئا كان ت ب دْا؛ للا ة أصل اأحهعماهش عَدت تَ نف غٍي ب لا ين ه
اسداد ال اعل ا ع ه. ين ت
ش رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد
عغ النجاو رحمد ي حف جاتي عالر ال هع بٍي ت
ط3
1403هش 1/
334
2/
106
السٍياف
وتح ال ها
ش 1/
424
3/
130 ابن يعيش وتح ا ل
ش 7/
88 ابن ع و وتح ا جمل
ش
1/
372 رب حيان ا دلس ف الهلييل اله ميل
ش 6/
113
. ُن حمل عغ السبٍّف ةتةعو
ةالهئديتش رلر ية َن ز:د ُااما؟ لر يَ ة
ن ة ُااما رخ . :ن ت
ش
ابن النداس الهعليئق عغ ا ئت
ش 1/
393
. (
)
(
207
)
ف كل النسخش "... ضمٍيا ته ال تتة عا تع حبٍّف". الهصعيح تن رتثلت ا ال ف حاُ ا ا فلف تن
َحة و مت َا؛ ن ال اغل ُذا كان ضمٍيا ته ال تتة عا تع حبٍّف َب عَ ف ا غ ه عن ا عملة عغ الضمٍي
ا ه ل أ غٍي د ن س يل ُد ذ ت ا فلف ال ا سألق بعل ا ا سألق ا ل تن ا ساال ا خمس ف
ا جم عق الثا يق. :ن ت
ش
: ال او وتح ها حيب ش406
ابن ع و ا ئت
ش 1/
91
ابن
النداس الهعليئق عغ ا ئت
ش1/
393 (
212
)
" " ف."ك"ش ز:د (
213
) ف "د"ش ةإن (
214
)
. ف "ر"ش حمل (
215
)
" " ف ."ك"ش ز:د (
216
)
.ف "د"ش ي ق (
217
)
َ الهئديت ف ا عالق ا ل ش رلر يَضو تت و ز:دْ ا رخ ؟ لر يَضوتتبو ة رخ . الهئديت ف ا عالق الثا يق ُن حَ مَ لو
َ عغ ا ه ل ةن بو َ ش رلر يَ ةن ز:دْ ا ُاامْا رخ ؟ لر يَ ةن ة ُاامْا رخ . ُن حمل عغ السبٍّف ةتةعو
ةالهئديتش رلر ية َن ز:د ُااما؟ لر يَ ة
ن ة ُااما رخ . :ن ت
ش
ابن النداس الهعليئق عغ ا ئت
ش 1/
393
. (
218
)
. ف "ك"ش أ سأت (
220
)
حبا تعليل ب از الا ف ا سألق الثالثق تن ا ساال ا خمس تن ا جم عق الثا يق. :ن ت
ش
: حيب ال ها
ش
2/
367
ابن الستاج ا ص ه
ش 2/
241
السٍياف وتح ال ها
ش 3/
119
130
ابن يعيش وتح
ا ل
ش 7/
88
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
109
. (
221
)
. ف "د"ش ةي 89 90
(
223
)
.ف "د"ش د
(
224
)
."ُ ل ش " ا ع ه" حاُط تن "د
(
225
)
.ف "ك"ش لسبٍّف
(
226
)
. ف "ر"ش يعد
(
227
) ف "ك"ش ظن ُع. د ُد
(
228
)
باز تج ء ةاعل ت ع ه "عد ةئد" ضمٍيين ته لٍن تة ه دت دَ ا عنى احهعماه تجاز ٌّ ؛ ن
ال اعل ا ع ه ةً ما أ بةد رن يك ا ت ب دين ةإذا عَدت َ ال اعلة سَ ة صاو عادتْ ا تعد تْ ا ف آن
احد الا تداه؛ ن تن عَدت َ ويئا كان ت ب دْا؛ للا ة أصل اأحهعماهش عَدت تَ نف غٍي ب لا ين ه
اسداد ال اعل ا ع ه. ين ت
ش رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد
عغ النجاو رحمد ي حف جاتي عالر ال هع بٍي ت
ط3
1403هش 1/
334
2/
106
السٍياف
وتح ال ها
ش 1/
424
3/
130 ابن يعيش وتح ا ل
ش 7/
88 ابن ع و وتح ا جمل
ش
1/
372 رب حيان ا دلس ف الهلييل اله ميل
ش 6/
113
. (
229
)
اتهنع ا سألق ن سئديتاا حٍن َ وعت ا غ هت عن حمالْ عغ الضمٍي ا ن ا ش ضَتَ َ ز:دْ ا ا
ضَتَبَ ة سئديتاا حٍن وَةوعت ت حمالْ ع .غ الضمٍي ا سهحي ا تة اش ضَتَبَ ة ز:د ضَتَبَ ة ر ش ضَتَ َ ز:د سَ ة
ة ي ال ب ا ه تَعَد ى ةتعولة ا ضمتت ا ه ل ُل ظاات ا تمهنع ف بميع رب ا العتبيق ف ال ب
الثاني تَعَد ى ةتعولة ال اات ُل ضمٍي ا ه ل ا تمهنع ف غٍي با (ظَن )؛ ا يلن علي ف ال ب ٍن-
ما حبا-
تن بَ عولت ا ع هت ةاعال ف ا عنى حاُّ ال اعل رن يةغايت ا ع ه ف الل مل ا عنى ر ف الل مل
ةئط. ين ت
ش
حيب : ال ها
ش 2/
366
ابن الستاج ا ص ه
ش 2/
121
241 ابن يعيش وتح ا ل
ش
7/
88
ابن ع و
وتح ا جمل
ش 1/
372
التض ف وتح التض ف عغ الكاةيق
ش 4/
169
ابن النداس
الهعليئق عغ ا ئت
ش 1/
393
رب حيان ا دلس ف
الهلييل اله ميل
ش 6/
112
357
. (
230
)
.ف "د" "ك"ش ي ضح
(
231
)
. ُ ل ش "يد" حاُط تن "ر". ودت ف " " "ك"ش ب
(
232
)
. سم ا ساال الع ت بدمد هللا ع ف "د"ش " هللا رعلر بال ا ُلي ا تبع ا آ صغ هللا عغ حيد ا تدمد آل صعب حلر
." ف "ظ"ش " هللا حبدا تعال رعلر. سم ا ساال الع ت بدمد هللا ع 90
(
234
)
.ف " "ش ناد
(
235
)
ا رب ناو ا عسن بن صاف البغداد ةلد ف بغداد ب ا ن أ كان تة هَ َ ن تنْا ف العل بَتَاَ ف الند
تَ َتَ ةي ح ى صاو ر ح رال طبئه لَئ عَ س تَ لتكَ النداِ. تن ت ن اس ش ا عا ف الند
ا ئه د ف اله ت:ف رحل ا عا ف تعليل الئتاءات الع ت ل ع ت تساال احو تَ و كَلَ َا ف 90 العتبي ق حم ااا ا ساال العَ و ت ا و ةهوعتبَات ُل ا و عَ و ت طةبع تدئ ئق تع ب ا ابن بت ت عه ا. س ف ف
دت ا حنق568
ه. سن ت ستبمه ف
ش عغ بن ا عسن بن ابق هللا ابن عسا ت ساو:خ دت ا سدئيا
ش
عمت بن غتاتق العمت داو ال ت للطباعق الن ت اله زيع
بٍي ت
1415هش
13
/
71
عغ بن
ي حف الئ طي ُ با الت اِ عغ ر با النداِ سدئيا
ش
تدمد رب ال ضل ُبتااير داو ال ت العتِي
الئااتِ تفحسق ال هع الثئاةيق بٍي ت ط1
1406هش 1/
340 عمت بن رحمد بن اب ق هللا
العئيغ ابن العدير بغيق الطلع ف ساو:خ حلع سدئيا ش
ح يل زكاو داو ال ت بٍي ت د.ط د.تش
5/
2390 اليماني ُواوِ الهعيٍن ش91. :ن ت
ش عبدهللا بن بَت ت بن عبد
ا جباو ا ئدس ف
ابن بَت ت ب ا
ا ساال الع ت سدئيا
ش
تدمد الدال داو الب اات دت ا
ط1
1418ه ش3 ُل ن ايق ا ساال ش92
. (
236
)
ف " "ش " هللا حبدا تعال رعلر بال ا . سَر ذلك ا عمد هلل صغ هللا عغ حيد ا تدمد عغ
آل صعب ربمعٍن حَ ل رَ تسليما". ثر هع الناسخ سدت اش "باهلل ُن تت عيناك تا هب يد ال ئٍي
ُل غ تان ت أ ". ف "ك"ش بئلر
.ِال ئٍي ُل هللا بة دت ت عبد العن:ن عطيق حم د
." ف "د"ش " هللا رعلر بال ا ُلي ا تبع ا آ صغ هللا عغ حيد ا تدمد آل صعب حلر
." ف "ظ"ش " هللا حبدا تعال رعلر. :قائمة املصادر واملراجع (8)
ابن تالك وتح التس يل سدئياش عبدالتحمن السيد
تدمد بد ا خه ن هجت للطباعق
الن ت اله زيع اإلعالن
ط1
1410اا. (9)
ابن تَضَاء الئتطٍّف التد عغ النداِ سدئيا
ش
ِتدمد ُبتااير البنا داو اأعه ا الئاات
ط1
1399
.ه (9)
ابن تَضَاء الئتطٍّف التد عغ النداِ سدئيا
ش
ِتدمد ُبتااير البنا داو اأعه ا الئاات
ط1
1399
.ه (
10
)
ابن ا ا ا او
اعحياض ال تط عغ ال تط سدئيا ش عبد ال هاح ا عم ز طباعق ن ت
داو عم او عم ان ط1
1406
.ه (
10
)
ابن ا ا ا او
اعحياض ال تط عغ ال تط سدئيا ش عبد ال هاح ا عم ز طباعق ن ت
داو عم او عم ان ط1
1406
.ه (
11
)
ابن ا ا ا او ا ساال الس ت:ق ف الند
سدئيا ش عغ حسٍن الب ا ن ت داو
طيبق
للن ت اله زيع الت:اض
د.ط د.ت . (
11
)
ابن ا ا ا او ا ساال الس ت:ق ف الند
سدئيا ش عغ حسٍن الب ا ن ت داو
طيبق
للن ت اله زيع الت:اض
د.ط د.ت . (
12
) ابن ا ا ا او ر ضح ا سالك نسخق خطيق تد ظق ف ت هبق عاطف رةند ف
ُصطنب ه بحي يا سد وُرش2442
. ً (
12
) ابن ا ا ا او ر ضح ا سالك نسخق خطيق تد ظق ف ت هبق عاطف رةند ف
ُصطنب ه بحي يا سد وُرش2442
. :قائمة املصادر واملراجع (1)
ُبتااير بن تدمد بن عبد هللا
ابن
ت لح ا ئ د ا وود ف ذ ت رصعا اإلتا رحمد
سدئيا
ش
عبد
التحمن بن حليمان العثيمٍن ت هبق التود الت:اض ط1
1410ه. (1)
ُبتااير بن تدمد بن عبد هللا
ابن
ت لح ا ئ د ا وود ف ذ ت رصعا اإلتا رحمد
سدئيا
ش
عبد
التحمن بن حليمان العثيمٍن ت هبق التود الت:اض ط1
1410ه. (2)
ُبتااير بن ت س ى ال اطٍّف
ا ئاصد ال اةيق ف وتح ا خالصق الكاةيق سدئيا ش عبد التحمن
العثيمٍن زتالا تع د البد ث العلميق
ُحياء الحياث اإلحالتي
باتعق ر الئتى ت ق ا تتق
ط1
1428ه . ُ (2)
ُبتااير بن ت س ى ال اطٍّف
ا ئاصد ال اةيق ف وتح ا خالصق الكاةيق سدئيا ش عبد التحمن
العثيمٍن زتالا تع د البد ث العلميق
ُحياء الحياث اإلحالتي
باتعق ر الئتى ت ق ا تتق
ط1
1428ه . ُ (3)
ابن حجت رحمد بن عغ العسئالني الدُّ وَو الكاتنق ف رعيان ا ااق الثاتنق سدئيا
ش
تدمد حيد
باد ا عا تطبعق ا دني الئااتِ ط2
. د.ت (3)
ابن حجت رحمد بن عغ العسئالني الدُّ وَو الكاتنق ف رعيان ا ااق الثاتنق سدئيا
ش
تدمد حيد
باد ا عا تطبعق ا دني الئااتِ ط2
. د.ت (4)
ابن حجت رحمد بن عغ العسئالني ُ ب
اء الغمت بأبناء العمت سدئ يا
حسن حبء ف ن
ُحياء الحياث اإلحالتي ف ا جلس ا عغ لل ئ ن اإلحالتيق
ت ت
د.ط
1389ه. اإاإ
(5)
ابن حميد تدمد بن عبد هللا النجد السعع ال ابلق عغ ضتااح ا عنابلق سدئيا
ش
ب ت بن
عبد هللا رب ز:د
عبد التحمن بن حليمان العثيمٍن تفحسق التحالق بٍي ت
ط1
1416
.ه (5)
ابن حميد تدمد بن عبد هللا النجد السعع ال ابلق عغ ضتااح ا عنابلق سدئيا
ش
ب ت بن
عبد هللا رب ز:د
عبد التحمن بن حليمان العثيمٍن تفحسق التحالق بٍي ت
ط1
1416
.ه 91 (6)
ابن ع و ا ئت ت سدئيا
ش رحمد ا ج او عبد هللا ا جب و تطبعق العاني بغداد
ط1
1391
.ه (7)
ابن ع و تة ثةل ا ئت ت سدئيا
ش
ِصالح حعد ا ليطي داو اآلةاق العتبيق الئاات
ط1
1427
.ه (8)
ابن تالك وتح التس يل سدئياش عبدالتحمن السيد
تدمد بد ا خه ن هجت للطباعق
الن ت اله زيع اإلعالن
ط1
1410اا. :قائمة املصادر واملراجع (
19
)
رب العالء رحمد بن عبد هللا بن حليمان الهن خ
ا عت
وحالق ا الا ق سدئيا
ش
عبد العن:ن
ا يمنف داو ال هع العلميق بٍي ت لبنان
ط1
1424ه. (
20
) رب ب ت عمت بن عثمان بن ُنبي حيب : ال ها سدئيا
ش
عبد السال تدمد ااو ن ت هبق
ِا خا ج الئاات
ط3
1408ه. (
21
)
رب ب ت تة دَ م د بن الس ت الوبَغودَ اد ابن الستاج ا ص ه ف الند سدئيا
ش
عبد ا عسٍن
ال هغ تفحسق التحالق بٍي ت
ط1
1405ه. (
22
)
رب حيان ا دلس ف ف الهلييل اله ميل ف وتح
ها التس يل سدئيا ش حسن اندا داو
الئلر
داو ن ز ُوبيليا
ط1
1419ه. (
23
)
رب حيان ا دلس ف
اوت اف الضت تن لسان العت سدئيا
ش
وبع عثمان تدمد ت هبق
ِا خا ج الئاات
ط1
1418
اا. (
24
) رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد عغ النجاو رحمد ي حف
جاتي عالر ال هع بٍي ت
ط3
1403ه. (
25
) رب حعيد ا عسن بن عبد هللا بن ا تزبان السٍياف وتح ال ها سدئيا
ش
رحمد حسن ت دل
عغ حيد عغ داو ال هع العلميق بٍي ت
ط1
2008
. (
26
) رحمد بن رحمد بن عبد هللا بن تدمد
الغتبويينف عن ان الدوايق ةيمن عةتتفَ تن العلماء ف ا ااق
السابعق ببجايق سدئيا
ش
عاده ض تن وات داو اآلةاق ا جديدِ بٍي ت
ط2
1979
. (
27
)
سئي الدين تدمد بن هجتس السالتيابن واةع ال ةيات سدئيا
ش
صا ح ت د عباس ِ او ع اد
تعت ف تفحسق التحالق بٍي ت
ط1
1402ه. (
28
)
باله الدين
عبد التحمن بن رِي ب ت
ِالسي طي بغيق ال عاِ ف طبئات اللغ :ٍن الندا
سدئيا
ش تدمد رب ال ضل ُبتااير ا هبق الع ت:ق صيدا لبنان. د.ط د.ت
(
29
) ب اد ي حف العتبا ظااتِ اأوهغاه ف العتبيق وحالق تابسهٍي ا جاتعق ا ود يق كليق
الدواحات العليا
ا ودن
1412
.ه ا ت داو ال ه
ا اإلحالتي الئااتِ ط2
1413ه. (
19
)
رب العالء رحمد بن عبد هللا بن حليمان الهن خ
ا عت
وحالق ا الا ق سدئيا
ش
عبد العن:ن
ا يمنف داو ال هع العلميق بٍي ت لبنان
ط1
1424ه. :قائمة املصادر واملراجع ً (
13
)
ابن ا ا ا او وحالق ف
"س بي الن ع ف ُعتا "ةضالْ لغقْ خالةْا ريضْا الر بتًّا
سدئيا ش
حسن ت س ى ال
اعت ن ت داو ا وُر عم ان ط1
1404ه (
13
)
ابن ا ا ا او وحالق ف
"س بي الن ع ف ُعتا "ةضالْ لغقْ خالةْا ريضْا الر بتًّا
سدئيا ش
حسن ت س ى ال
اعت ن ت داو ا وُر عم ان ط1
1404ه (
14
)
ابن ا ا ا او تسألق ا ع مق ف سل ٍي ُت:ع ف ُ ل تعال ش»
ُتن
َ َوَحو م
الل ت
َُتت:ع
َتت ن
َا و ةدو ست نتٍن«
( ا عتافش
56
) سدئيا ش
عبد ال هاح ا عم ز داو عم او عم ان ط1
د.ت (
15
)
ابن ا ا ا او تغنف اللبيع عن هع ا عاو:ع سدئيا
ش
تازن ا باوك تدمد عغ حمد
الله
داو ال ت دت ا ط6
1985
. (
15
)
ابن ا ا ا او تغنف اللبيع عن هع ا عاو:ع سدئيا
ش
تازن ا باوك تدمد عغ حمد
الله
داو ال ت دت ا ط6
1985
. (
16
) ابن ا ا ا او تغنف اللبيع نسخق خطيق تد ظق ف ا هبق السليما يق الئديمق
ُصطنب ه
ست يا سد وُرش696
ه1 .ر (
16
) ابن ا ا ا او تغنف اللبيع نسخق خطيق تد ظق ف ا هبق السليما يق الئديمق
ُصطنب ه
ست يا سد وُرش696
ه1 .ر (
17
) رب البيكات عبد
التحمن
بن تدمد
ابن ا باو اإل اف ف تساال ا خالف بٍن الند :ٍن
الب ت:ٍن الك ةيٍن سدئيا
ش
تدمد تديف الدين عبد ا عميد ا هبق الع ت:ق بٍي ت
ط1
1407ه. (
17
) رب البيكات عبد
التحمن
بن تدمد
ابن ا باو اإل اف ف تساال ا خالف بٍن الند :ٍن
الب ت:ٍن الك ةيٍن سدئيا
ش
تدمد تديف الدين عبد ا عميد ا هبق الع ت:ق بٍي ت
ط1
1407ه. (
18
) رب البئاء ري بن ت س ى ا عسينف ال الكليا ت اعهنى ب ش عد ان دو يش تدمد 92 93
ا ت داو ال ه
ا اإلحالتي الئااتِ ط2
1413ه. :قائمة املصادر واملراجع (
40
) عبدهللا بن عبد
التحمن ابن عئيل ا ساعد عغ تس يل ال ااد سدئيا ش تدمد كاتل بتكات
ن ت باتعق ر الئتى طباعق داو ال ت دت ا داو ا دني
ِبد
ط1
1400
-
1405ه. (
41
) عبيد هللا بن رحمد
ا وبيغ السب ف
ابن رِي التبيع البسيط ف وتح بمل النباج
سدئيا
ش
عياد بن عيد الثبي ف داو الغت اإلحالتي بٍي ت
ط1
1407ه. (
30
) ا عسن بن ُاحر بن عبد هللا ا تاد ا جنى ا
لداني ف حت ف ا عاني سدئيا
ش
ةخت الدين
ُبا ِ تدمد دير ةاضل داو ال هع العلميق بٍي ت
ط1
. د.ت
(
31
) ا عسن بن ُاحر بن عبد هللا
ا تاد س ضيح ا ئاصد ا سالك ب تح رل يق ابن تالك
سدئيا
ش
عبد التحمن عغ حليمان داو ال ت العتِي
بٍي ت
ط1
1428اا. (
32
)
خالد بن عبد هللا ا جتبا
اله ت:ح بمضم ن اله ضيح ف الند داو ال هع العلميق بٍي ت
ط1
1421
.ه (
33
) ال يخ تدمد بن ت ط بن حسن الدتياطي
ا خضت حاويق ا خضت عغ وتح ابن
عئيل ل يق ابن تالك تطبعق
ت ط الباِي ا علٍّف
د.ط د.ت. (
33
) ال يخ تدمد بن ت ط بن حسن الدتياطي
ا خضت حاويق ا خضت عغ وتح ابن
عئيل ل يق ابن تالك تطبعق
ت ط الباِي ا علٍّف
د.ط د.ت. (
34
) صالح الدين خليل بن ريبك ال د رعيان الع ت رع ان الن ت سدئيا
ش
عغ رب ز:د (
34
) صالح الدين خليل بن ريبك ال د رعيان الع ت رع ان الن ت سدئيا
ش
عغ رب ز:د
زتالا داو ال ت بٍي ت دت ا
ط1
1418اا. (
35
) صالح الدين خليل بن ريبك ال د ال اف بال ةيات سدئيا
ش رحمد ا اؤ ط
ستكي
ٍت ط داو ُحياء الحياث ب ي ت د.ط1420ه. (
36
) عبدالباق بن عبد
ا جيد اليماني ُواوِ الهعيٍن ف ستابر النداِ اللغ :ٍن سدئيا
ش عبد ا جيد
ديا تت ن ا لك ةي ل للبد ث الدواحات اإلحالتيق السع ديق
ط1
1406ه
(
37
) عبد
هللا بن بَت ت بن عبد ا جباو ا ئدس ف ابن بَت ت ب ا ا ساال الع ت سدئيا
ش
تدمد الدال
داو الب اات دت ا
ط1
1418ه. :قائمة املصادر واملراجع (
20
) رب ب ت عمت بن عثمان بن ُنبي حيب : ال ها سدئيا
ش
عبد السال تدمد ااو ن ت هبق
ِا خا ج الئاات
ط3
1408ه. (
21
)
رب ب ت تة دَ م د بن الس ت الوبَغودَ اد ابن الستاج ا ص ه ف الند سدئيا
ش
عبد ا عسٍن
ال هغ تفحسق التحالق بٍي ت
ط1
1405ه. (
22
)
رب حيان ا دلس ف ف الهلييل اله ميل ف وتح
ها التس يل سدئيا ش حسن اندا داو
الئلر
داو ن ز ُوبيليا
ط1
1419ه. (
23
)ا ا ل
ر
س ئ ا
ال
ل ا
ا ت اف الض
ه ق
ث ا ر
د و ز ُوبي ي
9ه. (
23
)
رب حيان ا دلس ف
اوت اف الضت تن لسان العت سدئيا
ش
وبع عثمان تدمد ت هبق
ِا خا ج الئاات
ط1
1418
اا. (
24
) رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد عغ النجاو رحمد ي حف
ال ال
ا
ط3
1403 (
23
)
رب حيان ا دلس ف
اوت اف الضت تن لسان العت سدئيا
ش
وبع عثمان تدمد ت هبق
ِا خا ج الئاات
ط1
1418
اا. (
24
) رب ز ت:ا يديى بن ز:اد الديلمف ال تاء تعاني الئتآن سدئيا
ش
تدمد عغ النجاو رحمد ي حف
جاتي عالر ال هع بٍي ت
ط3
1403ه. (
26
) رحمد بن رحمد بن عبد هللا بن تدمد
الغتبويينف عن ان الدوايق ةيمن عةتتفَ تن العلماء ف ا ااق
السابعق ببجايق سدئيا
ش
عاده ض تن وات داو اآلةاق ا جديدِ بٍي ت
ط2
1979
. (
27
)
سئي الدين تدمد بن هجتس السالتيابن واةع ال ةيات سدئيا
ش
صا ح ت د عباس ِ او ع اد
تعت ف تفحسق التحالق بٍي ت
ط1
1402ه. (
28
)
باله الدين
عبد التحمن بن رِي ب ت
ِالسي طي بغيق ال عاِ ف طبئات اللغ :ٍن الندا
سدئيا
ش تدمد رب ال ضل ُبتااير ا هبق الع ت:ق صيدا لبنان. :قائمة املصادر واملراجع د.ط د.ت
(
29
) ب اد ي حف العتبا ظااتِ اأوهغاه ف العتبيق وحالق تابسهٍي ا جاتعق ا ود يق كليق
الدواحات العليا
ا ودن
1412
.ه 93 (
30
) ا عسن بن ُاحر بن عبد هللا ا تاد ا جنى ا
لداني ف حت ف ا عاني سدئيا
ش
ةخت الدين
ُبا ِ تدمد دير ةاضل داو ال هع العلميق بٍي ت
ط1
. د.ت
(
31
) ا عسن بن ُاحر بن عبد هللا
ا تاد س ضيح ا ئاصد ا سالك ب تح رل يق ابن تالك
سدئيا
ش
عبد التحمن عغ حليمان داو ال ت العتِي
بٍي ت
ط1
1428اا. (
32
)
خالد بن عبد هللا ا جتبا
اله ت:ح بمضم ن اله ضيح ف الند داو ال هع العلميق بٍي ت
ط1
1421
.ه
(
33
) ال يخ تدمد بن ت ط بن حسن الدتياطي
ا خضت حاويق ا خضت عغ وتح ابن
عئيل ل يق ابن تالك تطبعق
ت ط الباِي ا علٍّف
د.ط د.ت. (
34
) صالح الدين خليل بن ريبك ال د رعيان الع ت رع ان الن ت سدئيا
ش
عغ رب ز:د
زتالا داو ال ت بٍي ت دت ا
ط1
1418اا. (
35
) صالح الدين خليل بن ريبك ال د ال اف بال ةيات سدئيا
ش رحمد ا اؤ ط
ستكي
ٍت ط داو ُحياء الحياث ب ي ت د.ط1420ه. (
36
) عبدالباق بن عبد
ا جيد اليماني ُواوِ الهعيٍن ف ستابر النداِ اللغ :ٍن سدئيا
ش عبد ا جيد
ديا تت ن ا لك ةي ل للبد ث الدواحات اإلحالتيق السع ديق
ط1
1406ه
(
37
) عبد
هللا بن بَت ت بن عبد ا جباو ا ئدس ف ابن بَت ت ب ا ا ساال الع ت سدئيا
ش
تدمد الدال
داو الب اات دت ا
ط1
1418ه. (
38
) عبدالح بن رحمد بن تدمد ابن العماد ا عنبغ ولوات اللاع ف رخباو تن ذاع سدئيا
ش
تدم د ا و اؤ ط داو ابن ثٍي دت ا-
بٍي ت
ط1
1406ه. (
39
) عبدالتحمن بن رِي ب ت بن تدمد السي طي امع ال اتع ف وتح بمع ا ج اتع سدئيا ش
عبد العاه حالر ت ت ن ت داو البد ث : العلميق الك
د.ط
1394
-
1400ه. :قائمة املصادر واملراجع /
(
51
) ُاحر بن عغ بن تدمد البطلي س ف
ال او وتح ها حيب : سدئيا
ش
عن:نِ اللبياني
رطت حق د ه وا باتعق طيبق
ا دينق ِا ن و
السع ديق د.ط
1434
.ه
(
52
) تدمد بن ُبتااير ا علٍّف
ابن النداس الهعليئق عغ ا ئت وتح ا ئت ا سم ى الهعليئق (
44
) عغ بن ا عسن بن ابق هللا ابن عسا ت
ساو:خ دت ا سدئيا
ش
عمت بن غتاتق العمت داو
ال ت للطباعق
الن ت اله زيع
د.ط
1415ه. (
44
) عغ بن ا عسن بن ابق هللا ابن عسا ت
ساو:خ دت ا سدئيا
ش
عمت بن غتاتق العمت داو
ال ت للطباعق
الن ت اله زيع
د.ط
1415ه. (
45
)
عغ بن تدمد بن عغ ا عضتتي اإلوبيغ
ابن خت ف وتح بمل
النباج سدئيا ش حل ى
تدمد عت ن ت باتعق ر الئت ى
د.ط
1419ه. ب
ت ت زيع د.5ه. (
45
)
عغ بن تدمد بن عغ ا عضتتي اإلوبيغ
ابن خت ف وتح بمل
النباج سدئيا ش حل ى
تدمد عت ن ت باتعق ر الئت ى
د.ط
1419ه. (
45
)
عغ بن تدمد بن عغ ا عضتتي اإلوبيغ
ابن خت ف وتح بمل
النباج سد
تدمد عت ن ت باتعق ر الئت ى
د.ط
1419ه. (
46
)
عغ بن تفتن بن تةدَ م د ا و عَ ضوتَتتي اإلوبيغ
ابن ع و وتح ا ئت ت نسخق خطيق تد ظق
ف ا خنا ق العاتق بالتباط
سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
47
) عغ بن ي حف الئ طي
ُ با الت اِ عغ ر با النداِ سدئيا
ش
تدمد رب ال ضل ُبتااير داو
ال ت العتِي الئااتِ تفحسق ال هع الثئاةيق بٍي ت ط1
1406ه. (
48
)
عغ ة دِ يل ابن ا ا ا او ش آثاو تلاب الند عمادِ وف ن ا هبات
باتعق (
48
)
عغ ة دِ يل ابن ا ا ا او ش آثاو تلاب الند عمادِ وف ن ا هبات
باتعق
ا لك حع د الت:اض
د.ط
1404ه. (
49
)
عمت بن رحمد بن ابق هللا العئيغ ابن العدير بغيق الطلع ف س
او:خ حلع سدئيا
ش
ح يل
زكاو داو ال ت بٍي ت. :قائمة املصادر واملراجع (
48
)
عغ ة دِ يل ابن ا ا ا او ش آثاو تلاب الند عمادِ وف ن ا هبات
باتعق
ا لك حع د الت:اض
د.ط
1404ه. (
49
)
عمت بن رحمد بن ابق هللا العئيغ ابن العدير بغيق الطلع ف س
او:خ حلع سدئيا
ش
ح يل
زكاو داو ال ت بٍي ت. د.ط د.ت
(
50
)عمت بن َُديد بن عبد
هللا الئَ لَمو طا ا عن حاويق ابن َُديد عغ ر ضح ا سالك نسخق (
42
) عثمان بن عمت ابن ا عابع اإليضاح ف وتح ا ل سدئيا
ش ت س ى بنا الع ليغ تطبعق
العاني بغداد
د.ط
1982ه. (
43
)
ع ا و الدين ابن ا ا ا او - حياس تنهج ال ند ال ت ق العا يق لل ها
بٍي ت ط1
1989
. (
44
) عغ بن ا عسن بن ابق هللا ابن عسا ت
ساو:خ دت ا سدئيا
ش
عمت بن غتاتق العمت داو
ال ت للطباعق
الن ت اله زيع
د.ط
1415ه. (
45
)
عغ بن تدمد بن عغ ا عضتتي اإلوبيغ
ابن خت ف وتح بمل
النباج سدئيا ش حل ى
تدمد عت ن ت باتعق ر الئت ى
د.ط
1419ه. (
46
)
عغ بن تفتن بن تةدَ م د ا و عَ ضوتَتتي اإلوبيغ
ابن ع و وتح ا ئت ت نسخق خطيق تد ظق
ف ا خنا ق العاتق بالتباط
سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
47
) عغ بن ي حف الئ طي
ُ با الت اِ عغ ر با النداِ سدئيا
ش
تدمد رب ال ضل ُبتااير داو
ال ت العتِي الئااتِ تفحسق ال هع الثئاةيق بٍي ت ط1
1406ه. (
48
)
عغ ة دِ يل ابن ا ا ا او ش آثاو تلاب الند عمادِ وف ن ا هبات
باتعق
ا لك حع د الت:اض
د.ط
1404ه. (
49
)
عمت بن رحمد بن ابق هللا العئيغ ابن العدير بغيق الطلع ف س
او:خ حلع سدئيا
ش
ح يل
زكاو داو ال ت بٍي ت. د.ط د.ت
(
50
) عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق
خط ت
ط
ق تد ظق ف ا هبق السليما يق
ُصطنب ه
ست يا سد وُرش1327
1
. :قائمة املصادر واملراجع (
38
) عبدالح بن رحمد بن تدمد ابن العماد ا عنبغ ولوات اللاع ف رخباو تن ذاع سدئيا
ش
تدم د ا و اؤ ط داو ابن ثٍي دت ا-
بٍي ت
ط1
1406ه. 94
(
38
) عبدالح بن رحمد بن تدمد ابن العماد ا عنبغ ولوات اللاع ف رخباو تن ذاع سدئيا
ش
تدم د ا و اؤ ط داو ابن ثٍي دت ا-
بٍي ت
ط1
1406ه. (
39
) عبدالتحمن بن رِي ب ت بن تدمد السي طي امع ال اتع ف وتح بمع ا ج اتع سدئيا ش
عبد العاه حالر ت ت ن ت داو البد ث : العلميق الك
د.ط
1394
-
1400ه. (
40
) عبدهللا بن عبد
التحمن ابن عئيل ا ساعد عغ تس يل ال ااد سدئيا ش تدمد كاتل بتكات
ن ت باتعق ر الئتى طباعق داو ال ت دت ا داو ا دني
ِبد
ط1
1400
-
1405ه. (
41
) عبيد هللا بن رحمد
ا وبيغ السب ف
ابن رِي التبيع البسيط ف وتح بمل النباج
سدئيا
ش
عياد بن عيد الثبي ف داو الغت اإلحالتي بٍي ت
ط1
1407ه. 94 (
42
) عثمان بن عمت ابن ا عابع اإليضاح ف وتح ا ل سدئيا
ش ت س ى بنا الع ليغ تطبعق
العاني بغداد
د.ط
1982ه. (
43
)
ع ا و الدين ابن ا ا ا او - حياس تنهج ال ند ال ت ق العا يق لل ها
بٍي ت ط1
1989
. (
44
) عغ بن ا عسن بن ابق هللا ابن عسا ت
ساو:خ دت ا سدئيا
ش
عمت بن غتاتق العمت داو
ال ت للطباعق
الن ت اله زيع
د.ط
1415ه. (
45
)
عغ بن تدمد بن عغ ا عضتتي اإلوبيغ
ابن خت ف وتح بمل
النباج سدئيا ش حل ى
تدمد عت ن ت باتعق ر الئت ى
د.ط
1419ه. (
46
)
عغ بن تفتن بن تةدَ م د ا و عَ ضوتَتتي اإلوبيغ
ابن ع و وتح ا ئت ت نسخق خطيق تد ظق
ف ا خنا ق العاتق بالتباط
سد وُرش511
ل ا ت وِ ف تع د ا خط طات ُحياء الحياث
اإلحالتي بجاتعق ر الئتى ف ت ق ا تتق ه52
. (
47
) عغ بن ي حف الئ طي
ُ با الت اِ عغ ر با النداِ سدئيا
ش
تدمد رب ال ضل ُبتااير داو
ال ت العتِي الئااتِ تفحسق ال هع الثئاةيق بٍي ت ط1
1406ه. :قائمة املصادر واملراجع د.ط د.ت (
50
) عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق
خط ت
ط
ق تد ظق ف ا هبق السليما يق
ُصطنب ه
ست يا سد وُرش1327
1
. /
(
51
) ُاحر بن عغ بن تدمد البطلي س ف
ال او وتح ها حيب : سدئيا
ش
عن:نِ اللبياني
رطت حق د ه وا باتعق طيبق
ا دينق ِا ن و
السع ديق د.ط
1434
.ه
(
52
) تدمد بن ُبتااير ا علٍّف
ابن النداس الهعليئق عغ ا ئت وتح ا ئت ا سم ى الهعليئق
سدئيا ش ِخٍي عبد التاض ف عبد اللطيف ت هبق داو النتان للن ت اله زيع ا دينق ا ن و
ط1
1426ه. (
50
) عمت بن َُدت يد بن عبد هللا الئَ لَمو طا ا عن ي حاويق ابن َُدت يد عغ ر ضح ا سالك نسخق
خط ت
ط
ق تد ظق ف ا هبق السليما يق
ُصطنب ه
ست يا سد وُرش1327
1
. / 95 (
53
) تدمد بن رحمد بن عثمان بن ُايماز
اللاٍّف
ساو:خ اإلحال َ َ ةيات ا ااٍي ا عال سدئيا ش
ب او ع اد تعت ف داو الغت اإلحالتي بٍي ت
ط1
2003
. (
54
) تدمد بن وا ت بن رحمد ابن وا ت ة ات ال ةيات سدئيا
ش
ُحسان عباس داو صادو
بٍي ت
ط1
1973
1974 (
55
) تدمد بن عبد التحمن بن تدمد
السخا الض ء الالتع ال الئتن الهاحع تن وات داو
ت هبق ا عياِ بٍي ت. (
56
) تدمد بن عبد هللا الطائي ا جياني
ابن تالك وتح الكاةيق ال اةيق سدئيا عبد ا نعر رحمد
ات:د تت ن البدس العلمف ُحياء الحياث اإلحالتي بكليق ال تيعق الدواحات اإلحالتيق بجاتعق
ر الئتى
ت ق ا تتق
ط1
1402ه. (
57
) تدمد بن عبد هللا الطائي ا جياني تس يل ال ااد س ميل ا ئاصد سدئيا
ش
تدمد كاتل بتكات داو
ال ها العتِي للطباعق الن ت
بٍي ت
1387ه (
58
) تدمد بن عبد هللا بن العباس ابن ال و اق علل الند سدئيا
ش
تدم د باحر تدمد
الدو يش ت هبق التود الت:اض
ط1
1420اا. (
59
)
تدمد بن عغ اليمنف ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق
بٍي ت. :قائمة املصادر واملراجع د.ط د.ت (
59
)
تدمد بن عغ اليمنف ال كاني البدو الطالع بمداحن تن بعد الئتن السابع داو ا عتةق
بٍي ت. د.ط د.ت (
60
) تدمد بن تدمد بن عبد ا لك ا او ا تا ء ف ابن عبد ا لك الليل اله ملق ل هاِي
ا ص ه ال لق سدئيا
ش
ُحسان عباس تدمد بن وت: ق ِ او ع اد تعت ف داو الغت
اإلحالتي
بٍي ت ط1
2012
. (
60
) تدمد بن تدمد بن عبد ا لك ا او ا تا ء ف ابن عبد ا لك الليل اله ملق ل هاِي
ا ص ه ال لق سدئيا
ش
ُحسان عباس تدمد بن وت: ق ِ او ع اد تعت ف داو الغت
اإلحالتي
بٍي ت ط1
2012
. (
61
)
تدمد بن يعئ ال ٍي زآباد البلغق ف ستابر رامق الند اللغق سدئيا
ش
تدمد ا ت
: تطبعق ال ي ل الك
ط1
. د.ت (
61
)
تدمد بن يعئ ال ٍي زآباد البلغق ف ستابر رامق الند اللغق سدئيا
ش
تدمد ا ت
: تطبعق ال ي ل الك
ط1
. د.ت 9
(
62
)
تدمد عبد ا خالا ة اوس ها حيب : تطبعق السعادِ الئاات ِط1
1395
.ه
(
63
)
تدمد تد ظ
ستابر ا فل ٍن اله نسيٍن داو الغت اإلحالتي بٍي ت
ط2
1994
. (
64
) اظت ا جيش تدمد بن ي حف بن رحمد سم يد الئ اعد ب تح تس يل ال ااد سدئيا
ش
عغ
تدمد ةاخت زتالا ِداو السال للطباعق الن ت اله زيع الحيبمق الئاات
ط1
1428ه. 96 (
65
)
جر الدين تدمد بن ا عسن التض ف ا ححياباذ وتح التض ف عغ الكاةيق سدئيا ي حف حسن
عمت
تطابع ال ت ق بٍي ت ن ت باتعق بنغاز ليبيا. د.ط د.ت
(
66
)
ابن ع و وتح ا جمل سدئيا
ش
صاحع رب بناح ا صل
د.ط
1402
.ه
(
67
) يعيش بن عغ بن يعيش ا علٍّف ابن يعيش وتح ا ل تِ هبق ا هنٍّف الئاات. د.ط د.ت
(
68
) ي حف بن تغت بتد ا عن ي ابن تغت بتد ا ه ل ال اف ا سه ف بعد ال اف سدئيا
ش
تدمد تدمد رتٍن ال يئق ا ت:ق العاتق لل ها. :قائمة املصادر واملراجع الئااتِ د.ط د.ت
(
69
)
ي حف بن حسن ال الح ا عنبغ
ابن ا وتبويَد ا ج ات ا نضد ف طبئات تهأخت رصعا
رحمد سدئيا
ش عبد
التحمن بن حليمان العثيمٍن ت هبق العبيكان الت:اض
ط1
1421ه. (
70
)
ي حف عبد التحمن الضبع
ابن ا ا رثت ف الند العتِي
داو ا عديس ت ت
ط1
1998
. (
65
)
جر الدين تدمد بن ا عسن التض ف ا ححياباذ وتح التض ف عغ الكاةيق سدئيا ي حف حسن
عمت
تطابع ال ت ق بٍي ت ن ت باتعق بنغاز ليبيا. د.ط د.ت
(
66
)
ابن ع و وتح ا جمل سدئيا
ش
صاحع رب بناح ا صل
د.ط
1402
.ه
(
67
) يعيش بن عغ بن يعيش ا علٍّف ابن يعيش وتح ا ل تِ هبق ا هنٍّف الئاات. د.ط د.ت
(
68
) ي حف بن تغت بتد ا عن ي ابن تغت بتد ا ه ل ال اف ا سه ف بعد ال اف سدئيا
ش
تدمد تدمد رتٍن ال يئق ا ت:ق العاتق لل ها. الئااتِ د.ط د.ت 97
|
https://openalex.org/W4200322855
|
https://www.degruyter.com/document/doi/10.1515/9783110745832-013/pdf
|
English
| null |
12 New approaches in the Renaissance
|
De Gruyter eBooks
| 2,021
|
cc-by
| 10,035
|
Open Access. © 2021 Philipp Roelli, published by De Gruyter.
This work is licensed under the
Creative Commons Attribution 4.0 International License.
https://doi.org/10.1515/9783110745832-013 1 See OED (s. v. ‘renaissance’).
2 See Eisenstein (1979); White (2017).
3 More on these matters in chap. 13 §3.
4 For this topic, see Kristeller (1961). 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. The This work is licensed under the 291 Humanist Latin Renaissance may, indeed, be said to depend in a certain sense on the fall of the
Byzantine Empire and the immigration of Greek scholars to Italy in the decades
before it. These scholars were heirs of the Palaeologan ‘Renaissance’, which re-
vived learning in the last two centuries of Byzantium and produced scholars like
Manuel Chrysolaras (1353–1417), Johannes Argyropoulos (ca. 1415–1487), Con-
stantine Lascaris (1434–1501), or Cardinal Bessarion (1403–1472), who all emi-
grated to Italy and sparked enthusiastic interest there for Greek Antiquity – in-
cluding its literature, superstitions, magic, and also science. Some humanists
not only used Greek words and phrases in their texts – which was already a com-
mon practice among some mediaeval writers – but even penned entire texts in
Classical Greek. Despite some fourteenth-century precursors such as Francesco
Petrarca (1304–1374) or Coluccio Salutati (1331–1406), Renaissance humanism
starts as a large scale phenomenon in the many small fifteenth-century Italian re-
publics and was fuelled by the Greek émigrés. The patronage of the arts by Cosimo
de’ Medici (1389–1464) in Florence was especially important.5 Among other
things, he sponsored a Platonic academy led by Marsilio Ficino (1433–1499; see
§4 below). Beginning in the second half of the fifteenth century, the movement
moved across the Alps – early on it arrived in Vienna6 – and took roots there in
the sixteenth century, especially in Germany and the Netherlands. Cardinal Nico-
laus Cusanus (1401–1464) was one of the early adopters; but, despite hailing from
Kues (near Luxembourg), he characteristically spent most of his later life south of
the Alps. This chapter begins with Renaissance humanism’s approach to Latin
(§2), then the most important currents of thought are introduced: hermetic neo-
Platonism (§3), magia naturalis (§4), and mathematical theology (§5), leading to
‘universal science’ (§6). The next chapter treats the Scientific Revolution, which
can be seen as a synthesis of the Aristotelianism discussed in the previous chapter
and the Renaissance currents discussed here. §2 Already in the later Middle Ages, people who practised good Latin tended to
have studied at universities,7 in contrast to the earlier Middle Ages, when basic
Latin training was usually acquired at ecclesiastical grammar schools. 5 On which see Hankins (1990).
6 Overfield (1984: 102–103).
7 See Korenjak (2016: 15). 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. Sarton (1929: 76) Sarton (1929: 76) §1 The term ‘Renaissance’, intended as a rebirth of Antiquity, was first used in art,
although as late as the mid-sixteenth century, by Giorgio Vasari.1 Moreover, it was
only Jacob Burckhardt’s epoch-making work Die Cultur der Renaissance in Italien
(1860) that paved the way for the introduction of a completely new epoch in all fa-
cets of life emerging in fifteenth-century Italy, one that tried to renew and emulate
Roman Antiquity. His approach has been much criticised in the past few decades;
indeed, even the fact that many leading Renaissance men were members of the
clergy should have alerted scholars to the fact that they did not intend to resurrect
ancient Rome in all its facets, which would have included pagan religion. Even
so, it does still make sense to have a new epoch begin in fifteenth century, as in
this time many external parameters changed that were bound to influence peo-
ple’s perception of themselves and their relation to the past. The most important
of these were the immigration of Greeks to Italy (in 1453 Constantinople fell to the
Ottomans); the invention of the printing press (Gutenberg from 1455),2 accelerat-
ing the circulation of new ideas decidedly; the new republican state forms in Italy,
in which a new, flourishing literate middle class engaged in trade; and, in connec-
tion with this knowledge of foreign places, the reconquista of the Iberian peninsu-
la (completed in 1492). This led to the age of discoveries: Bartolomeu Dias sailed
around Africa in 1488, Columbus landed in the New World in 1492, Vasco da
Gama reached India in 1498, and Fernão de Magalhães, finally, sailed around the
world in 1522.3 The term ‘Renaissance humanism’ is used in order to emphasise the new pic-
ture of man emerging in this time.4 This entailed the possibility of forming man to
true humanity by means of classical studies (and, in contrast to earlier similar at-
tempts, outside the Church). Humanism was at its core a rhetorical and pedagogi-
cal movement, seeking to move away from ‘un-Latin’ and unrhetorical scholastic
language and back to Ciceronian purity of language and thought. Many huma-
nists were themselves teachers or wrote textbooks and translations intended to
supplant the ‘barbarous’, ‘mediaeval’ material available. A high appreciation of
Greek culture came as a by-product of emulating classical Roman erudition. 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. Over the
centuries it would seem that – although slowly and far from linearly – proficiency
in Latin retreated to ever higher intellectual strata of society, which can be viewed
in connection with the emergence of vernacular languages that drifted further
and further from Latin, gradually replacing Latin in more and more facets of 12 New approaches in the Renaissance 292 life, and, finally, precipitating the end of Latin’s predominance altogether (see
chap. 14 below). Renaissance humanists were not content with university Latin
and studied classical rhetoric; they became especially critical of the twelfth-cen-
tury way of translating verbatim from Greek, which hurt Latin syntax and accord-
ing to them produced obscuritas. Thus, the Renaissance translator Argyropoulos
writes in his Praefatio in librum Phisicorum (Venetiis, 1496 edition), fol. 3v, about
his new translation: life, and, finally, precipitating the end of Latin’s predominance altogether (see
chap. 14 below). Renaissance humanists were not content with university Latin
and studied classical rhetoric; they became especially critical of the twelfth-cen-
tury way of translating verbatim from Greek, which hurt Latin syntax and accord-
ing to them produced obscuritas. Thus, the Renaissance translator Argyropoulos
writes in his Praefatio in librum Phisicorum (Venetiis, 1496 edition), fol. 3v, about
his new translation: invenies, certo scio, faciliores nunc cognitu sententias omnes eius quas perobscuras olim inter-
pretandi modus ille rudis reddebat. pretandi modus ille rudis reddebat. ‘you will, I do not doubt it, find all his [Aristotle’s] thoughts, which that uncultivated way of
translating rendered so obscurely in the past, to be of easier understanding.’ Some humanists even wrote treatises on how to translate from Greek. Leonardo
Bruni, De interpretatione,8 believed that Aristotle wrote in excellent Greek style
and had been abused by mediaeval Latin translators.9 The discussion of how to
translate remained very much alive in the centuries to come. In 1531, Juan Luis
Vives reached the other extreme, accusing Aristotle himself of obscuritas, wor-
sened by the translators who ‘did not leave it Greek and did not make it Latin’,
and by scholasticism (De disciplinis, ed. Vigliano, pp. 8 [L]ibros in greco plenos elegantie, plenos suavitatis, plenos inestimabilis cuiusdam decoris (‘In
Greek the books are full of elegance, full of subtlety, full of a certain invaluable grace’; §2, ed. Viti,
p. 74). Viti’s edition also prints Bruni’s interesting forewords; Kuhlmann (2002) re-evaluates Bru-
ni’s new approach. 9 In fact, the works of Aristotle we possess today were rather terse lecture notes; his works meant
for wider circulation are, unfortunately, lost. On this topic in general, see Pym (1998). 8 [L]ibros in greco plenos elegantie, plenos suavitatis, plenos inestimabilis cuiusdam decoris (‘In
Greek the books are full of elegance, full of subtlety, full of a certain invaluable grace’; §2, ed. Viti,
p. 74). Viti’s edition also prints Bruni’s interesting forewords; Kuhlmann (2002) re-evaluates Bru-
ni’s new approach.
9 In fact, the works of Aristotle we possess today were rather terse lecture notes; his works meant
for wider circulation are, unfortunately, lost. On this topic in general, see Pym (1998). 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. […] Aristotle is drawn by the in-
terpreter where he could never have expected to end up, so much so that Aristotle was pub-
licly said among them [the scholastics] in a not at all ignorant way (as is otherwise their
wont) to have a waxen nose that he turns whither he will.’11 Although there is no doubt that some late scholastic authors wrote complicated
treatises with little actual content in what the humanists must have perceived as
horrible Latin, it is on the other hand just as easy to hide a lack of understanding
under a veil of classicist rhetoric. As in so many things humanist, Francesco Pe-
trarca led the way. Trying to prove that Plato is to be preferred over Aristotle, he
already inveighed against insanum et clamosum scolasticorum vulgus (‘the insane
and noisy rabble of the scholastics’).12 Petrarch preferred a rhetorical Wissen-
schaftsmodell to the then usual scholastic one (Kessler 1978: 198). In other words,
he goes back to the classical Roman lack of interest in science in favour of oratory. A typical humanist rant against scholastic language and thought can be found
in Lorenzo Valla’s Repastinatio dialecticae et philosophiae. Among many other
things, Valla claims that the suffix ‑tas is abused by scholastics: (4, ed. Zippel,
p. 30): Although there is no doubt that some late scholastic authors wrote complicated
treatises with little actual content in what the humanists must have perceived as
horrible Latin, it is on the other hand just as easy to hide a lack of understanding
under a veil of classicist rhetoric. As in so many things humanist, Francesco Pe-
trarca led the way. Trying to prove that Plato is to be preferred over Aristotle, he
already inveighed against insanum et clamosum scolasticorum vulgus (‘the insane
and noisy rabble of the scholastics’).12 Petrarch preferred a rhetorical Wissen-
schaftsmodell to the then usual scholastic one (Kessler 1978: 198). In other words,
he goes back to the classical Roman lack of interest in science in favour of oratory. A typical humanist rant against scholastic language and thought can be found
in Lorenzo Valla’s Repastinatio dialecticae et philosophiae. Among many other
things, Valla claims that the suffix ‑tas is abused by scholastics: (4, ed. Zippel,
p. 30): Nulla nomina in ‘itas’ descendere a substantivis sed ab adiectivis, nec his omnibus. 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. 77–78): Sed ut Aristotelis obscuritas multum nocuit artibus, sic horum in Aristotelem interpretationes
artes omnes peruerterunt: non potuerunt recte Aristotelem exponere, et haec ipsa difficultas te-
meritatem atque impudentiam exacuebat, ut tanto magis auderet quisque pro interpretamento
adferre quicquid in mentem uenisset, quo minus refelli ac confutari posset inter tantas tene-
bras: et (quemadmodum uulgo dicunt) perturbatus amnis quaestui erat piscantibus: […] Ver-
sus est male ab imperitis, qui dum in latinum transferunt, nec latinum fecerunt nec reliquerunt
graecum; […] tractus ab expositore quo nunquam se Aristoteles uenturum potuit suspicari. Vt
iam etiam uulgo inter eos non omnino, ut solent, inscite – Aristoteles dicatur habere nasum cer-
eum, quem quilibet quo uelit flectat pro libito. ‘But as much as Aristotle’s obscurity damaged the arts a lot, so the interpretations of Aristot-
le by these men perverted all arts: they could not expound Aristotle correctly, and this diffi-
culty aggravated their rashness and impudence, so much so that the more anybody dared to
bring forward whatever he had in mind as interpretation, the less he could be disproved or 8 [L]ibros in greco plenos elegantie, plenos suavitatis, plenos inestimabilis cuiusdam decoris (‘In
Greek the books are full of elegance, full of subtlety, full of a certain invaluable grace’; §2, ed. Viti,
p. 74). Viti’s edition also prints Bruni’s interesting forewords; Kuhlmann (2002) re-evaluates Bru-
ni’s new approach. 9 In fact, the works of Aristotle we possess today were rather terse lecture notes; his works meant
or wider circulation are, unfortunately, lost. On this topic in general, see Pym (1998). 293 Humanist Latin Humanist Latin refuted within so much darkness. As people say: troubled rivers bring gain to fishermen.10
[…] He was translated badly by inexperienced men, who while transferring the content into
Latin, did not make it Latin but did not leave it Greek either. 10 This saying is not found in Walther (1963–1986). The idea seems to be that fish are more easily
caught in the turbulent waters. 11 Vives attacked the Paris scholastics of his own day strongly in his In pseudodialecticos (ed.
Fantazzi). More on Valla’s, Vives’s, and other prominent humanists’ attacks against Aristotle and
his followers in Rummel (1995: 153–192). caught in the turbulent waters.
11 Vives attacked the Paris scholastics of his own day strongly in his In pseudodialecticos (ed.
Fantazzi). More on Valla’s, Vives’s, and other prominent humanists’ attacks against Aristotle and
his followers in Rummel (1995: 153–192).
12 De sui ipsius et multorum ignorantia 4, ed. Buck, p. 112. Kessler (1978) evaluated Petrarch as a 10 This saying is not found in Walther (1963–1986). The idea seems to be that fish are more easily
caught in the turbulent waters.
11 Vives attacked the Paris scholastics of his own day strongly in his In pseudodialecticos (ed.
Fantazzi). More on Valla’s, Vives’s, and other prominent humanists’ attacks against Aristotle and
his followers in Rummel (1995: 153–192).
12 De sui ipsius et multorum ignorantia 4, ed. Buck, p. 112. Kessler (1978) evaluated Petrarch as a
historian and philologist and reached mixed conclusions. Petrarch had no interest whatsoever for
natural sciences and has to be seen mostly in a rhetorical, humanist context. 14 We will review some ‘antibarbarus’ literature from later times in chap. 14 §11 below. 12 New approaches in the Renaissance 12 New approaches in the Renaissance does not use these terms, then they cannot be formed from nouns [in “proper” Latin], finally
they cannot even be formed from all adjectives but only from those in ‑us (second declen-
sion) – although not even of all of them – and in ‑er (same declension), and those in ‑is (third
declension), and some others, but not all.’ 13 Elegantiae II, ed. Garin, p. 602. 13 Elegantiae II, ed. Garin, p. 602. 12 New approaches in the Renaissance
From the scientific point of view the Renaissance was not a renaissance. Quid, quod ab isto ‘ens’ faciunt ‘entitas’ (ut de hac quoque materia nunc disputem) qualia mul-
ta alia, ut a ‘quid’ ‘quiditas’, a ‘per se’ ‘perseitas’, ab ‘hecce’ ‘hecceitas’ et cetera, e barbarie
quodam gurgustio prolata? Nam primum hec ab Aristotele non traduntur, deinde a substanti-
vis deduci nequeunt: ‘ens’ autem et ‘quid’ substantiva sunt; postremo nec ab omnibus adiecti-
vis, nisi ab iis que exeunt in ‘us’, que sunt secunde declinationis (quanquam nec ista omnia),
aut in ‘er’ eiusdem declinationis, et que in ‘is’ tertie, et in quasdam alias litteras, non omnes ta-
men. ‘That no nouns in -itas can be formed from nouns, but only from adjectives and not even
from all of them. Why, that they [the scholastics] derive entitas from the word ens – let me now enter upon this
topic too – as well as many other cases such as quiditas from quid, perseitas from per se,
haecceitas from haecce, and so on, acquired from some barbarian hovel. For, firstly Aristotle 10 This saying is not found in Walther (1963–1986). The idea seems to be that fish are more easily
caught in the turbulent waters. 11 Vives attacked the Paris scholastics of his own day strongly in his In pseudodialecticos (ed. Fantazzi). More on Valla’s, Vives’s, and other prominent humanists’ attacks against Aristotle and
his followers in Rummel (1995: 153–192). 12 De sui ipsius et multorum ignorantia 4, ed. Buck, p. 112. Kessler (1978) evaluated Petrarch as a
historian and philologist and reached mixed conclusions. Petrarch had no interest whatsoever for
natural sciences and has to be seen mostly in a rhetorical, humanist context. 12 De sui ipsius et multorum ignorantia 4, ed. Buck, p. 112. Kessler (1978) evaluated Petrarch as a
historian and philologist and reached mixed conclusions. Petrarch had no interest whatsoever for
natural sciences and has to be seen mostly in a rhetorical, humanist context. 294 Later (5, p. 36) he claims that fine scholastic terminology is meaningless: Inter ‘essentiam’ et ‘esse’ nihil interesse et item in ceteris, ut inter ‘voluntatem’ et ipsum ‘velle’. ‘That there is no difference between essentia and esse, and similarly in other cases such as
voluntas and velle.’ Both these points overshoot the target: it will have become obvious that a detailed
terminology is fundamental for scientific thought, and there are even classical ex-
ceptions to his linguistic point (necessitas from necesse, civitas from cives). None-
theless, such bold claims helped to make people more aware of their language,
and considering to what parts of speech suffixes can be appended is an important
linguistic achievement. Among authors like Valla, the prejudice of a dark middle
age between the Roman orators and themselves began to be felt. For instance,
Valla saw Isidore as indoctorum arrogantissimus (‘the most arrogant of the unedu-
cated’).13 Needless to say, these humanist polemicists did not make any signifi-
cant scientific discoveries themselves – even less so than their much-admired Ci-
cero (see chap. 8 §7). It was from these people that the idea of an unadulterated
Latinity, allowing the use exclusively of what can be shown to be extant in Cicero,
began; pupils of the humanist gymnasium had to put up with it as late as the
twentieth century.14 But some early humanists, such as Angelo Poliziano (1454–
1494), already saw that a complete emulation of Cicero, prohibiting all words and
expressions not found in him, was not a good idea. He points out in a letter to
Paulo Cortesi (ed. Garin, p. 902): Mihi certe quicumque tantum componunt ex imitatione, similes esse vel psittaco vel picae vi-
dentur, proferentibus, quae non intelligunt. Nihil ibi verum, nihil solidum, nihil efficax. Non ex-
primis, inquit aliquis, Ciceronem. Quid tum? non enim sum Cicero; me tamen, ut opinor, expri-
mo. ‘It seems to me that those who compose only through imitating are similar to parrots or mag-
pies: they express what they do not understand. There is nothing true, nothing solid, noth-
ing powerful in them. One says: “you do not express yourself like Cicero.” And so? I am not
Cicero; it seems to me that I should express myself as myself!’ 295 Humanist Latin New grammars (late mediaeval modist speculative grammar theory was among
the main targets of the humanists)15 and new dictionaries were necessary to teach
the new language. 15 See Overfield (1984: 75–86).
16 Quotation from Cicero, De oratore II.7(30), ed. Kumaniecki, p. 115.
17 Further on the Cornucopiae: Furno (1995).
18 Some examples are studied by Overfield (1984: 120–142), e. g. ‘many Germans resented the
smug sense of superiority exuded by the Italians’ (141).
19 Already Olschki pointed out: ‘Es ist klar, dass man mit dem relativ beschränkten Sprach- und
Stilschatz Ciceros nicht den ungeheuren Wissensschatz beherrschen konnte, den die Gelehrten
der Renaissance aus den entferntesten Gebieten der Kultur- und Naturgeschichte zusammenge-
tragen hatten’ (‘It is clear that with Cicero’s relatively limited vocabulary and style, it was not pos-
sible to master the immense wealth of knowledge that the scholars of the Renaissance had gath-
ered from the most remote areas of cultural and natural history’; 1922: 71). The same assessment
is made by Stotz (1996–2004: I, §67.11 = vol. 1, pp. 166–167) and Korenjak (2016: 11). Later (5, p. 36) he claims that fine scholastic terminology is meaningless: The most successful, and at the same time an unusual and ex-
treme one, was Nicolaus Perotti’s Cornucopiae (1506). It started as a commentary
on Martial but grew into a full dictionary of Classical Latin. A list at the beginning
tells the reader where to find which word in the big tome. Thus, scientia is found
in column 1019, part D, while commenting on Martial’s Epigramma I.3 (modern
numbering) Argiletanas mavis habitare tabernas: Nescio autem ex ne & scio componitur. Scire autem proprie est rem ratione & per causam cog-
noscere a quo Scientia dicitur rerum quae sunt inmutabili ratione comprehensio. Cicero Ars
enim eorum est quae sciuntur. Oratoris autem omnis actio opinionibus non scientia contine-
tur.16 Ponitur autem frequenter scio pro cognosco intelligo a quo fit Scisco inchoatiuum: & par-
ticipium sciens: & Scienter aduerbium. ‘The word nescio is composed of ne and scio. Scire properly is to know something rationally
and through its causes, which is why scientia is said to be the understanding by reason of
things that are unchangeable. Cicero says: for art is of things that are known. But all actions
of an orator depend on [his audience’s] opinion, not on knowledge [scientia]. Scio is also of-
ten used instead of cognosco or intelligo; thus an inchoative form scisco is formed, besides a
participle sciens and an adverb scienter.’ The examples and usages are exclusively classical: for instance, under oratio the
author does not mention that the word also means ‘prayer’ in Christian Latin.17
The sixteenth and seventeenth centuries will see more balanced dictionaries, be-
ginning with Calepinus (used in chap. 2 §5 above). Humanist Latin by no means supplanted scholastic Latin in the centuries
following the humanist Renaissance. Later (5, p. 36) he claims that fine scholastic terminology is meaningless: After sometimes rather bilious strife be-
tween humanist poets and scholastic scholars,18 a kind of demarcation of compe-
tences largely prevailed: the former in rhetoric, speeches, poetry, and the like; the
latter in universities and science.19 The two registers, humanist and scholastic, of- 12 New approaches in the Renaissance 296 ten coexisted much more closely than one tends to realise, as in Pico della Miran-
dola’s (1463–1494) famous Oratio de dignitate hominis, written in very humanistic
Latin in 1486 and intended as an introduction to his much longer catalogue of
nine hundred theses in normal ‘scholastic’ Latin (Conclusiones nongentae).20 Pico
may have been more aware of the relativity of such language registers due to his
familiarity with Greek and Hebrew. In a letter to Ermolao Barbaro, he points out
(ed. Garin, p. 818): quid prohibet hosce philosophos, quos nuncupatis barbaros, conspirasse in unam dicendi nor-
mam, apud eos non secus sanctam ac habeatur apud vos romana? quid prohibet hosce philosophos, quos nuncupatis barbaros, conspirasse in unam dicendi nor-
mam, apud eos non secus sanctam ac habeatur apud vos romana? ‘what forbids those philosophers whom you call barbarians having conspired to use a single
linguistic norm as sacred to them as to you the Roman tongue?’ ‘what forbids those philosophers whom you call barbarians having conspired to use a single
linguistic norm as sacred to them as to you the Roman tongue?’ Pico goes on to quote the antique ‘noble savage’, the Scythian Anacharsis: Pico goes on to quote the antique ‘noble savage’, the Scythian Anacharsis: Ἀνάχαρσις παρ’ Ἀθηναίοις σολοικίζει, Ἀθηναῖοι δὲπαρὰΣκύθαις. ‘Anacharsis speaks badly for the Athenians, the Athenians for the Scythians.’ 20 Editions: Garin; Biondi. More on Pico’s ‘double tongue’ in Moss (2003: 67–70). He concludes (pp. 820–822): Scribat Lucretius de natura, de Deo, de providentia, scribat de eisdem ex nostris quispiam, scri-
bat Ioannes Scotus et quidem carmine ut sit ineptior. Dicet Lucretius rerum principia atomos et
vacuum, Deum corporeum, rerum nostrarum inscium, temere omnia fortuito occursu corpuscu-
lorum ferri, sed haec latine dicet eleganter. Dicet Ioannes quae natura constant, sua materia
specieque constitui, esse Deum separatam mentem, cognoscentem omnia, omnibus consulen-
tem. […] At dicet insulse, ruditer, non latinis verbis. Quaeso, quis in dubium revocet, uter poeta
melior, uter philosophus? ‘Let Lucretius write about nature, God, Providence; let someone of ours [a Christian] write
about the same things, let us say Duns Scotus, and he is not so poetically minded. Lucretius
will say that atoms and emptiness are the principles of things, God corporeal and not caring
about our matters, that everything happens by chance collision of particles, but he says it in
elegant Latin. Duns will say that what exists in nature is made up of matter and form, that
God is a mind separate from it, who knows all, takes counsel about everything. […] But he
says it in a tasteless, rude manner, in words that are not Latin. I ask: who would doubt who
is the better poet, who the better philosopher?’ Pico could say such things and get away with them among humanists, because he
penned them in very rhetorical humanist Latin. In general, in later times the two
approaches have to be seen rather as two different registers adapted for different
uses than as exclusive types of ‘good’ and ‘bad’ Latin. Thus, although curricula
shifted toward a greater importance of studia humaniora at German universities in 297 Humanist Latin the later 1510s, scientific study, its sources and goals, hardly changed.21 Human-
ism was a rhetorical, not a scientific movement. Indeed, Thorndike (1943) draws
very negative conclusions about humanism’s impact on science: he sees human-
ists as uniform and backward with their wish to imitate Roman classical times. Science was still predominantly done at Aristotelian universities, such as Padua
(especially the natural sciences). Private academies and societies would only be-
come important in science in the seventeenth century.22 As Eugenio Garin puts it:
‘Aristotelica, dunque, rimaneva, almeno per buona parte l’indagine scientifica,
l’ossatura del sapere’ (‘Scientific investigation, therefore, remained Aristotelian,
at least for the most part, the skeleton of knowledge’; 2009: 35). 21 See Overfield (1984: 298–299).
22 On these see Biagioli (2002).
23 For some notes on the language of medicine, including exceptions to the above statement,
especially Vesalius, see chap. 21 below. The interest in antique science that was significant in
many fields would hardly seem typically humanist; it was already common among many mediae-
val scholars. In law, for instance, authors such as Hugo Donellus (1527–1591) speak of ‘legal hu-
manism’. Their main point was to go back to the antique texts, disregarding mediaeval commen-
tators.
24 Rummel (1995: 195) rightly speaks of the ‘third option’ besides scholasticism and humanism.
25 Compare German Humanismus vs Geisteswissenschaft. Indeed, the former’s meaning is a re-
cent acquisition, coined by Niethammer (1808). 25 Compare German Humanismus vs Geisteswissenschaft. Indeed, the former’s meaning is a re-
cent acquisition, coined by Niethammer (1808). 23 For some notes on the language of medicine, including exceptions to the above statement,
especially Vesalius, see chap. 21 below. The interest in antique science that was significant in
many fields would hardly seem typically humanist; it was already common among many mediae-
val scholars. In law, for instance, authors such as Hugo Donellus (1527–1591) speak of ‘legal hu-
manism’. Their main point was to go back to the antique texts, disregarding mediaeval commen-
tators. 22 On these see Biagioli (2002). 24 Rummel (1995: 195) rightly speaks of the ‘third option’ besides scholasticism and humani 21 See Overfield (1984: 298–299). He concludes (pp. 820–822): In general, Mediaeval Latin is divided from Neo-Latin precisely on the grounds
of a more classicist approach to language from the fifteenth century onward. In po-
etry, for instance, the difference is often striking. In contrast, the extent to which
the humanist movement influenced scientific writing seems to depend quite a lot
on the science in question. In less ‘humanistic’ sciences (so to speak) such as the
natural sciences or medicine (see chap. 21 below), clear humanist linguistic influ-
ence is rare;23 in some of them, a change of style around the sixteenth century can
be observed, although not toward a humanist style but toward a Euclidean axio-
matic approach, for example in mathematics and physics, but also in Spinoza’s
ethics.24 In other sciences, a more pretentious, rhetorical style did come to be ex-
pected, for instance in philology or literary studies, indeed in the traditional hu-
man sciences. Philosophical scholasticism may be said to have become more con-
scious of its possible fallacies as a result of the attacks of the humanists. It was to
remain important and was to produce a new flowering in Spanish neo-scholasti-
cism (discussed in chap. 11 §7 above), which had, among other things, important
contributions to make to the formation of international law. Incidentally, human-
ism and the human sciences bear a similar name by historical accident only,25 but
it has recently been pointed out that the humanist Poliziano can be seen as the ori- 23 For some notes on the language of medicine, including exceptions to the above statement,
especially Vesalius, see chap. 21 below. The interest in antique science that was significant in
many fields would hardly seem typically humanist; it was already common among many mediae-
val scholars. In law, for instance, authors such as Hugo Donellus (1527–1591) speak of ‘legal hu-
manism’. Their main point was to go back to the antique texts, disregarding mediaeval commen-
tators. Rummel (1995: 195) rightly speaks of the ‘third option’ besides scholasticism and humanism. 24 Rummel (1995: 195) rightly speaks of the ‘third option’ besides scholasticism and humanism. 25 Compare German Humanismus vs Geisteswissenschaft. Indeed, the former’s meaning is a re-
cent acquisition coined by Niethammer (1808) 24 Rummel (1995: 195) rightly speaks of the third option besides scholasticism and humanism. 25 Compare German Humanismus vs Geisteswissenschaft. Indeed, the former’s meaning is a re-
cent acquisition, coined by Niethammer (1808). He concludes (pp. 820–822): 12 New approaches in the Renaissance 298 ginator of a special kind of humanist science.26 He proposes in his Panepistemon
a new classification of the sciences into speculativa (approximately natural sci-
ences), practica (approximately artes mechanicae), and rationalis (encompassing
historia ad fidem, distinguished from historia fabulosa, which is not scientific; dia-
lectic, rhetoric, and poetry). This adds the scientific study of history to the usual tri-
vium, but we have seen (chap. 9 §1) that a similar approach can already be traced in
Augustine. Sarton (1929: 80) had a point when he called Renaissance humanists ‘soph-
ists’ with few exceptions. But even if humanism as a rhetorical movement had lit-
tle impact on science, the renewed interest in Antiquity also led some humanists
to seek out new ways in scientific inquiry. It will become apparent that the Renais-
sance Platonist movement did provide some new methodological impulses to
science. §3 The Renaissance had a clear preference for Plato over Aristotle. Renaissance
scholars meant to reintroduce what was lost from Antiquity, such as Platonism,
but this grew into something quite new. In the case of Renaissance Platonism,
antique neo-Platonism grew into hermetic neo-Platonism with a penchant
toward pantheism and a special interest in magic.27 The priest Marsilio Ficino
(1433–1499) translated all the extant Platonic dialogues into Latin for the first
time at Lorenzo de’ Medici’s Platonic academy in Florence; he also translated
many neo-Platonic works, such as Plotinus’ Enneads and the Corpus Hermeti-
cum.28 He delved so far into neo-Platonist occultism that he had to write an Apo-
logia justifying his interests.29 The Corpus Hermeticum in particular came to enjoy
huge prestige as prisca sapientia (a term dear to Ficino) and an Egyptian precursor
of ancient Greek learning, especially at Italian Renaissance academies; this lasted
at least until the philologist Isaac Casaubon was able to date this ‘ancient’ wis-
dom to Late Antiquity in 1614. The Corpus Hermeticum’s main theme is the unity
of all things that the hermeticist should find, especially his own with God. The re-
sult is quite far from science as we understand it; Sarton (1929: 79) would speak
of a ‘superficial mixture of ideas too vague to be of real value’. Neo-Platonism,
which had already been largely incorporated into the thinking of the Church
Fathers in a ‘purified’ form (i. e. 26 Edelheit (2015).
27 The seminal study on this is Yates (1964).
28 Greek text ed. Nock & Festugière.
29 Details in Thorndike (1923–1958: 4:562). He concludes (pp. 820–822): minus its all too ‘pagan’ and ‘superstitious’ consti-
tuents) and thus heavily influenced the Middle Ages and scholasticism, returned
now in its unadulterated late antique form – including the traits that the Church 299 Hermetic neo-Platonism Fathers had found unacceptable. Interestingly, these were mostly non-scientific
ones, as we would say today (using e. g. the criteria in part 1): theurgy, demonol-
ogy, magic, secrecy (lack of sharing results with the uninitiated), or number mys-
ticism. Thus, Renaissance science with its turn to ‘Platonism’ (through neo-Pla-
tonism) was rather a step back in terms of scientific testability and transparency. We can quote a later, but very characteristic author: Giordano Bruno. He is no-
table for his philosophical poems (Francofurti, 1591) written in clear imitation of
Lucretius. De monade, numero et figura studies the first ten numbers as metaphy-
sical entities and their magical and philosophical meanings in very obscure and
often purposefully ambiguous language, of which the following sentence describ-
ing the circle can serve as an example (De monade, numero et figura 2, lines 4–14,
ed. Fiorentino, vol. 1.2, p. 335): Hoc de fonte fluunt primoque parente, figurae
Clarandaeque forum ipsius iustumque tribunal
5
Conquirunt, facie inque sua spectantur adauctae,
In faciemque suam degliscunt omnia tandem:
Illius ut crescit surgens in imagine horizon
Amplius a nostris se quando sensibus effert,
Illius ut formam capiunt attrita recessu
10
Corpora ad obtutum, quando momenta perire
Cuspidis expertum est, laterum discrimina vultus
Amittunt rerum, in speciem cita principiorum,
Quo amplius in nihilum ad oculos solvenda fatiscunt. ‘From this source and first ancestor [i. e. the circle] they [i. e. mathematical shapes] flow forth,
the figures that are to be explained search it out as its marketplace and its just tribunal. When increased, they are seen in its surface; toward its surface they all un-grow30 at length. The horizon grows in its image as it widens further when it removes itself from our senses. 31 Von Samsonow et al. (1991: 226–227) believe this to be a chiffre for a geometric method. 30 Deglisco is a very uncommon word, not found in dictionaries. 32 On which see Zambelli (2007). The form magica is also used with the same meaning.
33 The latter is forbidden as sorcery in Islam (Quran, Sura 2.102). The Arabic roots of magia natu-
ralis and their effects on the Latin world merit a profounder study than Saif (2015).
34 See Thorndike (1923–1958: 3:346), quoting De universo I.1.43, 1674 edition, vol. 1, p. 648: in ea
parte naturalis scientiae, quae vocatur magica naturalis (‘in that part of natural science that is
called natural magic’). 0
12 New approaches in the Renaissance 300 12 New approaches in the Renaissance §4 One way in which the new Renaissance outlook did profit science was through
its stress on experimentalism, which went under the heading of magia natura-
lis.32 Arabic sources already differentiated between ‘natural’ and demonic ma-
gic,33 only the first of which is licit. The Latins took this over; the Latin term is
used as generally known since at least William of Auvergne (d. 1249).34 Giambat-
tista della Porta’s (1535?–1615) huge encyclopaedia, Magia naturalis (1558), shows
the wide range of phenomena that could be subsumed under this heading: a lot of
medicine, magnetism, poisons, witches, invisible writing, and much more. This
natural magic is eulogised by Cornelius Agrippa of Nettesheim (1486–1535) as (De
occulta philosophia I.2, ed. Compagni, p. 86): Magica facultas, potestatis plurimae compos, altissimis plena mysteriis, profundissimam re-
rum secretissimarum contemplationem, naturam, potentiam, qualitatem, substantiam et virtu-
tem totiusque naturae cognitionem complectitur et quomodo res inter se differunt et quomodo
conveniunt nos instruit, hinc mirabiles effectus suos producens, uniendo virtutes rerum per ap-
plicationem earum ad invicem et ad sua passa congruentia, inferiora superiorum dotibus ac
virtutibus passim copulans atque maritans: haec perfectissima summaque scientia, haec altior
sanctiorque philosophia, haec denique totius nobilissimae philosophiae absoluta consumma-
tio. ‘The magic faculty, compounded of most powers, full of highest mysteries, comprises the
most profound contemplation of secret things, nature, potency, quality, substance and vir-
tue, and the knowledge of all nature. It instructs us how things differ among one another
and how they come together in order to produce their miraculous effects by unifying the vir-
tues of things by applying them to one another and to their congruent passive sides, by here
and there coupling and marrying the lower things to the gifts and virtues of the upper [hea-
venly]. This is the most perfect and highest science, this is the higher and holier philosophy,
this, finally, is the absolute consummation of the most noble philosophy.’ This passage is quite typical in many ways: the complicated, hymnic language,
the emphasis on mystery but also the lack of interest in scientific step-by-step ex-
planations. Other authors were less cautious and did study illicit, demonic magic
as well. Authors who engaged in it often fared badly: Giordano Bruno was burned
at the stake for heresy in 1600; John Dee (1527–1608/1609) had to face a life of
hardship. He concludes (pp. 820–822): Apparently, worn-off bodies take something like its form in departure, when it experienced
the perishing of the thrust of the spear,31 and the looks of things lose the difference of their
sides, then cite them to the realm of ideas where visible things decay the more into nothing-
ness.’ 10 The poems are in general very hard to understand: often it is hard to tell whether
the author is speaking about geometric constructions or about metaphysical enti-
ties: in fact, it would seem that he usually intends both. The vocabulary is rela-
tively normal; the obscurity comes rather from the often ambiguous syntactic
nexuses and the precise meaning of the often poetically circumscribed terms. Se-
crecy was certainly intended. Bruno also wrote more accessible works in Italian. 0
12 New approaches in the Renaissance Not such extreme adepts, but rather experimentally minded scientists
who coupled magical ‘virtues’ and higher and lower influences, were to have last- This passage is quite typical in many ways: the complicated, hymnic language,
the emphasis on mystery but also the lack of interest in scientific step-by-step ex-
planations. Other authors were less cautious and did study illicit, demonic magic
as well. Authors who engaged in it often fared badly: Giordano Bruno was burned
at the stake for heresy in 1600; John Dee (1527–1608/1609) had to face a life of
hardship. Not such extreme adepts, but rather experimentally minded scientists
who coupled magical ‘virtues’ and higher and lower influences, were to have last- 301 Magia naturalis ing influence. Indeed, one of the main points of Thorndike’s magnum opus (1923–
1958) was to show the kinship between such magic and the rise of experimental
science; a point that has today become a commonplace. In this split of magia into
magic proper (magia ritualis vel daemonica) and magia naturalis, which becomes
a part of scientia, the formative rôle of the Church should not be overlooked: it
was the Church that made magia refrain from accepting demonic (unscientific)
powers as ‘mechanisms’ – the same sound guiding influence it had already exer-
cised in Late Antiquity on neo-Platonism. On the other hand, licit magia natura-
lis’s experimental and mechanistic tendencies greatly benefited the development
of natural experimental science. Apart from authors on magia naturalis, a new type of technician and practi-
cal scientist becomes more common in the fourteenth and fifteenth century; for
the first time, some of these practitioners did not hail from Latinate society. Leo-
nardo da Vinci (1452–1519), for instance, was self-taught in Latin, and his theo-
retical works did not find as great a resonance as his famous art. Others are still
understudied, such as the Venetian engineer Giovanni Fontana (ca. 1395–ca. 1455);35 his works on war machines, mnemotechnics, and clocks still exist only
in manuscript form, but a work of his on machines has recently been edited
(Liber instrumentorum iconographicus, ed. Kranz). He also wrote an encyclopae-
dia of natural philosophy that was printed in 1544. Niccolò Tartaglia wrote a
treatise on ballistics in Italian (La nova scientia, 1537). Natural scientists of the
following period were to profit from their new devices and discoveries. Galileo
(chap. 35 See Clagett (1976) on his life and works. 0
12 New approaches in the Renaissance 13 §4) was to fit well into this type of practically minded scientist and en-
gineer. A brief look at the language used by two important authors will now be taken. Hieronymus Cardanus (1501–1576) stood between different worlds: he was a
scientist in the new spirit (see next chapter) and was interested in natural magic,
Lullian combinatorics, and new scientific devices; his style is sometimes quite in
the vein of the humanism of his time, but his many compendious works look
rather scholastic in nature. There are some 130 printed works of his; they treat pro-
blems in mathematics, physics, medicine, astrology, philosophy, religion, and
music. His main contributions were mathematical and medical. His mathematical
main work is called the Ars magna (Norimbergae, 1545), echoing the title of Lullus’
main work (see §5 below). This work presents for the first time general solutions to
polynomial equations of degrees 3 and 4, although these formulas were not dis-
covered by Cardano himself. His large, encyclopaedic compendium De subtilitate
treats ‘subtlety’ in twenty-one books (1st ed., Norimbergae, 1551). He aims to ex- 12 New approaches in the Renaissance 302 plain difficult and refined things in nature, man, the senses, the soul, science, de-
monology, theology; the epilogue adds (Basileae, 1554 edition, p. 560): quaedam
ob raritatem, quaedam ob difficultatem adiecimus (‘we added some things because
of their rarity, some because of their difficulty’). His goal is explained thus (I.1, ed. Nenci, p. 53):36 Propositum nostri negocii in hoc opere est, de subtilitate tractare. Est autem subtilitas ratio
quaedam, qua sensibilia a sensibus, intelligibilia ab intellectu, difficile comprehenduntur. […] Idque solum apertum et facile videri potest, quod in unaquaque disciplina est obscurissi-
mum. […] Cum enim scribentes in quatuor laborent generibus, rerum obscuritate, incertorum
dubitatione, causarum inventione, rectaque earum explicatione, omnia haec hoc in libro cu-
mulatius habentur. […] Quaedam etiam cum desierint, aut nuper sint inventa, nominibus aut
carent, aut nomina rebus ipsis. Porro nomina invenire novis rebus, et senescente lingua, diffi-
cillimum est. […] Constat ergo subtilitas in tribus, substantiis, accidentibus, ac repraesentatio-
nibus. ‘The purpose of our work is to treat “subtlety”. Subtlety is an approach by which sensible
things are understood by the senses with difficulty, intelligible things by the intellect. […]
And it alone is capable of making openly and easily visible what is most difficult in each dis-
cipline. 36 This is a modern edition of books I–VII; see also the edition project at http://www.cardano.
unimi.it. 37 The approach to natural science is analysed by Schütze (2000). 36 This is a modern edition of books I–VII; see also the edition project at http://www.cardano.
unimi.it.
37 The approach to natural science is analysed by Schütze (2000). 0
12 New approaches in the Renaissance […] Now, as those who write struggle with four problems: obscurity of things, doubt-
ing of uncertain things, finding of causes, their correct explanation: all of these are con-
tained in this one book together. […] Some things that are missing were either recently
discovered, or lack names, or the names lack things. But it is very hard to invent names for
new things in a language that is growing old. […] Thus, “subtlety” consists of three spheres:
substances, accidents, and representations.’ The author thus believes that explaining difficult things in all sciences in one
book will help to make them plainer. He is also an early scientific voice perceiving
Latin as growing too old for scientific use. The book contains many geometric fig-
ures and some sketches of ‘subtle’ devices.37 Julius Caesar Scaliger (1484–1558)
wrote a 1,128-page ‘review’ (Exoticarum, first published 1557) of the work, dis-
agreeing with some things and elaborating on others, which shows a spirit of
scientific discussion. §5 A related undercurrent with a long past but becoming influential only during
Renaissance times is mathematical theology of a ‘Pythagorean’ kind. Its kin-
ship to hermeticism and neo-Platonism is apparent – Proclus had already tried to
mathematicise theology in his Elementa theologica – but its line of development
leads elsewhere. Authors of this kind had a tendency to coin unusual vocabulary 303 Mathematical theology for their unusual thoughts. In fact, this – as we might call it – ‘lower’ neo-Platon-
ism had already reached some individuals before Ficino’s translations, presum-
ably through Arabic intermediaries or direct contact. An influx of Christianised
Qabalistics already arose in some authors in the thirteenth century. This kind of
thought typically sought correspondences or ‘sympathies’ between different le-
vels of reality. Examples are the Byzantine demonologist Michael Psellos (1017/
1018–ca. 1078), who had direct access to the Greek sources, the eschatologist Joa-
chim of Fiore (ca. 1135–1202) in his Liber figurarum (ed. Tonelli),38 the Catalan
missionary Raimundus Lullus (ca. 1232–1316), or alchemists such as John of
Rupescissa (1310–ca. 1370). Fig. 20: Figura A from Lull’s Art (Moguntina edition, vol. 1, after p. 432). Fig. 20: Figura A from Lull’s Art (Moguntina edition, vol. 1, after p. 432). Lullus worked on his ars lulliana for much of his life, a combinatoric system that
he believed would be able to reform the sciences, especially the science of theol-
ogy, and become their common foundation. 38 On this topic, see Guerrini (2016). 0
12 New approaches in the Renaissance In various forms of movable figures
with epithets of God (one of them is depicted in fig. 20), he believed he was able to
form the basis of a ‘scientific’ theology that would prove the Trinity and the Incar- 12 New approaches in the Renaissance 304 nation of Christ to non-Christians. His system seems to have been inspired by Qa-
balah and mystic Islam. Lull’s ars is apparently meant to be able to classify all
strata of being, but it does not work purely mechanically;39 the name ars (not
scientia) may have been chosen in order to stress its practical aspects of bringing
the artista toward God. Leibniz was to be impressed by this art and would develop
it further, although in a mathematical not a theological way, into the science of
combinatorics. Its details remain – despite repeated new attempts to explain it by
its author – rather obscure. It would seem that the obvious closeness of mystic
currents in all three Abrahamitic religions that Lullus had apparently noted stems
from their common neo-Platonist ingredients. They also form the basis of Lullus’
system. This system would fail to qualify as scientific according to our criteria, but
this important precursor of Renaissance Platonism used a very special language
that is worth noting in this context. He coined terms that might be called hyper-
scholasticisms, such as unificentia, bonificentia, aeternificentia,40 and constructed
a fully fledged system of derivations in groups of divine attributes. Thus, bonitas
becomes the set of bonificativus, bonificabilis, and bonificare:41 that through
which bonitas happens, that which can receive it, and the act of bestowing it. Thus, the typically scholastic suffixes ‑ivus and ‑bilis can be used freely to build
up a linguistic system mirroring Lullus’ conception of how things are. This may be
the boldest mediaeval attempt at Latin language engineering. The following ex-
ample illustrates both his language and his main idea (Ars generalis ultima 1, ed. Madre, ROL 14, pp. 5–6): Quoniam intellectus humanus est ualde plus in opinione, quam in scientia constitutus, ex eo
quia quaelibet scientia habet sua principia propria, et diuersa a principiis aliarum scientiarum,
idcirco requirit et appetit intellectus, quod sit una scientia generalis ad omnes scientias. Et hoc
cum suis principiis generalibus, in quibus principia aliarum scientiarum particularium sint im-
plicita et contenta, sicut particulare in uniuersali. 39 He says: absque ratione artista non potest bene uti ista arte (‘without reason the artist cannot
use this art well’; Ars generalis ultima 13, ed. Madre, ROL 14, p. 524). Platzek (1962) is a good intro-
duction to this difficult author.
40 Liber de scientia perfecta dist. 1, ed. Stöhr, ROL 1, vol. 1, pp. 224–225.
41 Ars generalis ultima IV.3, ed. Madre, ROL 14, p. 31. 41 Ars generalis ultima IV.3, ed. Madre, ROL 14, p. 31. 40 Liber de scientia perfecta dist. 1, ed. Stöhr, ROL 1, vol. 1, pp. 224–225. 0
12 New approaches in the Renaissance Ratio huius est, ut cum ipsis principiis alia
principia subalternata sint et ordinata, et etiam regulata, ut intellectus in ipsis scientiis quies-
cat per uerum intelligere, et ab opinionibus erroneis sit remotus et prolongatus. Per hanc qui-
dem scientiam possunt aliae scientiae faciliter acquiri. ‘As the human intellect is much more grounded in opinion than in science, due to the fact
that every science has its own principles, differing from those of the other sciences, therefore
the intellect requires and desires one science general to all other sciences. And this science
ought to have its own general principles, in whose principles those of the other particular
sciences be implicit and contained, as particulars are contained in universals. The reason for 305 Mathematical theology
305 Mathematical theology
305 this is that by these principles others will be subordinate to them and be put in line and also
be ruled, so that the intellect will find peace in these sciences understanding the truth and
that it can be removed and separated from wrong opinions. By means of this [general]
science the other sciences can then easily be acquired.’ this is that by these principles others will be subordinate to them and be put in line and also
be ruled, so that the intellect will find peace in these sciences understanding the truth and
that it can be removed and separated from wrong opinions. By means of this [general]
science the other sciences can then easily be acquired.’ It may be important in this context that Lullus was a layman, an outsider among
university people, who came to scholarly writing only after undergoing a mysti-
cal experience. What Lullus may have groped his way toward is the idea that for-
malisation (in the form of mathematics as well as language engineering) can in-
deed be a general foundation for all sciences. There was serious opposition to
Lullus’ innovations in university circles, and even the Inquisition took an interest
in him. It may be important in this context that Lullus was a layman, an outsider among
university people, who came to scholarly writing only after undergoing a mysti-
cal experience. What Lullus may have groped his way toward is the idea that for-
malisation (in the form of mathematics as well as language engineering) can in-
deed be a general foundation for all sciences. 42 διάλογος is, of course, derived from δια-, not δύο. But Cusanus did not invent the word: Wy
cliffe had already used it as a work title: Trialogus, ed. Lahey; see Werner (1999). 42 διάλογος is, of course, derived from δια-, not δύο. But Cusanus did not invent the word: Wy-
cliffe had already used it as a work title: Trialogus, ed. Lahey; see Werner (1999).
43 An important tool for studying these currents is Hanegraaff (2006).
44 Kullmann (1974, 1998) shows the many links between modern science and Aristotelian meth-
ods.
45 See Uebinger (1895: esp. 403–414). Regiomontanus, De triangulis, proved that Cusanus’ con-
structions were wrong. 45 See Uebinger (1895: esp. 403–414). Regiomontanus, De triangulis, proved that Cusanus’ con-
structions were wrong. y
g
y
43 An important tool for studying these currents is Hanegraaff (2006). 44 Kullmann (1974, 1998) shows the many links between modern science and Aristotelian meth
ods. 0
12 New approaches in the Renaissance There was serious opposition to
Lullus’ innovations in university circles, and even the Inquisition took an interest
in him. Another author in this tradition is Cardinal Nicolaus Cusanus (1401–
1464), who had read Lullus and also coined unusual words, although he did not
create a system from them. Often they are also attempts to name the unnameable
(God), such as his possest (of God being the coincidentia of actus and potentia,
posse and esse), or his referring to God as tricausalis. A matter of questionable
Greek is his trialogus, a dialogue between three people.42 Other authors who con-
tinued to spin such ideas further include magicians such as Giordano Bruno and
John Dee. §6 Similar hermeticist neo-Platonist currents43 can still pop up among scientific
disciplines today – as in Rudolf Steiner’s (1861–1925) ‘spiritual science’, Carl Gus-
tav Jung’s (1875–1961) depth psychology, or Fritjof Capra’s (1939–) Taoist Phy-
sics – usually controversial and often spurned by scientists in the fields in ques-
tion. What unites these authors can be described as introspection, mysticism,
esotericism, and holistic approaches, all of which have been alien to university
scholarship and science, whose methodology stayed largely Aristotelian through
the Renaissance and still is so today.44 Another point that unites at least some of
the authors mentioned is their dabbling or even outright failures in generally ac-
knowledged science: Lullus was convinced that his ars nova could revolutionise
all sciences, and Cusanus believed he had squared the circle.45 This universalist §6 Similar hermeticist neo-Platonist currents43 can still pop up among scientific
disciplines today – as in Rudolf Steiner’s (1861–1925) ‘spiritual science’, Carl Gus-
tav Jung’s (1875–1961) depth psychology, or Fritjof Capra’s (1939–) Taoist Phy-
sics – usually controversial and often spurned by scientists in the fields in ques-
tion. 0
12 New approaches in the Renaissance What unites these authors can be described as introspection, mysticism,
esotericism, and holistic approaches, all of which have been alien to university
scholarship and science, whose methodology stayed largely Aristotelian through
the Renaissance and still is so today.44 Another point that unites at least some of
the authors mentioned is their dabbling or even outright failures in generally ac-
knowledged science: Lullus was convinced that his ars nova could revolutionise
all sciences, and Cusanus believed he had squared the circle.45 This universalist 12 New approaches in the Renaissance 306 approach continued to flourish in the age of the Scientific Revolution as a kind of
science which tried to produce a synthesis of the Platonic Renaissance science
and the ‘new’ science (treated in the next chapter); among these authors, Kircher
believed he had deciphered the Egyptian hieroglyphs. This approach typically
made use of the Latin language, of tables and figures and systems of characters
and signs of all kinds, though less of mathematical ones. These authors mix ‘Wis-
senschaftlichkeit und stilistische Höhenflüge’ (‘scientificity and stylistic flights of
fancy’; Korenjak 2016: 246). Some among them would become part of the scienti-
fic mainstream, such as Copernicus, whose motivation for putting the Sun at the
centre of the universe was at least in part a kind of mystical Sun-theology,46 or the
hermeticist Kepler (on these authors, see chap. 13).47 On the whole, the Renaissance movement did change the outlook on many
things, but (Thorndike 1923–1958: 4:4) much of all this was a somewhat superficial phenomenon and not so extensive as it ap-
peared on the surface. The scholastic method was kept up at numerous universities. Medie-
val Latin and Arabic authors continued to pour from the printing press. The authors of this mystic-holistic and Platonic ‘science’ were much less numer-
ous than the countless ‘Aristotelians’ at the universities since the thirteenth cen-
tury. They can be arranged in a web of dependencies in a way that the authors
from before and during the Scientific Revolution studied in the next chapter could
not. This provides a hint to the fact that this Platonic scientific ‘underground’
movement was of a much more limited scope than the Aristotelian ‘mainstream’
science, although, of course, the two movements were linked. The authors at the
bottom right in figure 21 were, in fact, also important exponents of the Scientific
Revolution. 47 e. g. Kepler’s Harmonices mundi (Lincii, 1619) is of a hermeticist nature. 46 e. g. the hymnic formulation in De revolutionibus I.10 quoted in chap. 13 §4 below. 48 The fourteenth-century Oxford calculators were encountered above (chap. 11 §5); for arith-
metic, some more texts will be met below (chap. 20 §2). 0
12 New approaches in the Renaissance Experimentalism and magia naturalis and a new return to Greek nat-
ural science were major ingredients in the Scientific Revolution discussed in the
next chapter. We might consider having the period of Renaissance science end in
AD 1543, the annus mirabilis (Sarton 1929: 86) in which several groundbreaking
works for science were published. Relation to criteria for science
307 307 Fig. 21: Tentative web of influences among some important hermetic-Platonist authors. Fig. 21: Tentative web of influences among some important hermetic-Platonist authors. Relation to criteria for science §7 Despite the Renaissance’s importance as a literary movement, it failed to alter
the sciences to the same depth. Especially in the natural sciences, universities
kept using scholastic methods and language; indeed, it will be seen below
(chap. 18) that the language employed changed much less dramatically in the fif-
teenth century than it had with the advent of scholasticism in the thirteenth. It
might be argued that the new forms of Renaissance Platonism stimulated mathe-
matical approaches and magia naturalis experimental ones, thus paving the way
for the Scientific Revolution treated in the next chapter. This is true to some ex-
tent, but mathematics progressed to its significant advances in the sixteenth cen-
tury largely in traditional settings; indeed, a chain of important precursors from
the twelfth to the fifteenth century can easily be named.48 Precursors for the ex-
perimental method among the ‘magicians’ of the pre-humanist centuries can also
be found abundantly, as Thorndike’s monumental work (1923–1958) shows. 12 New approaches in the Renaissance 308 While rhetorically minded Renaissance humanism had its influence on some con-
comitants of science but was not a scientific movement at all, the other current we
have described, Platonic Renaissance hermeticism, can be said to have had a sys-
tematic method (I) and to have built a coherent system (IV), but it was one that fo-
cused too much on the greater patterns only (II) and largely lacked testability (III). It did not ‘walk’ (as Galen put it). A larger community was certainly active at the
Aristotelian universities than at the Renaissance private academies (V). As al-
ready stressed, some authors moved in both worlds, some also used both kinds of
Latin (humanist and scholastic) for different purposes, and in some authors math-
ematical formalisation advanced significantly – but these are the very authors
usually included in the Scientific Revolution to be studied in the next chapter. Thus, the quotation from Sarton at the beginning of this chapter is largely con-
firmed: the Renaissance was not so much one of science, but some of its new
ideas – especially mathematical and experimental magia naturalis – would still
bear fruit in the Scientific Revolution. Relation to criteria for science While rhetorically minded Renaissance humanism had its influence on some con-
comitants of science but was not a scientific movement at all, the other current we
have described, Platonic Renaissance hermeticism, can be said to have had a sys-
tematic method (I) and to have built a coherent system (IV), but it was one that fo-
cused too much on the greater patterns only (II) and largely lacked testability (III). It did not ‘walk’ (as Galen put it). A larger community was certainly active at the
Aristotelian universities than at the Renaissance private academies (V). As al-
ready stressed, some authors moved in both worlds, some also used both kinds of
Latin (humanist and scholastic) for different purposes, and in some authors math-
ematical formalisation advanced significantly – but these are the very authors
usually included in the Scientific Revolution to be studied in the next chapter. Thus, the quotation from Sarton at the beginning of this chapter is largely con-
firmed: the Renaissance was not so much one of science, but some of its new
ideas – especially mathematical and experimental magia naturalis – would still
bear fruit in the Scientific Revolution.
|
https://openalex.org/W4381165060
|
https://www.frontiersin.org/articles/10.3389/fgwh.2023.1177718/pdf
|
English
| null |
Editorial: Sex differences in adiposity and cardiovascular disease
|
Frontiers in global women’s health
| 2,023
|
cc-by
| 1,141
|
Editorial on the Research Topic
Sex differences in adiposity and cardiovascular disease © 2023 Janorkar and Marañón. This is an open-
access article distributed under the terms of the The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. The Research Topic Sex Differences in Adiposity and Cardiovascular Disease was proposed a
few months before the COVID pandemic by AJ from the University of Mississippi Medical
Center (USA) and ROM from the Universidad Nacional de Tucuman (Argentina) with the
goal to aid the understanding of the role of the body fat constitution as a cardiovascular risk
factor, and whether this risk is similar for men and women. In addition to this, to determine
if sex steroids play a specific role either in the health of females and males. Today, although there is progress in the study of the effect of sex hormones on the
adipocyte’s metabolism and distribution, its role is still unclear in cardiovascular and
metabolic diseases. Controversial evidence suggests that different fat types contribute to
the high risk of cardiovascular events (1). Thus, visceral fat is meant to contribute to a
deleterious cardiovascular effect, while subcutaneous fat has the opposite function (2). In
this hypothesis, sex hormones may play a vital role, increasing or decreasing the fat mass
in the mentioned depots and contributing to the fat metabolism’s equilibrium. However,
the role of androgens in females, and estrogens in males, need further investigation. g
g
g
In our proposal, we received six original research manuscripts that indirectly fill the aim
of our topic and contribute to the leading journal Frontiers in Global Women’s Health. Of
100% of the manuscript submitted to our topic, 67% were accepted, and 33% were rejected. The case-control study by Zhao et al. examines the association between hypertension and its
control with atrial fibrillation and how the biological sex affects this association. The original
research study by Khatami et al. explored whether iron biomarkers mediate sex differences in
NT-proBNP levels. Authors investigated, using linear regression analyses, the association of
sex and iron biomarkers with NT-proBNP levels, independent of adjustment for potential
confounders. Another original research study by Li et al. TYPE Editorial
PUBLISHED 19 June 2023
DOI 10.3389/fgwh.2023.1177718 EDITED AND REVIEWED BY
Sanne Peters,
Imperial College London, United Kingdom
*CORRESPONDENCE
Rodrigo O. Marañón
rmaranon@fm.unt.edu.ar
RECEIVED 01 March 2023
ACCEPTED 07 June 2023
PUBLISHED 19 June 2023
CITATION
Janorkar A and Marañón RO (2023) Editorial:
Sex differences in adiposity and cardiovascular
disease. Front. Glob. Womens Health 4:1177718. doi: 10.3389/fgwh.2023.1177718
COPYRIGHT
© 2023 Janorkar and Marañón. 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. Amol Janorkar
1 and Rodrigo O. Marañón
2,3* Amol Janorkar
1 and Rodrigo O. Marañón
2,3* 1Department of Biomedical Materials Science, University of Mississippi Medical Center, Jackson, MS,
United States, 2Department of Physiology, Faculty of Medicine, Universidad Nacional de Tucuman, San
Miguel de Tucumán, Argentina, 3National Council on Scientific and Technical Research (CONICET),
Tucuman, Argentina COPYRIGHT Editorial on the Research Topic
Sex differences in adiposity and cardiovascular disease Frontiers in Global Women’s Health Imperial College London, United Kingdom KEYWORDS adiposity, sex differences, cardiovascular disease, women health, sex hormones Front. Glob. Womens Health 4:1177718. doi: 10.3389/fgwh.2023.1177718 Editorial on the Research Topic
Sex differences in adiposity and cardiovascular disease examined the effects of age and
sex on outcomes of coronary artery bypass grafting (CABG) and percutaneous coronary
intervention (PCI) in non-ST-segment elevation acute coronary syndrome (NSTE-ACS)
patients with the three-vessel disease (TVD) and the interaction between treatment and
age or sex in NSTE-ACS and TVD. Finally, the cohort study by Huang et al. showed the
sex difference temporal trends of short-term and long-term mortality after CAD
admission in China. It was based on 11 years of data from a sample of 24,432 people in
the largest cardiovascular disease center in South China. Frontiers in Global Women’s Health frontiersin.org 01 Janorkar and Marañón 10.3389/fgwh.2023.1177718 10.3389/fgwh.2023.1177718 2. Agrawal S, Klarqvist MD, Diamant N, Stanley TL, Ellinor PT, Mehta NN, et al.
BMI-adjusted
adipose
tissue
volumes
exhibit
depot-specific
and
divergent
associations with cardiometabolic diseases. Nat Commun. (2023) 14(1):266. doi: 10.
1038/s41467-022-35704-5 Author contributions AJ and ROM acted as editors of all the manuscripts submitted
to
the
Research
Topic
Sex
differences
in
adiposity
and
cardiovascular disease. Both authors approved the final version of
the manuscript for publication. All authors contributed to the
article and approved the submitted version. Publisher’s note This work was supported by the National Scientific and
Technical
Research
Council—Argentina,
Department
of
Physiology of the Faculty of Medicine, Instituto Superior de
Investigaciones Biológicas (INSIBIO), and the Secretariat of the
Science, Art and Technological Innovation of the Universidad
Nacional
de
Tucumán,
Tucumán,
Argentina
(CONICET-
PIP1220200103303CO). 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. Acknowledgments In this experience, we learned to manage different situations
involved during the publication process, where, in the present
day, the offer to publish and review manuscripts is increasing. The limitation was finding reviewers for the specific field. As editors, we want to thank the excellent people from the
Frontiers staff, and without their collaboration, our work would
have been impossible. We had a great experience with different
authors worldwide, and we thank all the reviewers and staff for
their help and support in this endeavor. Also, we want to thank
Frontiers
for
their
trust
in
us
and
for
letting
us
work
independently and professionally. We are very grateful for the
opportunity to work with all of you. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. 1. Fitzgerald SJ, Janorkar AV, Barnes A, Maranon RO. A new approach to study the
sex differences in adipose tissue. J Biomed Sci. (2018) 25(1):89. doi: 10.1186/s12929-
018-0488-3 Janorkar and Marañón References 1. Fitzgerald SJ, Janorkar AV, Barnes A, Maranon RO. A new approach to study the
sex differences in adipose tissue. J Biomed Sci. (2018) 25(1):89. doi: 10.1186/s12929-
018-0488-3 Frontiers in Global Women’s Health 02 frontiersin.org
|
https://openalex.org/W3030487456
|
https://europepmc.org/articles/pmc7259665?pdf=render
|
English
| null |
The ultrastructural characteristics of bile canaliculus in porcine liver donated after cardiac death and machine perfusion preservation
|
PloS one
| 2,020
|
cc-by
| 8,992
|
PLOS ONE PLOS ONE RESEARCH ARTICLE Editor: Kourosh Saeb Parsy, University of
Cambridge, UNITED KINGDOM Editor: Kourosh Saeb Parsy, University of
Cambridge, UNITED KINGDOM
Received: December 14, 2019
Accepted: May 14, 2020
Published: May 29, 2020 Received: December 14, 2019
Accepted: May 14, 2020
Published: May 29, 2020 Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0233917 Copyright: © 2020 Ishihara 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. Introduction The shortage of brain dead donors for liver transplantation is a serious problem worldwide
[1]. Although donors with circulatory arrest have the potential to expand the transplanted liver
pool [2,3], post-circulatory arrest liver grafts induce high rates of primary nonfunction and
ischemia-reperfusion injury after transplantation [4]. In particular, a high risk of acute and
chronic rejection, including ischemic bile duct damage, and biliary complications has been
reported [5]; thus, the development of liver graft preservation methods after circulatory arrest
is required to overcome these problems [1]. Yo Ishihara1☯¤, Hiroki BochimotoID1,2☯*, Daisuke KondohID3, Hiromichi ObaraID4,
Naoto Matsuno1,5 Yo Ishihara1☯¤, Hiroki BochimotoID1,2☯*, Daisuke KondohID3, Hiromichi ObaraID4,
Naoto Matsuno1,5 Yo Ishihara1☯¤, Hiroki BochimotoID1,2☯*, Daisuke KondohID3, Hiromichi ObaraID4,
Naoto Matsuno1,5 1 Department of Transplantation Technology and Therapeutic Development, Asahikawa Medical University,
Asahikawa, Japan, 2 Division of Aerospace Medicine, Department of Cell Physiology, The Jikei University
School of Medicine, Minato-ku, Japan, 3 Laboratory of Veterinary Anatomy, Obihiro University of Agriculture
and Veterinary Medicine, Obihiro, Japan, 4 Department of Mechanical Engineering, Tokyo Metropolitan
University, Hachioji, Japan, 5 Department of Surgery, Asahikawa Medical University, Asahikawa, Japan ☯These authors contributed equally to this work. ¤ Current address: Shonan Kamakura General Hospital, Kamakura, Japan
* botimoto@jikei.ac.jp Abstract Citation: Ishihara Y, Bochimoto H, Kondoh D,
Obara H, Matsuno N (2020) The ultrastructural
characteristics of bile canaliculus in porcine liver
donated after cardiac death and machine perfusion
preservation. PLoS ONE 15(5): e0233917. https://
doi.org/10.1371/journal.pone.0233917 The effects of each type of machine perfusion preservation (MP) of liver grafts donated after
cardiac death on the bile canaliculi of hepatocytes remain unclear. We analyzed the intracel-
lular three-dimensional ultrastructure of the bile canaliculi and hepatocyte endomembrane
systems in porcine liver grafts after warm ischemia followed by successive MP with modified
University of Wisconsin gluconate solution. Transmission and osmium-maceration scanning
electron microscopy revealed that lumen volume of the bile canaliculi decreased after warm
ischemia. In liver grafts preserved by hypothermic MP condition, bile canaliculi tended to
recover in terms of lumen volume, while their microvilli regressed. In contrast, midthermic
MP condition preserved the functional form of the microvilli of the bile canaliculi. Machine
perfusion preservation potentially restored the bile canaliculus lumen and alleviated the ces-
sation of cellular endocrine processes due to warm ischemia. In addition, midthermic MP
condition prevented the retraction of the microvilli of bile canaliculi, suggesting further miti-
gation of the damage of the bile canaliculi. donated after cardiac death and machine perfusion
preservation. PLoS ONE 15(5): e0233917. https://
doi.org/10.1371/journal.pone.0233917
Editor: Kourosh Saeb Parsy, University of
Cambridge, UNITED KINGDOM
Received: December 14, 2019
Accepted: May 14, 2020
Published: May 29, 2020
Peer Review History: PLOS recognizes the
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author
responses alongside final, published articles. The
editorial history of this article is available here:
https://doi.org/10.1371/journal.pone.0233917 Data Availability Statement: All relevant data are
within the paper. Data Availability Statement: All relevant data are
within the paper. Data Availability Statement: All relevant data are
within the paper. Funding: This work was supported by the Grants-
in-Aid for Scientific Research (C) (KAKENHI) PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 1 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion No.17K10503 of Japan Society for the Promotion
of Science to N.M. URL of the funder’s website:
http://www.jsps.go.jp/english/. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the
manuscript. In addition, all of the authors received
no salary from any funder of this research. Existing cold storage organ preservation techniques fail to preserve marginal donor grafts
[6]. On the other hand, machine perfusion (MP) of post-circulatory arrest donor liver grafts
has been reported to have numerous advantages [6–25], and the optimal conditions of MP,
including perfusion temperature, oxygenation status, flow rate, steady flow and pulsatile flow,
have been discussed [26–33]. Recently, hypothermic MP (HMP) has been established to main-
tain the functions of liver grafts, and its application in clinical practice has begun [34–46]. On
the other hand, warm perfusion has also been reported as an advanced MP method that main-
tains the liver graft functions [2,38,47–56]. Warm perfusion had introduced to maintain liver
grafts at a more physiologic temperature compared with HMP to offers the opportunity to
assess and possibly repair a metabolically active liver graft. Our previous reports indicated that
the midthermic MP (MMP), one type of warm perfusion [57], reduces the hepatocellular
enzyme release [58,59]. In addition, we confirmed that hepatocytes of DCD liver grafts after
MMP retain a functional ultrastructure compared to HMP, by using the observation method
of scanning electron microscopy after osmium-maceration (OM-SEM) [60,61]; ultrastructural
characteristics of hepatocytes are reported to reflect the function of the transplanted liver [60]. Competing interests: The authors have declared
that no competing interests exist. One of the important physiological functions of hepatocytes is the production and secretion
of bile [62]. For liver grafts, MP has the potential to not only inhibit the development of post-
transplant biliary complications, including ischemic cholangiopathy [17,63–67], but also to
protect the bile canaliculus [68]. We evaluated the ultrastructural changes in the bile canaliculi
and hepatocytes around them at four hours after HMP or MMP using OM-SEM and transmis-
sion electron microscopy (TEM). Animals We purchased domestic female pigs (cross-bred Large White, Landrace, and Duroc pigs; age,
2–3 months; body weight, approximately 25 kg) from Taisetsusanroku-sya Co., Ltd. (Asahi-
kawa, Japan). The pigs were kept in a well-ventilated room with a 12-h light: dark cycle, con-
trolled temperature and humidity, and ad libitum access to food and water. All experiments
were performed according to the Guide for the Care and Use of Laboratory Animals at Asahi-
kawa Medical University, and the procedures were approved by the Institutional Animal Eth-
ics Committee of the Clinical Research Center, Asahikawa Medical University (permit no. 14172). Data Availability Statement: All relevant data are
within the paper. As a result, the bile canaliculi that regressed one hour after
warm ischemia showed a strong tendency to recover after MP, especially in MMP, suggesting
the preventative effects of HMP and MMP on bile canaliculi-related functions in liver grafts. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Machine perfusion preservation Livers harvested from pigs were connected and perfused with a MP system (Fig 1), as
described previously [61]. The system was composed of two separate circulating perfusion cir-
cuits, which had a roller pump, for the hepatic artery (HA) and portal vein (PV). Each circuit
had a flow meter and a pressure sensor, allowing pulsatile and non-pulsatile flow, respectively. Additionally, an oxygenator was installed in the the upstream of the circuits for the PV and
HA were connected via plastic connectors to each of the hepatic vessels. The MP systems had
waterproof thermocouples that measured the solution and organ temperatures, and a dissolved
oxygen meter. The flow conditions and temperatures of the preservation solution were
recorded by a system-installed computer. In the systems, the organ chamber temperature was
controlled by ice-cold water and a heat exchanger. The flow rate was mainly set to 0.22 mL/
min/g for the PV and 0.06 mL/min/g for the HA, as described previously [61]. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 2 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Preparation and preservation of the liver donated after cardiac death
Pigs were used as liver graft donors. These pigs were intubated and ventilated with inhalation
anesthesia by isoflurane (Forane; Abbott, Japan), and laparotomized. Immediately after lapa-
rotomy, liver tissue samples from the liver surface were obtained by biopsy as a control. Then
the pigs were intravenously injected with potassium chloride to induce circulatory arrest fol-
lowed by the withdrawal of ventilation, as described previously [61]. The time point of the
Fig 1. Schematic representation of the continuous machine perfusion system. https://doi.org/10.1371/journal.pone.0233917.g001
p PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Preparation and preservation of the liver donated after cardiac death During warm
ischemia, hepatic artery and portal vein were isolated to connect with organ flush lines, and at
60 minutes of warm ischemia, the liver tissue samples were obtained from distinct regions of
the liver surface. Immediately after tissue sampling, the liver grafts were harvested and subse-
quently flushed with Euro-Collins solution via the HA and PV routes at 8˚C on the back table. After the initial flushing, the flush routes were connected to the perfusion preservation
machine and the liver grafts were continuously perfused for four hours with modified Univer-
sity of Wisconsin gluconate solution (sodium gluconate 17.5 g, KH2PO4 3.4 g, trehalose 10 g,
glutathione 0.9 g, adenosine 1.3 g, HEPES 4.7 g, penicilline 200,000 U, dexamethasone 16 mg,
MgSO4 1 g, caffeine 4 g, polyethylene glycol 10 g, and glycine 1 g per 1 L). The liver grafts were
conserved at a constant temperature of 8˚C as HMP (n = 4) or gradually warmed from 8˚C to
22˚C during perfusion as MMP (n = 5). After MP, the liver tissue samples were biopsied from
th
ll
f
d
i
f
ft
f
i
h
Li
l bl
k
i
di t l Transmission electron microscopy The liver tissue samples were trimmed into small blocks and fixed with 2% glutaraldehyde and
2% paraformaldehyde in 0.1 M phosphate buffer (PB) for two hours at 4˚C. After fixation, the
blocks were washed 3 times with PB containing 7.5% sucrose and post-fixed with 1% osmium
tetroxide (OsO4) in PB for two hours at 4˚C. After washing thoroughly with PB containing
7.5% sucrose, the blocks were dehydrated with a graded series of ethanol. After dehydration,
the samples were transferred in propylene oxide, infiltrated and then embedded in epoxy resin
(Epon 812). Ultrathin section (80 nm thick) were cut, stained with uranyl acetate and lead cit-
rate, and observed using an HT7700 transmission electron microscope (Hitachi High Technol-
ogies, Tokyo, Japan). Preparation and preservation of the liver donated after cardiac death Pigs were used as liver graft donors. These pigs were intubated and ventilated with inhalation
anesthesia by isoflurane (Forane; Abbott, Japan), and laparotomized. Immediately after lapa-
rotomy, liver tissue samples from the liver surface were obtained by biopsy as a control. Then
the pigs were intravenously injected with potassium chloride to induce circulatory arrest fol-
lowed by the withdrawal of ventilation, as described previously [61]. The time point of the PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 3 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion induction of circulatory arrest was defined as 0 minutes of warm ischemia. During warm
ischemia, hepatic artery and portal vein were isolated to connect with organ flush lines, and at
60 minutes of warm ischemia, the liver tissue samples were obtained from distinct regions of
the liver surface. Immediately after tissue sampling, the liver grafts were harvested and subse-
quently flushed with Euro-Collins solution via the HA and PV routes at 8˚C on the back table. After the initial flushing, the flush routes were connected to the perfusion preservation
machine and the liver grafts were continuously perfused for four hours with modified Univer-
sity of Wisconsin gluconate solution (sodium gluconate 17.5 g, KH2PO4 3.4 g, trehalose 10 g,
glutathione 0.9 g, adenosine 1.3 g, HEPES 4.7 g, penicilline 200,000 U, dexamethasone 16 mg,
MgSO4 1 g, caffeine 4 g, polyethylene glycol 10 g, and glycine 1 g per 1 L). The liver grafts were
conserved at a constant temperature of 8˚C as HMP (n = 4) or gradually warmed from 8˚C to
22˚C during perfusion as MMP (n = 5). After MP, the liver tissue samples were biopsied from
the well-perfused region of graft surface in each group. Liver sample blocks were immediately
fixed with an appropriate fixative for the analysis, as described below. The degree of biliary
injury at four hours after MP were evaluated based on the alkaline phosphatase level in perfus-
ate collected from the suprahepatic vena cava of liver grafts in each MP group, as described
previously [69]. These alkaline phosphatase data were presented as the mean ± SEM, and
unpaired two-tailed t-tests were used to compare the significance of differences between
groups A and B. induction of circulatory arrest was defined as 0 minutes of warm ischemia. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Changes in the ultrastructure of the bile canaliculi after warm ischemia The continuous hypoxic exposure of liver grafts induced by one hour of warm ischemia caused
the cessation of bile production and morphological abnormalities of the bile canaliculi. After
warm ischemia, OM-SEM revealed the large vacuoles in hepatocytes (Fig 3A, colored red), as
described previously. Although the bile canaliculi seemed normal at low magnification (Fig
3A), the cross-sectional area of the lumen of the bile canaliculi after warm ischemia tended to
become smaller in comparison to controls (Figs 2B and 3B). In contrast with the controls,
small stacks of Golgi apparatus were rarely detected around these bile canaliculi (Figs 2B and
3B). The small vacant spaces around the bile canaliculi were observed similarly to controls,
and these space corresponded to the cytoplasmic area without any endomembrane organelles
observed by TEM (Fig 3B and 3C). These findings showed that warm ischemia causes ultra-
structural destruction of bile canaliculi and the related intracellular subsets, reflecting the
decreased bile production induced by hypoxic exposure. Ultrastructure of the normal bile canaliculi observed by OM-SEM and TEM First, we established the overall shape of bile canaliculus by OM-SEM and TEM. In control liv-
ers, OM-SEM revealed that hepatocytes mutually formed the bile canaliculi with microvilli
between the plasma membranes of contiguous hepatocytes (Fig 2A and 2B). The bile canaliculi
were often accompanied by several small stacks of Golgi apparatus around the cytoplasm of
the constituent hepatocytes (Fig 2B). The small vacant spaces without any other endomem-
brane organelles around the bile canaliculi were often found (Fig 2B). The corresponding find-
ings were also obtained by TEM observation (Fig 2C). The vacant space around the bile
canaliculi observed by OM-SEM corresponded to the cytoplasmic region without endomem-
brane organelles (Fig 2C). These findings showed that the details and three-dimensional con-
formation of the bile canaliculi and the related intracellular components could be visualized by
OM-SEM with complementary TEM observation. Osmium-maceration for SEM For SEM observation, the osmium maceration method was applied to the liver tissue samples,
as described previously [61]. In brief, liver samples cut into small pieces were fixed with 0.5%
glutaraldehyde and 0.5% paraformaldehyde in PB for 30 min at 4˚C. After fixation, the liver
blocks were directly immersed in 1% OsO4 in PB for four hours at 4˚C. The samples were then
washed thoroughly with PB and transferred into dimethyl sulfoxide solution in order of 25 to
50% each for 30 min for cryoprotection. The samples were then frozen on a deeply chilled alu-
minum metal plate with liquid nitrogen, and cracked into two particles with a screwdriver and
a hammer. After freeze cracking, the samples were thawed in 50% dimethyl sulfoxide solution,
washed thoroughly in PB and transferred into 0.1% diluted OsO4 in PB for 96 hours at around
20˚C under light for maceration. After the maceration period, the samples were immersed
into 1% OsO4 in PB for one hour for post-fixation and then thoroughly washed with PB. The
samples were transferred into 1% tannic acid in PB for two hours, subsequently washed with
PB, and then immersed in 1% OsO4 in PB for one hour for conductive staining. The liver sam-
ples were dehydrated in graded series of ethanol and immersed in tert-butyl alcohol. After
freezing, the samples were lyophilized in an ES2030 freeze-dryer (Hitachi Koki Co., Ltd.,
Tokyo, Japan). The dried specimens were then mounted onto a metal plate and lightly coated PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 4 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion with platinum-palladium in an E1010 ion sputtering device (Hitachi Koki). These finally pro-
cessed specimens were observed in secondary electron-mode by a field emission S4100 scan-
ning electron microscope (Hitachi High Technologies). PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Recovery of the ultrastructure of bile canaliculi by HMP and MMP Even after liver graft preservation with four hours of HMP or MMP, almost no bile was col-
lected from the bile ducts of the liver grafts. However, ultrastructural restoration of the bile
canaliculi was found to have occurred, particularly after MMP. After four hours of HMP, OM-SEM revealed swollen mitochondria in many hepatocytes
(Fig 4A). The cross-sectional area of the lumen of the bile canaliculi after HMP was restored
from the changes that were observed in warm ischemia (Figs 3B and 4B); however, the restora-
tion was decreased in comparison to that in the controls (Figs 2B and 4B). This restoration of
the bile canaliculi was more clearly indicated by TEM (Fig 4C), whereas the microvilli in the
bile canaliculi tended to regress after HMP (Fig 4C). OM-SEM revealed that the hepatocytes after MMP included macro-autophagosomes and
functional forms of mitochondria (Fig 5A), indicating that MMP was more protective for
hepatocytes than HMP, as described previously. Additionally, after MMP, the cross-sectional
area of the lumen of the bile canaliculi almost recovered to the control level (Fig 5B and 5C),
and the microvilli in the bile canaliculi were also maintained (Fig 5B and 5C). 5 / 17 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Fig 2. The ultrastructure of the bile canaliculi in porcine hepatocytes of the control liver. (A and B) Representative hepatocytes and bile canaliculi were observed by
SEM in osmium-macerated control porcine liver graft samples. The partial area indicated in A was further photographed at a higher magnification (B). (C) Typical bile
canaliculi were identified in the ultrathin sections of the Epon 812-embedded control liver tissue. Bile canaliculi are colored green. Arrows indicate the Golgi apparatus,
and asterisks indicate vacant spaces without any other endomembrane organelles around the bile canaliculi. Bars = 1 μm. https://doi.org/10.1371/journal.pone.0233917.g002 Fig 2. The ultrastructure of the bile canaliculi in porcine hepatocytes of the control liver. (A and B) Representative hepatocytes and bile canaliculi were observed by
SEM in osmium-macerated control porcine liver graft samples. The partial area indicated in A was further photographed at a higher magnification (B). (C) Typical bile
canaliculi were identified in the ultrathin sections of the Epon 812-embedded control liver tissue. Bile canaliculi are colored green. Arrows indicate the Golgi apparatus,
and asterisks indicate vacant spaces without any other endomembrane organelles around the bile canaliculi. Bars = 1 μm. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Recovery of the ultrastructure of bile canaliculi by HMP and MMP https://doi.org/10.1371/journal.pone.0233917.g002 6 / 17 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Fig 3. Changes in the ultrastructure of the bile canaliculi in porcine hepatocytes after warm ischemia. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples after warm ischemia for 60 minutes. The partial area indicated in A was further photographed at a
higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue, huge vacuoles are colored red. Asterisks indicate vacant space without any other
endomembrane organelles around the bile canaliculi. (C) Typical bile canaliculi were identified in the ultrathin sections of Epon 812-embedded tissues from liver graft
samples after warm ischemia for 60 minutes. Bile canaliculi are colored green and asterisks indicate the vacant space without any other endomembrane organelles around
the bile canaliculi. Bars = 1 μm. Fig 3. Changes in the ultrastructure of the bile canaliculi in porcine hepatocytes after warm ischemia. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples after warm ischemia for 60 minutes. The partial area indicated in A was further photographed at a
higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue, huge vacuoles are colored red. Asterisks indicate vacant space without any other
endomembrane organelles around the bile canaliculi. (C) Typical bile canaliculi were identified in the ultrathin sections of Epon 812-embedded tissues from liver graft
samples after warm ischemia for 60 minutes. Bile canaliculi are colored green and asterisks indicate the vacant space without any other endomembrane organelles around
the bile canaliculi. Bars = 1 μm. https://doi.org/10.1371/journal.pone.0233917.g003 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 7 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Fig 4. The ultrastructural changes of the bile canaliculi in porcine liver grafts preserved by HMP. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples preserved by HMP for 4 h after 60 minutes of warm ischemia. The partial area indicated in A was
further photographed under higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue. Asterisks indicate vacant space without any other
endomembrane organelles around the bile canaliculi. https://doi.org/10.1371/journal.pone.0233917.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Recovery of the ultrastructure of bile canaliculi by HMP and MMP (C) Typical bile canaliculi were identified in the ultrathin sections of Epon 812-embedded tissues from liver graft
samples preserved by HMP for 4 h after 60 minutes of warm ischemia. Bile canaliculi are colored green. Asterisks indicate the vacant space without any other
endomembrane organelles around the bile canaliculi. Bars = 1 μm. https://doi org/10 1371/journal pone 0233917 g004 Fig 4. The ultrastructural changes of the bile canaliculi in porcine liver grafts preserved by HMP. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples preserved by HMP for 4 h after 60 minutes of warm ischemia. The partial area indicated in A was
further photographed under higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue. Asterisks indicate vacant space without any other
endomembrane organelles around the bile canaliculi. (C) Typical bile canaliculi were identified in the ultrathin sections of Epon 812-embedded tissues from liver graft
samples preserved by HMP for 4 h after 60 minutes of warm ischemia. Bile canaliculi are colored green. Asterisks indicate the vacant space without any other
endomembrane organelles around the bile canaliculi. Bars = 1 μm. https://doi.org/10.1371/journal.pone.0233917.g004 8 / 17 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Fig 5. The ultrastructural changes of the bile canaliculi in porcine liver grafts preserved by MMP. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples preserved by MMP for 4 h after 60 minutes of warm ischemia. The partial area indicated in A was
further photographed under higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue, huge vacuoles are colored red. Asterisks indicate vacant
space without any other endomembrane organelles around the bile canaliculi. (C) Typical bile canaliculi were identified in the ultrathin sections of the Epon
812-embedded tissues from liver graft samples preserved by MMP for 4 h after 60 minutes of warm ischemia. Bile canaliculi are colored green. Asterisks indicate vacant
space without any other endomembrane organelles around the bile canaliculi. Bars = 1 μm. Fig 5. The ultrastructural changes of the bile canaliculi in porcine liver grafts preserved by MMP. (A and B) Representative hepatocytes and bile canaliculi were
observed by SEM in osmium-macerated porcine liver graft samples preserved by MMP for 4 h after 60 minutes of warm ischemia. https://doi.org/10.1371/journal.pone.0233917.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Discussion In the present study, the porcine bile canaliculi after warm ischemia and after HMP and MMP
preservation were analyzed by OM-SEM and complementary TEM methods. Okouhchi et al. [70] analyzed the ultrastructure of the rat liver after HMP preservation by OM-SEM, but did
not describe the bile canaliculi. Our previous study using OM-SEM [61] described the bile can-
aliculi after MMP preservation under low magnification, but the detailed ultrastructure of the
bile canaliculi was not established at that stage. The present study revealed the detailed ultra-
structural changes of the bile canaliculi after both HMP and MMP preservation. Morphological abnormalities of the bile canaliculi after warm ischemia were alleviated by
subsequent MP, and this preventative effect was greater in MMP than in HMP. These findings
were consistent with previous physiological reports [2,38,47–56], which suggested that the
ultrastructure of the bile canaliculi is important from both morphological and functional
perspectives. One hour of warm ischemia was associated with regression of the bile canaliculus lumen;
this change is consistent with our previous findings [61]. Moussa et al. [71] also reported the
regression and disappearance of the bile canaliculi during warm ischemia based on observa-
tion by TEM. These findings may reflect autophagic changes in hepatocytes after warm ische-
mia suppressed the intracellular trafficking pathway [72]. In addition, small stacks of Golgi
apparatus around the bile canaliculi were not found after warm ischemia. This distorted
arrangement of Golgi apparatus was probably caused by hypoxia because the hepatocellular
polarity is maintained by mitochondrial energy [73,74]. On the other hand, the organelle-poor
cytoplasmic region of hepatocytes around the bile canaliculi and functional microvilli in the
bile canaliculi was retained, even after warm ischemia. The accumulation of actin, a cyto-
plasmic component concentrated in the peri-biliary region and microvilli, occurs in hepato-
cyte cytoplasm around the bile canaliculi [75], and the actin in this region is disrupted under
reperfusion rather than during warm ischemia [76], although the microtubules in hepatocytes
are distorted after warm ischemia [77]. The present study supported the opinion that warm
ischemia does not largely affect the cytoplasmic components around the bile canaliculi. The cross-sectional area of the bile canaliculus lumen in liver grafts that regressed from
warm ischemia tended to recover after both HMP and MMP preservation, suggesting the
resumption of the secretion of bile canaliculi contents, namely bile salts. Recovery of the ultrastructure of bile canaliculi by HMP and MMP The partial area indicated in A was
further photographed under higher magnification (B). Bile canaliculi are colored green, nuclei are colored blue, huge vacuoles are colored red. Asterisks indicate vacant
space without any other endomembrane organelles around the bile canaliculi. (C) Typical bile canaliculi were identified in the ultrathin sections of the Epon
812-embedded tissues from liver graft samples preserved by MMP for 4 h after 60 minutes of warm ischemia. Bile canaliculi are colored green. Asterisks indicate vacant
space without any other endomembrane organelles around the bile canaliculi. Bars = 1 μm. https://doi.org/10.1371/journal.pone.0233917.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 9 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Correspondingly, the value of alkaline phosphatase in perfusate after four hours of MMP (18.0
±3.1 IU/L) was significantly lower in comparison to HMP (26.3±1.0 IU/L) (S1 Fig), indicating
that MMP suppressed the increased biliary enzyme release in comparison to the HMP. Never-
theless, after preservation with both HMP and MMP, small stacks of Golgi apparatus were
rarely detected around the bile canaliculi (Figs 4B and 5B). These findings indicated that, MP
preservation, especially MMP, of liver grafts after warm ischemia enabled the ultrastructure of
the bile canaliculi to be maintained and restored. Correspondingly, the value of alkaline phosphatase in perfusate after four hours of MMP (18.0
±3.1 IU/L) was significantly lower in comparison to HMP (26.3±1.0 IU/L) (S1 Fig), indicating
that MMP suppressed the increased biliary enzyme release in comparison to the HMP. Never-
theless, after preservation with both HMP and MMP, small stacks of Golgi apparatus were
rarely detected around the bile canaliculi (Figs 4B and 5B). These findings indicated that, MP
preservation, especially MMP, of liver grafts after warm ischemia enabled the ultrastructure of
the bile canaliculi to be maintained and restored. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 Discussion The secretion of bile
salts from hepatocytes into the bile canaliculi is an important factor for activating bile produc-
tion [78]. HMP and MMP did not seem to irregularly increase the bile duct pressure, although
irregular peri-biliary actin accumulation and biliary dilatation caused by the ischemia-reperfu-
sion treatment—reflecting increased bile duct pressure—has been reported [79]. These find-
ings suggested that the initial cold perfusion phase present in both MMP and HMP may
prevent mitochondrial damage in hepatocytes and lead to the resumption of bile salt secretion
[65]. The bile canaliculus microvilli after HMP preservation tended to regress more in compari-
son to after MMP. The ultrastructure of the microvilli in the bile canaliculi is associated with PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 10 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion the bile secretion function [73,80,81], and ultrastructural changes of bile canaliculus microvilli
during ischemia-reperfusion injury, drug injury, and cholestasis are characterized by micro-
villi withdrawal [81–87]. Retraction of the microvilli occurs due to damage of the cell mem-
brane forming the bile canaliculi by protonated hydrophobic bile salts [65,88]. Since HMP
consumes less oxygen than warm perfusion [89], hepatocytes may reduce ATP production due
to decreased metabolism during HMP preservation. ATP depletion in hepatocytes alters trans-
porter functions, such as the bile salt export pump and disrupts the balance between bile salts
and phospholipids [65]. It is therefore considered that the neutralization of salts by phospho-
lipids is weakened and that the cell membrane of the bile canaliculi may be more damaged
during HMP than during MMP. Actually, it was also confirmed that the level of ALP, a marker
of capillary bile duct damage, was lower in MMP than in HMP [90]. The present study was associated with several limitations. First, this study did not confirm
the ultrastructural changes in the bile canaliculi in the liver transplanted or monitored after
perfusion storage. Like a previous study of allogeneic liver transplantation of porcine liver
grafts under conditions that were similar to the conditions in this study [91], the present MMP
results are still in the preclinical stage. In addition, this study did not evaluate the effect in
reperfusion at normothermia Thus, the present results should be investigated by further stud-
ies using liver transplant models that are clinically suitable for transplantation and reperfusion
at normothermia ex situ. Supporting information S1 Fig. Changes in the perfusate enzymes after warm ischemia and subsequent preserva-
tion by HMP or MMP. The levels of alkaline phosphatase (ALP) in the perfusate at 4 hours
after hypothermic and midthermic machine perfusion preservation. Data are shown as the
mean ± SEM. Unpaired two-tailed t-tests were used (p<0.05). (TIF) Acknowledgments We thank all of the lab members and colleagues for their helpful suggestions and assistance in
the experiments. We are extremely grateful to Mr. Yoshiyasu Satake for carrying out all of the
research. Discussion Second, among the components of the biliary system, bile duct cells are very sensitive to
ischemia [4]; however, this study did not examine the bile ducts. Future studies are needed to
examine the ultrastructure of the bile duct in order to investigate the optimal conditions for
clinical transplantation [65]. In conclusion, MP preservation alleviated the cessation of intracellular trafficking processes
of hepatocytes caused by warm ischemia and restored the retracted bile canaliculus lumen. In
addition, MMP temperature conditions prevented the retraction of the microvilli in the bile
canaliculi by mitigating the damage to the cell membrane forming the bile canaliculi. In future
study, more clinically appropriate MP conditions to preserve the functions of the liver grafts
should be established by using normothermically reperfusion systems. To achieve the above
objectives, further physiological studies are required to reveal the ultrastructural changes in
liver constituent cells under various conditions, including different temperatures and different
levels of oxygenation, during perfusion storage. References 1. Dutkowski P, Linecker M, DeOliveira ML, Mu¨llhaupt B, Clavien P-A. Challenges to liver transplantation
and strategies to improve outcomes. Gastroenterology. 2015; 148: 307–23. https://doi.org/10.1053/j. gastro.2014.08.045 PMID: 25224524 2. Goldaracena N, Barbas AS, Selzner M. Normothermic and subnormothermic ex-vivo liver perfusion in
liver transplantation. Curr Opin Organ Transplant. 2016; 21: 315–21. https://doi.org/10.1097/MOT. 0000000000000305 PMID: 27093224 3. Okamura Y, Hata K, Tanaka H, Hirao H, Kubota T, Inamoto O, et al. Impact of Subnormothermic
Machine Perfusion Preservation in Severely Steatotic Rat Livers: A Detailed Assessment in an Isolated
Setting. Am J Transplant. 2017; 17: 1204–1215. https://doi.org/10.1111/ajt.14110 PMID: 27860296 4. Mourad MM, Algarni A, Liossis C, Bramhall SR. Aetiology and risk factors of ischaemic cholangiopathy
after liver transplantation. World J Gastroenterol. 2014; 20: 6159–69. https://doi.org/10.3748/wjg.v20. i20.6159 PMID: 24876737 5. Chan EY, Olson LC, Kisthard JA, Perkins JD, Bakthavatsalam R, Halldorson JB, et al. Ischemic cholan-
giopathy following liver transplantation from donation after cardiac death donors. Liver Transpl. 2008;
14: 604–10. https://doi.org/10.1002/lt.21361 PMID: 18433032 6. Vogel T, Brockmann JG, Pigott D, Neil DAH, Muthusamy ASR, Coussios CC, et al. Successful trans-
plantation of porcine liver grafts following 48-hour normothermic preservation. PLoS One. 2017; 12:
e0188494. https://doi.org/10.1371/journal.pone.0188494 PMID: 29176869 7. Tchilikidi KY. Liver graft preservation methods during cold ischemia phase and normothermic machine
perfusion. World J Gastrointest Surg. 2019; 11: 126–142. https://doi.org/10.4240/wjgs.v11.i3.126
PMID: 31057698 8. Banan B, Chung H, Xiao Z, Tarabishy Y, Jia J, Manning P, et al. Normothermic extracorporeal liver per-
fusion for donation after cardiac death (DCD) livers. Surgery. 2015; 158: 1642–50. https://doi.org/10. 1016/j.surg.2015.07.016 PMID: 26294088 9. Op den Dries S, Karimian N, Westerkamp AC, Sutton ME, Kuipers M, Wiersema-Buist J, et al. Normo-
thermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor
livers. Liver Transpl. 2016; 22: 994–1005. https://doi.org/10.1002/lt.24436 PMID: 26946466 10. Bral M, Gala-Lopez B, Bigam D, Kneteman N, Malcolm A, Livingstone S, et al. Preliminary Single-Cen-
ter Canadian Experience of Human Normothermic Ex Vivo Liver Perfusion: Results of a Clinical Trial. Am J Transplant. 2017; 17: 1071–1080. https://doi.org/10.1111/ajt.14049 PMID: 27639262 11. Monbaliu DR, Debbaut C, Hillewaert WJ, Laleman WJ, Sainz-Barriga M, Pirenne J, et al. Flow competi-
tion between hepatic arterial and portal venous flow during hypothermic machine perfusion preservation
of porcine livers. Int J Artif Organs. 2012; 35: 119–31. https://doi.org/10.5301/ijao.5000038 PMID:
22388941 12. Kim JS, Boudjema K, D’Alessandro A, Southard JH. Writing – original draft: Hiroki Bochimoto. Writing – original draft: Hiroki Bochimoto. Writing – review & editing: Yo Ishihara, Hiroki Bochimoto, Daisuke Kondoh, Hiromichi
Obara, Naoto Matsuno. Supervision: Naoto Matsuno. Supervision: Naoto Matsuno. Supervision: Naoto Matsuno. Author Contributions Conceptualization: Hiroki Bochimoto, Naoto Matsuno. 11 / 17 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion Funding acquisition: Naoto Matsuno. Investigation: Yo Ishihara, Hiroki Bochimoto, Daisuke Kondoh, Hiromichi Obara, Naoto
Matsuno. Resources: Yo Ishihara, Hiroki Bochimoto, Daisuke Kondoh, Hiromichi Obara, Naoto
Matsuno. References Machine perfusion of the liver: maintenance of
mitochondrial function after 48-hour preservation. Transplant Proc. 1997; 29: 3452–4. https://doi.org/
10.1016/s0041-1345(97)00975-5 13. Nasralla D, Coussios CC, Mergental H, Akhtar MZ, Butler AJ, Ceresa CDL, et al. A randomized trial of
normothermic preservation in liver transplantation. Nature. 2018; 557: 50–56. https://doi.org/10.1038/
s41586-018-0047-9 PMID: 29670285 14. Bessems M, Doorschodt BM, van Marle J, Vreeling H, Meijer AJ, van Gulik TM. Improved machine per-
fusion preservation of the non-heart-beating donor rat liver using Polysol: a new machine perfusion PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 12 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion preservation solution. Liver Transpl. 2005; 11: 1379–88. https://doi.org/10.1002/lt.20502 PMID:
16237689 15. Fondevila C, Hessheimer AJ, Maathuis M-HJ, Muñoz J, Taura´ P, Calatayud D, et al. Superior preserva-
tion of DCD livers with continuous normothermic perfusion. Ann Surg. 2011; 254: 1000–7. https://doi. org/10.1097/SLA.0b013e31822b8b2f PMID: 21862925 16. Jia J, Li J, Zhang S, Xie H, Zhou L, Zheng S. A promising ex vivo liver protection strategy: machine per-
fusion and repair. Hepatobiliary Surg Nutr. 2019; 8: 142–143. https://doi.org/10.21037/hbsn.2019.03.07
PMID: 31098362 17. Bellini MI, Nozdrin M, Yiu J, Papalois V. Machine Perfusion for Abdominal Organ Preservation: A Sys-
tematic Review of Kidney and Liver Human Grafts. J Clin Med. 2019; 8. https://doi.org/10.3390/
jcm8081221 PMID: 31443179 18. van Rijn R, van den Berg AP, Erdmann JI, Heaton N, van Hoek B, de Jonge J, et al. Study protocol for a
multicenter randomized controlled trial to compare the efficacy of end-ischemic dual hypothermic oxy-
genated machine perfusion with static cold storage in preventing non-anastomotic biliary strictures after
transplantation of liver gra. BMC Gastroenterol. 2019; 19: 40. https://doi.org/10.1186/s12876-019-
0956-6 PMID: 30866837 19. Burlage LC, Karimian N, Westerkamp AC, Visser N, Matton APM, van Rijn R, et al. Oxygenated hypo-
thermic machine perfusion after static cold storage improves endothelial function of extended criteria
donor livers. HPB (Oxford). 2017; 19: 538–546. https://doi.org/10.1016/j.hpb.2017.02.439 PMID:
28351756 20. Schlegel A, Rougemont O De, Graf R, Clavien PA, Dutkowski P. Protective mechanisms of end-ische-
mic cold machine perfusion in DCD liver grafts. J Hepatol. 2013; 58: 278–286. https://doi.org/10.1016/j. jhep.2012.10.004 PMID: 23063573 21. Jain S, Lee CY, Baicu S, Duncan H, Xu H, Jones JW, et al. Hepatic function in hypothermically stored
porcine livers: comparison of hypothermic machine perfusion vs cold storage. Transplant Proc. 2005;
37: 340–1. https://doi.org/10.1016/j.transproceed.2004.12.069 PMID: 15808637 22. Zhang Y, Zhang Y, Zhang M, Ma Z, Wu S. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 References https://doi.org/10.1111/tri.12344 PMID: 24797796 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 13 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion 33. Boteon YL, Afford SC. Machine perfusion of the liver: Which is the best technique to mitigate ischae-
mia-reperfusion injury? World J Transplant. 2019; 9: 14–20. https://doi.org/10.5500/wjt.v9.i1.14 PMID:
30697517 34. Li P, Liu YF, Yang L. Advantages of dual hypothermic oxygenated machine perfusion over simple cold
storage in the preservation of liver from porcine donors after cardiac death. Clin Transplant. 2015; 29:
820–828. https://doi.org/10.1111/ctr.12586 PMID: 26147375 35. Dutkowski P, Polak WG, Muiesan P, Schlegel A, Verhoeven CJ, Scalera I, et al. First Comparison of
Hypothermic Oxygenated PErfusion Versus Static Cold Storage of Human Donation After Cardiac
Death Liver Transplants: An International-matched Case Analysis. Ann Surg. 2015; 262: 764–70; dis-
cussion 770–1. https://doi.org/10.1097/SLA.0000000000001473 PMID: 26583664 36. van Rijn R, van Leeuwen OB, Matton APM, Burlage LC, Wiersema-Buist J, van den Heuvel MC, et al. Hypothermic oxygenated machine perfusion reduces bile duct reperfusion injury after transplantation of
donation after circulatory death livers. Liver Transpl. 2018; 24: 655–664. https://doi.org/10.1002/lt. 25023 PMID: 29369470 37. Dutkowski P, Furrer K, Tian Y, Graf R, Clavien P-A. Novel short-term hypothermic oxygenated perfu-
sion (HOPE) system prevents injury in rat liver graft from non-heart beating donor. Ann Surg. 2006;
244: 968–76; discussion 976–7. https://doi.org/10.1097/01.sla.0000247056.85590.6b PMID: 17122622 38. van Leeuwen OB, de Vries Y, Fujiyoshi M, Nijsten MWN, Ubbink R, Pelgrim GJ, et al. Transplantation
of High-risk Donor Livers After Ex Situ Resuscitation and Assessment Using Combined Hypo- and Nor-
mothermic Machine Perfusion: A Prospective Clinical Trial. Ann Surg. 2019; XX: 1. https://doi.org/10. 1097/SLA.0000000000003540 PMID: 31633615 39. Schlegel A, Kron P, Graf R, Dutkowski P, Clavien P-A. Warm vs. cold perfusion techniques to rescue
rodent liver grafts. J Hepatol. 2014; 61: 1267–75. https://doi.org/10.1016/j.jhep.2014.07.023 PMID:
25086285 40. Zeng X, Wang S, Li S, Yang Y, Fang Z, Huang H, et al. Hypothermic oxygenated machine perfusion
alleviates liver injury in donation after circulatory death through activating autophagy in mice. Artif
Organs. 2019; 1–13. https://doi.org/10.1111/aor.13525 PMID: 31237688 41. Zeng X, Li M, Fan X, Xue S, Liang W, Fang Z, et al. Hypothermic Oxygenated Machine Perfusion Allevi-
ates Donation After Circulatory Death Liver Injury Through Regulating P-selectin-dependent and -inde-
pendent Pathways in Mice. Transplantation. 2019; 103: 918–928. https://doi.org/10.1097/TP. 0000000000002621 PMID: 31033856 42. Schlegel A, Graf R, Clavien P-A, Dutkowski P. References Hypothermic machine perfusion reduces the incidences of
early allograft dysfunction and biliary complications and improves 1-year graft survival after human liver
transplantation: A meta-analysis. Medicine (Baltimore). 2019; 98: e16033. https://doi.org/10.1097/MD. 0000000000016033 PMID: 31169745 23. Guarrera J V., Estevez J, Boykin J, Boyce R, Rashid J, Sun S, et al. Hypothermic machine perfusion of
liver grafts for transplantation: technical development in human discard and miniature swine models. Transplant Proc. 2005; 37: 323–5. https://doi.org/10.1016/j.transproceed.2004.12.094 PMID:
15808631 24. Nassar A, Liu Q, Farias K, D’Amico G, Tom C, Grady P, et al. Ex Vivo Normothermic Machine Perfusion
Is Safe, Simple, and Reliable: Results From a Large Animal Model. Surg Innov. 2014;
1553350614528383-. https://doi.org/10.1177/1553350614528383 PMID: 24694840 25. Nickkholgh A, Nikdad M, Shafie S, Dezfouli SA, Mehrabi A, Eason JD, et al. Ex Situ Liver Machine Per-
fusion As An Emerging Graft Protective Strategy In Clinical Liver Transplantation: The Dawn of A New
Era. Transplantation. 2019; 1. https://doi.org/10.1097/TP.0000000000002772 PMID: 31022148 26. Barbas AS, Goldaracena N, Dib MJ, Selzner M. Ex-vivo liver perfusion for organ preservation: Recent
advances in the field. Transplant Rev (Orlando). 2016; 30: 154–60. https://doi.org/10.1016/j.trre.2016. 03.002 PMID: 27158081 27. Yuan X, Theruvath AJ, Ge X, Floerchinger B, Jurisch A, Garcı´a-Cardeña G, et al. Machine perfusion or
cold storage in organ transplantation: indication, mechanisms, and future perspectives. Transpl Int. 2010; 23: 561–70. https://doi.org/10.1111/j.1432-2277.2009.01047.x PMID: 20074082 28. Marecki H, Bozorgzadeh A, Porte RJ, Leuvenink HG, Uygun K, Martins PN. Liver ex situ machine perfu-
sion preservation: A review of the methodology and results of large animal studies and clinical trials. Liver Transpl. 2017; 23: 679–695. https://doi.org/10.1002/lt.24751 PMID: 28240817 29. Dutkowski P, de Rougemont O, Clavien P-A. Machine perfusion for “marginal” liver grafts. Am J Trans-
plant. 2008; 8: 917–24. https://doi.org/10.1111/j.1600-6143.2008.02165.x PMID: 18416733 30. Verhoeven CJ, Farid WRR, de Jonge J, Metselaar HJ, Kazemier G, van der Laan LJW. Biomarkers to
assess graft quality during conventional and machine preservation in liver transplantation. J Hepatol. 2014; 61: 672–84. https://doi.org/10.1016/j.jhep.2014.04.031 PMID: 24798616 31. Guarrera J V. Are We Emerging From the Ice Age of Liver Preservation? Am J Transplant. 2016; 16:
1647–8. https://doi.org/10.1111/ajt.13756 PMID: 26880347 32. Hessheimer AJ, Billault C, Barrou B, Fondevila C. Hypothermic or normothermic abdominal regional
perfusion in high-risk donors with extended warm ischemia times: impact on outcomes? Transpl Int. 2015; 28: 700–7. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 References Cryobiology. 2010; 60: 337–43. https://doi.org/10.1016/j.cryobiol.2010.03.005 PMID: 20233587 52. Vairetti M, Ferrigno A, Carlucci F, Tabucchi A, Rizzo V, Boncompagni E, et al. Subnormothermic
machine perfusion protects steatotic livers against preservation injury: a potential for donor pool
increase? Liver Transpl. 2009; 15: 20–9. https://doi.org/10.1002/lt.21581 PMID: 19109848 53. Horn C Von, Baba HA, Hannaert P, Hauet T, Leuvenink H, Paul A, et al. Controlled oxygenated rewarm-
ing up to normothermia for pretransplant reconditioning of liver grafts. Clin Transplant. 2017; 31: 1–7. https://doi.org/10.1111/ctr.13101 PMID: 28871615 54. Hoyer DP, Paul A, Luer S, Reis H, Efferz P, Minor T. End-ischemic reconditioning of liver allografts:
Controlling the rewarming. Liver Transpl. 2016; 22: 1223–30. https://doi.org/10.1002/lt.24515 PMID:
27398813 55. Goldaracena N, Echeverri J, Spetzler VN, Kaths JM, Barbas AS, Louis KS, et al. Anti-inflammatory sig-
naling during ex vivo liver perfusion improves the preservation of pig liver grafts before transplantation. Liver Transpl. 2016; 22: 1573–1583. https://doi.org/10.1002/lt.24603 PMID: 27556578 56. de Vries Y, Matton APM, Nijsten MWN, Werner MJM, van den Berg AP, de Boer MT, et al. Pretrans-
plant sequential hypo- and normothermic machine perfusion of suboptimal livers donated after circula-
tory death using a hemoglobin-based oxygen carrier perfusion solution. Am J Transplant. 2019; 19:
1202–1211. https://doi.org/10.1111/ajt.15228 PMID: 30588774 57. Karangwa SA, Dutkowski P, Fontes P, Friend PJ, Guarrera J V., Markmann JF, et al. Machine Perfu-
sion of Donor Livers for Transplantation: A Proposal for Standardized Nomenclature and Reporting
Guidelines. Am J Transplant. 2016; 1967: 2932–2942. https://doi.org/10.1111/ajt.13843 PMID:
27129409 58. Obara H, Matsuno N, Shigeta T, Hirano T, Enosawa S, Mizunuma H. Temperature controlled machine
perfusion system for liver. Transplant Proc. 2013; 45: 1690–2. https://doi.org/10.1016/j.transproceed. 2013.01.087 PMID: 23769025 59. Matsuno N, Obara H, Watanabe R, Iwata S, Kono S, Fujiyama M, et al. Rewarming preservation by
organ perfusion system for donation after cardiac death liver grafts in pigs. Transplant Proc. 2014; 46:
1095–8. https://doi.org/10.1016/j.transproceed.2013.12.035 PMID: 24815137 60. Meng L, Matsuno N, Watanabe K, Furukori M, Obara H, Bochimoto H, et al. Scanning Electron Micros-
copy Findings of Machine Perfused Liver Graft After Warm Ischemia Between Hypothermic and
Rewarming Machine Perfusion in Pigs. Transplant Proc. 2016; 48: 2467–2470. https://doi.org/10.1016/
j.transproceed.2016.03.059 PMID: 27742324 61. Bochimoto H, Matsuno N, Ishihara Y, Shonaka T, Koga D, Hira Y, et al. The ultrastructural characteris-
tics of porcine hepatocytes donated after cardiac death and preserved with warm machine perfusion
preservation. PLoS One. 2017; 12: e0186352. https://doi.org/10.1371/journal.pone.0186352 PMID:
29023512 62. References Hypothermic oxygenated perfusion (HOPE) protects
from biliary injury in a rodent model of DCD liver transplantation. J Hepatol. 2013; 59: 984–91. https://
doi.org/10.1016/j.jhep.2013.06.022 PMID: 23820408 43. Schlegel A, Muller X, Kalisvaart M, Muellhaupt B, Perera MTPR, Isaac JR, et al. Outcomes of DCD liver
transplantation using organs treated by hypothermic oxygenated perfusion before implantation. J Hepa-
tol. 2019; 70: 50–57. https://doi.org/10.1016/j.jhep.2018.10.005 PMID: 30342115 44. Dutkowski P, Schlegel A, de Oliveira M, Mu¨llhaupt B, Neff F, Clavien P-A. HOPE for human liver grafts
obtained from donors after cardiac death. J Hepatol. 2014; 60: 765–72. https://doi.org/10.1016/j.jhep. 2013.11.023 PMID: 24295869 45. Dutkowski P, Krug A, Krysiak M, Du¨nschede F, Seifert JK, Junginger T. Detection of mitochondrial elec-
tron chain carrier redox status by transhepatic light intensity during rat liver reperfusion. Cryobiology. 2003; 47: 125–42. https://doi.org/10.1016/j.cryobiol.2003.08.004 PMID: 14580847 46. Dondossola D, Lonati C, Zanella A, Maggioni M, Antonelli B, Reggiani P, et al. Preliminary Experience
With Hypothermic Oxygenated Machine Perfusion in an Italian Liver Transplant Center. Transplant
Proc. 2019; 51: 111–116. https://doi.org/10.1016/j.transproceed.2018.04.070 PMID: 30736971 47. Ciria R, Ayllon-Teran MD, Gonza´lez-Rubio S, Go´mez-Luque I, Ferrı´n G, Moreno A, et al. Rescue of Dis-
carded Grafts for Liver Transplantation by Ex Vivo Subnormothermic and Normothermic Oxygenated
Machine Perfusion: First Experience in Spain. Transplant Proc. 2019; 51: 20–24. https://doi.org/10. 1016/j.transproceed.2018.04.092 PMID: 30655130 48. de Vries Y, Berendsen TA, Fujiyoshi M, van den Berg AP, Blokzijl H, de Boer MT, et al. Transplantation
of high-risk donor livers after resuscitation and viability assessment using a combined protocol of oxy-
genated hypothermic, rewarming and normothermic machine perfusion: study protocol for a prospec-
tive, single-arm study (DHOPE-COR-NMP tri. BMJ Open. 2019; 9: e028596. https://doi.org/10.1136/
bmjopen-2018-028596 PMID: 31420387 49. Hoyer DP, Mathe´ Z, Gallinat A, Canbay AC, Treckmann JW, Rauen U, et al. Controlled Oxygenated
Rewarming of Cold Stored Livers Prior to Transplantation: First Clinical Application of a New Concept. Transplantation. 2016; 100: 147–52. https://doi.org/10.1097/TP.0000000000000915 PMID: 26479280 14 / 17 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion 50. Minor T, Efferz P, Fox M, Wohlschlaeger J, Lu¨er B. Controlled oxygenated rewarming of cold stored
liver grafts by thermally graduated machine perfusion prior to reperfusion. Am J Transplant. 2013; 13:
1450–60. https://doi.org/10.1111/ajt.12235 PMID: 23617781 51. Olschewski P, Gass P, Ariyakhagorn V, Jasse K, Hunold G, Menzel M, et al. The influence of storage
temperature during machine perfusion on preservation quality of marginal donor livers. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 References Imber CJ, St Peter SD, de Cenarruzabeitia IL, Lemonde H, Rees M, Butler A, et al. Optimisation of bile
production during normothermic preservation of porcine livers. Am J Transplant. 2002; 2: 593–9. https://doi.org/10.1034/j.1600-6143.2002.20703.x PMID: 12201359 63. Boteon YL, Boteon AP, Attard J, Wallace L, Bhogal RH, Afford SC. Impact of machine perfusion of the
liver on post-transplant biliary complications: A systematic review. World J Transplant. 2018; 8: 220–
231. https://doi.org/10.5500/wjt.v8.i6.220 PMID: 30370232 64. Schlegel A, Kron P, Dutkowski P. Hypothermic machine perfusion in liver transplantation. Curr Opin
Organ Transplant. 2016; 21: 308–14. https://doi.org/10.1097/MOT.0000000000000303 PMID:
26918882 65. Schlegel A, Dutkowski P. Impact of Machine Perfusion on Biliary Complications after Liver Transplanta-
tion. Int J Mol Sci. 2018; 19. https://doi.org/10.3390/ijms19113567 PMID: 30424553 66. Op den Dries S, Sutton ME, Karimian N, de Boer MT, Wiersema-Buist J, Gouw ASH, et al. Hypothermic
oxygenated machine perfusion prevents arteriolonecrosis of the peribiliary plexus in pig livers donated
after circulatory death. PLoS One. 2014; 9: e88521. https://doi.org/10.1371/journal.pone.0088521
PMID: 24551114 67. Westerkamp AC, Mahboub P, Meyer SL, Hottenrott M, Ottens PJ, Wiersema-Buist J, et al. End-ische-
mic machine perfusion reduces bile duct injury in donation after circulatory death rat donor livers inde-
pendent of the machine perfusion temperature. Liver Transpl. 2015; 21: 1300–11. https://doi.org/10. 1002/lt.24200 PMID: 26097213 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 15 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion 68. Bellomo R, Marino B, Starkey G, Fink M, Wang BZ, Eastwood GM, et al. Extended normothermic extra-
corporeal perfusion of isolated human liver after warm ischaemia: a preliminary report. Crit Care
Resusc. 2014; 16: 197–201. Available: http://www.ncbi.nlm.nih.gov/pubmed/25161022 69. Furukori M, Matsuno N, Meng LT, Shonaka T, Nishikawa Y, Imai K, et al. Subnormothermic Machine
Perfusion Preservation With Rewarming for Donation After Cardiac Death Liver Grafts in Pigs. Trans-
plant Proc. 2016; 48: 1239–43. https://doi.org/10.1016/j.transproceed.2015.12.076 PMID: 27320595 70. Okouchi Y, Sasaki K, Tamaki T. Ultrastructural changes in hepatocytes, sinusoidal endothelial cells and
macrophages in hypothermic preservation of the rat liver with University of Wisconsin solution. Virch-
ows Arch. 1994; 424: 477–84. https://doi.org/10.1007/BF00191432 PMID: 8032528 71. Moussa ME, Sarraf CE, Uemoto S, Sawada H, Habib NA. Effect of total hepatic vascular exclusion dur-
ing liver resection on hepatic ultrastructure. Liver Transpl Surg. 1996; 2: 461–467. S1527646596000512 [pii] 72. Khambu B, Li T, Yan S, Yu C, Chen X, Goheen M, et al. References Hepatic Autophagy Deficiency Compromises
Farnesoid X Receptor Functionality and Causes Cholestatic Injury. Hepatology. 2019; 69: 2196–2213. https://doi.org/10.1002/hep.30407 PMID: 30520052 73. Gissen P, Arias IM. Structural and functional hepatocyte polarity and liver disease. J Hepatol. 2015; 63:
1023–37. https://doi.org/10.1016/j.jhep.2015.06.015 PMID: 26116792 74. Treyer A, Mu¨sch A. Hepatocyte polarity. Compr Physiol. 2013; 3: 243–87. https://doi.org/10.1002/cphy. c120009 PMID: 23720287 75. Kachi K, Okanoue T, Morioka H, Ohta M, Ohta Y, Kanaoka H, et al. Immunoelectron microscopic locali-
zation of actin in normal and cholestatic rat hepatocytes. Gastroenterol Jpn. 1989; 24: 523–7. https://
doi.org/10.1007/bf02773879 PMID: 2680743 76. Benkoe¨l L, Dodero F, Hardwigsen J, Campan P, Botta-Fridlund D, Lombardo D, et al. Effect of ische-
mia-reperfusion on bile canalicular F-actin microfilaments in hepatocytes of human liver allograft: image
analysis by confocal laser scanning microscopy. Dig Dis Sci. 2001; 46: 1663–7. https://doi.org/10.1023/
a:1010693218680 77. Shinohara H, Tanaka A, Fujimoto T, Hatano E, Satoh S, Fujimoto K, et al. Disorganization of microtubu-
lar network in postischemic liver dysfunction: its functional and morphological changes. Biochim Bio-
phys Acta. 1996; 1317: 27–35. https://doi.org/10.1016/0925-4439(96)00031-2 78. Monbaliu D, Libbrecht L, De Vos R, Vekemans K, Walter H, Liu Q, et al. The extent of vacuolation in
non-heart-beating porcine donor liver grafts prior to transplantation predicts their viability. Liver Transpl. 2008; 14: 1256–65. https://doi.org/10.1002/lt.21513 PMID: 18756467 79. Yasui H, Yoshimura N, Kobayashi Y, Ochiai S, Matsuda T, Takamatsu T, et al. Microstructural changes
of bile canaliculi in canine liver: the effect of cold ischemia-reperfusion in orthotopic liver transplantation. Transplant Proc. 1998; 30: 3754–7. https://doi.org/10.1016/s0041-1345(98)01222-6 80. Tamaki I, Hata K, Okamura Y, Nigmet Y, Hirao H, Kubota T, et al. Hydrogen Flush After Cold Storage
as a New End-Ischemic Ex Vivo Treatment for Liver Grafts Against Ischemia/Reperfusion Injury. Liver
Transpl. 2018; 24: 1589–1602. https://doi.org/10.1002/lt.25326 PMID: 30120877 81. Cutrin JC, Cantino D, Biasi F, Chiarpotto E, Salizzoni M, Andorno E, et al. Reperfusion damage to the
bile canaliculi in transplanted human liver. Hepatology. 1996; 24: 1053–7. https://doi.org/10.1002/hep. 510240512 PMID: 8903374 82. Falasca L, Tisone G, Palmieri G, Anselmo A, Di Paolo D, Baiocchi L, et al. Protective role of taurourso-
deoxycholate during harvesting and cold storage of human liver: a pilot study in transplant recipients. Transplantation. 2001; 71: 1268–76. https://doi.org/10.1097/00007890-200105150-00015 PMID:
11397961 83. Nigmet Y, Hata K, Tamaki I, Okamura Y, Tsuruyama T, Miyauchi H, et al. PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 References Human Atrial Natriuretic Pep-
tide in Cold Storage of Donation After Circulatory Death Rat Livers: An Old but New Agent for Protecting
Vascular Endothelia? Transplantation. 2019; 103: 512–521. https://doi.org/10.1097/TP. 0000000000002552 PMID: 30461725 84. Okumura S, Uemura T, Zhao X, Masano Y, Tsuruyama T, Fujimoto Y, et al. Liver graft preservation
using perfluorocarbon improves the outcomes of simulated donation after cardiac death liver transplan-
tation in rats. Liver Transpl. 2017; 23: 1171–1185. https://doi.org/10.1002/lt.24806 PMID: 28650112 85. Walker RM, Racz WJ, McElligott TF. Scanning electron microscopic examination of acetaminophen-
induced hepatotoxicity and congestion in mice. Am J Pathol. 1983; 113: 321–30. Available: http://www. ncbi.nlm.nih.gov/pubmed/6650662 86. Baiocchi L, Tisone G, Russo MA, Longhi C, Palmieri G, Volpe A, et al. TUDCA prevents cholestasis and
canalicular damage induced by ischemia-reperfusion injury in the rat, modulating PKCalpha-ezrin path-
way. Transpl Int. 2008; 21: 792–800. https://doi.org/10.1111/j.1432-2277.2008.00682.x PMID:
18435680 PLOS ONE | https://doi.org/10.1371/journal.pone.0233917
May 29, 2020 16 / 17 PLOS ONE Ultrastructure of bile canaliculus after machine perfusion 87. Phillips MJ, Azuma T, Meredith S-LM, Squire JA, Ackerley CA, Pluthero FG, et al. Abnormalities in villin
gene expression and canalicular microvillus structure in progressive cholestatic liver disease of child-
hood. Lancet (London, England). 2003; 362: 1112–9. https://doi.org/10.1016/S0140-6736(03)14467-4 88. Hessheimer AJ, Ca´rdenas A, Garcı´a-Valdecasas JC, Fondevila C. Can we prevent ischemic-type bili-
ary lesions in donation after circulatory determination of death liver transplantation? Liver Transpl. 2016; 22: 1025–33. https://doi.org/10.1002/lt.24460 PMID: 27082839 89. Morito N, Obara H, Matsuno N, Enosawa S, Furukawa H. Oxygen consumption during hypothermic and
subnormothermic machine perfusions of porcine liver grafts after cardiac death. J Artif Organs. 2018;
21: 450–457. https://doi.org/10.1007/s10047-018-1063-0 PMID: 30046934 90. Bertone V, Tarantola E, Ferrigno A, Gringeri E, Barni S, Vairetti M, et al. Altered alkaline phosphatase
activity in obese Zucker rats liver respect to lean Zucker and Wistar rats discussed in terms of all puta-
tive roles ascribed to the enzyme. Eur J Histochem. 2011; 55: e5. https://doi.org/10.4081/ejh.2011.e5
PMID: 21556120 91. Shigeta T, Matsuno N, Obara H, Kanazawa H, Tanaka H, Fukuda A, et al. Impact of rewarming preser-
vation by continuous machine perfusion: improved post-transplant recovery in pigs. Transplant Proc. 2013; 45: 1684–9. https://doi.org/10.1016/j.transproceed.2013.01.098 PMID: 23769024 17 / 17
|
https://openalex.org/W4287548662
|
https://zenodo.org/records/4545725/files/QVC_V7.pdf
|
English
| null |
Quantum Vacuum Gravitation and Cosmology. Matter-Antimatter Antigravity.
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,020
|
cc-by
| 6,440
|
Abstract We show that the electromagnetic quantum vacuum plays a primary role in gravitation and cosmology. Photons are local oscillations of the vacuum field guided by a non-local vector potential wave function
and propagate at the speed imposed by the vacuum electric permittivity
0
and magnetic permeability
0
, which we demonstrate that they are intrinsic properties of the electromagnetic vacuum. The
electron-positron elementary charge derive naturally from the electromagnetic quantum vacuum and the
masses of all particles (leptons, mesons, baryons) and antiparticles are manifestations of the elementary
charges and their corresponding magnetic moments. We show that the gravitation constant G is also
related directly to the electromagnetic quantum vacuum putting in evidence the electromagnetic nature
of gravity. Furthermore, we draw that G is the same for matter and antimatter but gravitational forces
appear to be repulsive between particles and antiparticles since their masses bear opposite signs. d
h
di i
h
l
i
fi ld
i
h
h
i
l b
i f We show that the electromagnetic quantum vacuum plays a primary role in gravitation and cosmology. Photons are local oscillations of the vacuum field guided by a non-local vector potential wave function
and propagate at the speed imposed by the vacuum electric permittivity
0
and magnetic permeability 0
,
y
p
p
g
electron-positron elementary charge derive naturally from the electromagnetic quantum vacuum and the
masses of all particles (leptons, mesons, baryons) and antiparticles are manifestations of the elementary
charges and their corresponding magnetic moments. We show that the gravitation constant G is also
related directly to the electromagnetic quantum vacuum putting in evidence the electromagnetic nature
of gravity. Furthermore, we draw that G is the same for matter and antimatter but gravitational forces
appear to be repulsive between particles and antiparticles since their masses bear opposite signs. U d
th
diti
th
l
t
ti
t
fi ld
tit t th
h
i
l b
i f pp
p
p
p
pp
g
Under these conditions, the electromagnetic quantum vacuum field constitute the physical basis for a
unified field theory. Dark Energy and Dark Matter might originate from the electromagnetic quantum vacuum fluctuations. The calculated vacuum energy density, related to the cosmological constant considered responsible for
the cosmic acceleration, is in good agreement with the astrophysical observations. Quantum Vacuum Gravitation and Cosmology
Matter-Antimatter Antigravity Constantin Meis
National Institute for Nuclear Science and Technology
CEA – Saclay 91191 Gif-sur-Yvette, France. constantin.meis@cea.fr Abstract Finally, we advance the hypothesis that energy, matter and antimatter in the universe emerge
spontaneously from the quantum vacuum fluctuations as residues that remain stable in space. Following
this assumption, we present the principles upon which a new cosmological model based on the
electromagnetic quantum vacuum may be developed overcoming the well-known Big Bang issues. Key words: photons, electromagnetic quantum vacuum, dark light, gravitation, push gravity, antimatter,
antigravity, dark energy, cosmological constant, dark matter, elementary charges, mass-charge relation,
cosmology, unified field theory. Quantum Vacuum Gravitation and Cosmology
Matter-Antimatter Antigravity
Constantin Meis
National Institute for Nuclear Science and Technology
CEA – Saclay 91191 Gif-sur-Yvette, France.
constantin.meis@cea.fr Quantum Vacuum Gravitation and Cosmology
Matter-Antimatter Antigravity
Constantin Meis
National Institute for Nuclear Science and Technology
CEA – Saclay 91191 Gif-sur-Yvette, France. constantin.meis@cea.fr Introduction The CDM (Lambda – Cold Dark Matter) cosmological model is considered today as the most plausible
theory that has the merit to provide satisfactory answers to a large number of astrophysical observations. It is based on the enhanced Big Bang concept according to which the universe is homogeneous and
whose main components are Dark Matter and Dark Energy. The last one is associated to the
cosmological constant , which is considered responsible for the observed cosmic acceleration [1-4]. However, besides the difficulty to conceive that the whole universe emerged from a singular point, some
upsetting issues still remain such as, why did the Big Bang occurred and what happened before? Furthermore, in order to explain the horizon problem as well as the fact that the geometry of the universe
is Euclidean (flat), a cosmic inflation model has been introduced [5]. But, what are the physical reasons
that inflation occurred in the very first 10-30 seconds of the Big Bang expanding the universe at a
tremendous rate, many orders of magnitude faster than the speed of light in vacuum? What happened to
antimatter? Finally, quantum field theory fails by a factor of 10120 to give a precise estimation to the
cosmological constant [6, 7], the worst discrepancy between theory and observation in the history of
science. 1 The above drawbacks call the human intelligence to be humble in front of the immensity and complexity
of the still unknown universe. All the cosmological theories, including the one presented here, are
temporary attempts to explain the observations realized up to now using the present theoretical tools and
they are finally condemned to be replaced in the future according to the new observations to come. In what follows, we make an introduction to the fundamentals of Quantum Vacuum Gravitation and
Cosmology. The electromagnetic quantum vacuum, namely Dark Light, corresponds to the
electromagnetic field ground state and results naturally from Maxwell’s theory by considering the vector
potential quantization at a single photon level. It is a real field permeating all of space and has electric
potential dimension. We show that photons are oscillations of the electromagnetic vacuum field while
the lepton-antilepton elementary charges derive equally from this field. The gravitational constant G is a vacuum property having electromagnetic nature and is the same for
matter and antimatter and. 1. The electromagnetic quantum vacuum, the Dark Light 1. The electromagnetic quantum vacuum, the Dark Light
The quantized vector potential
( , )
k
r t
for a cavity free k-mode photon of the electromagnetic field
with angular frequency
k
and polarization (circular left or right) writes [8-14] The quantized vector potential
( , )
k
r t
for a cavity free k-mode photon of the electromagnetic field
with angular frequency
k
and polarization (circular left or right) writes [8-14] quantized vector potential
( , )
k
r t
for a cavity free k-mode photon of the electromagnetic fie with angular frequency
k
and polarization (circular left or right) writes [8-14] d polarization (circular left or right) writes [8-14]
ˆ
( , )
( , )
k
i k r
t
k
k
k
k
k
r t
e
cc
r t
(1) (1) where ˆk
is the polarization unit vector, k the wave vector with amplitude
2
k
where
k
is the polarization unit vector, k the wave vector with amplitude
2
/
k
k
,
k being here
the wavelength of the mode k, a phase parameter and cc the complex conjugate. where
k
is the polarization unit vector, k the wave vector with amplitude
2
/
k
k
,
k being here
the wavelength of the mode k, a phase parameter and cc the complex conjugate. The vector potential amplitude quantization constant may be positive or negative and is given by the wavelength of the mode k, a phase parameter and cc the complex conjugate. Th
i l
li d
i
i
b
i i
i the wavelength of the mode k, a phase parameter and cc the complex conjugate. The vector potential amplitude quantization constant may be positive or negative or potential amplitude quantization constant may be positive or negative and is given by
0
2
1
4
4
e
ec
(2) (2) (2) where e is the electron or positron charge, is Planck’s reduced constant(
/ 2 )
h
, c the speed of
light in vacuum,
1/137
is the fine structure constant and
0
the vacuum magnetic permeability. Introduction The masses of particles and antiparticles derive from elementary charges and
have opposite sign entailing repulsive gravitational forces between matter and antimatter. Fluctuations of the electromagnetic quantum vacuum generate transient states of photons composing the
Dark Energy and transient pairs of charges (particles) that might compose the Dark Matter. A small
fraction of the vacuum transient states might definitely remain stable in space composing the observed
energy and mass in the universe. 1. The electromagnetic quantum vacuum, the Dark Light It is straightforward to show that the vector potential function
( , )
k
r t
is a natural wave function for a
free k-mode photon satisfying the vector potential – energy (wave – particle) equation for the photon
0
( , )
( , )
k
k
i
r t
r t
H
t
(3)
where
0
i
c
H
(4) 0
( , )
( , )
k
k
i
r t
r t
H
t
(3) (3) where 0
i
c
H
(4) (4) The constants
and characterize respectively the energy and the vector potential field quantization for
a single photon state. The perfect symmetry between the photon energy
k
k
E
and the vector
potential amplitude
0k
k
expresses the simultaneous wave-particle nature of the single photon
through the vector potential – energy equation (3). The constants
and characterize respectively the energy and the vector potential field quantization for
a single photon state. The perfect symmetry between the photon energy
k
k
E
and the vector
potential amplitude
0k
k
expresses the simultaneous wave-particle nature of the single photon
through the vector potential – energy equation (3). It is worthy noticing that the amplitude of the electric field of a cavity free k-mode photon is proportional
to the square of the angular frequency [10, 12] 2
( , )
( , ) /
k
k
k
r t
r t
t
(5) (5) 2 Obviously, for
2
/
0
k
k
c
, that is for
k, the photon energy tends to zero and the
wavelength to infinity. 1. The electromagnetic quantum vacuum, the Dark Light However, for
0
k
the resulting electromagnetic field ground state does not
correspond to a perfectly empty space because the fundamental function
)
,
(
t
r
in the vector potential
expression (1) still subsists and becomes [14-17] *
0
0
0
ˆ
ˆ
i
i
e
e
(6) (6) (6) Hence, in total absence of energy, of electric and magnetic fields,
0
is the electromagnetic field ground
state, the electromagnetic quantum vacuum corresponding to light at zero frequency, “Dark Light”. It is a real cosmic field
kpermeating all of space and having electric potential nature according
to the physical dimensions of , that is Volt m-1 s2. The phase parametermay take any value and consequently the electromagnetic quantum vacuum is
composed of all possible functions
( , )
k
r t
corresponding to all modes and polarizations
ˆ
( , )
i k r
t
k
k
r t
e
cc
(7) (7) From equation (4), an angular frequency operator ~ can be readily defined [17] From equation (4), an angular frequency operator ~ can be readily defined [17] ic
(8) (8) ic
(8)
Using (3) and (8) we get the equation governing the fundamental function
( , )
k
r t
of the vector
potential in vacuum Using (3) and (8) we get the equation governing the fundamental function
( , )
k
r t
of the vector
potential in vacuum ( , )
( , )
( , )
k
k
k
i
r t
r t
r t
t
(9) (9) Obviously, according to the last equation, photons are generated by the action of the angular frequency
operator~ upon the vacuum function
( , )
k
r t
creating a real vector potential
( , )
k
r t
of a k-mode
photon. Consequently, photons are local oscillations of the electromagnetic quantum vacuum over a
wavelength, with circular polarisation (left or right), guided by a non-local vector potential wave
function [10, 13, 14]. 1. The electromagnetic quantum vacuum, the Dark Light The vacuum electric permittivity
0
and magnetic permeability
0
are intrinsic physical characteristics
of the electromagnetic vacuum and are expressed through the fundamental physical constants
, ,
and the elementary charge e 3
0
2
e
;
2
0
4
e
(10) (10) Considering the product of the last expressions 2
0
0
2
4
1
e
c
(11) (11) it is straightforward to obtain the velocity of light in vacuum c. it is straightforward to obtain the velocity of light in vacuum c. 2. The electromagnetic quantum vacuum fluctuations, from QED to cosmology 2.1 Transient photons from the electromagnetic quantum vacuum. Dark Energy and the cosmic acceleration. Quantum field theory fails to evaluate the vacuum energy density by a factor of 120 orders of magnitude. In fact, the zero-point energy levels of all fields, corresponding to the fundamental eigenstate of the
harmonic oscillator Hamiltonian, yields an infinite vacuum energy density which when upper limited to
Planck’s energy
19
( 10
)
GeV gives the “astronomic” value ~
110
3
10
J m, while the observed one is
roughly
10
3
10
J m
[1]. 3 This unphysical theoretical result, the worst ever in the history of science, is mainly due to the
mathematical ambiguity during the field quantization procedure, according to the harmonic oscillator
model, consisting of replacing commuting classical variables of momentum and position by non-
commuting quantum mechanics Hermitian operators [10, 14, 18]. Electromagnetic waves are not
composed of harmonic oscillators and no experiment has ever demonstrated that a single photon state is
a harmonic oscillator. It has to be emphasized that, in contrast with material oscillators, the zero-point
energy in quantum field theory resulting from the harmonic oscillator Hamiltonian does not correspond
to a physical state and this is what indeed the astrophysical observations confirm. Conversely, it has been demonstrated that the electromagnetic quantum vacuum complements naturally
the normal ordering Hamiltonian in QED and describes a real physical vacuum state in both classical
and quantum electromagnetism [10, 11, 14]. We have seen that photons are local oscillations of the
electromagnetic quantum vacuum field propagating at the speed imposed by its intrinsic properties, the
vacuum electric permittivity
0and magnetic permeability
0
. On the other hand, from Heisenberg’s
energy-time uncertainty relation we deduce directly that the vector potential amplitude is also subject to
a fluctuation uncertainty [10, 16, 17] 0
k
k
E
t
t
(12) (12) Consequently, due to the electromagnetic vacuum fluctuations, space is permanently full of transient
photons at all frequencies underlying the cosmic radiation background. Consequently, due to the electromagnetic vacuum fluctuations, space is permanently full of transient
photons at all frequencies underlying the cosmic radiation background. Obviously, according to the uncertainty relation, the lower the energy the longer the transient photons
lifetime. 2. The electromagnetic quantum vacuum fluctuations, from QED to cosmology Assuming that the probability for a transient k-mode photon spontaneous creation is
proportional to
/
k
m
e
, where
k
is the transient photon electric field amplitude generated in space and
m
a mean photon electric field amplitude over the electromagnetic spectrum, and using (5) we get the
electromagnetic vacuum energy density [19] 2
2
4
/
3
4
0
0
2
3
2
3
0
2
2
m
m
vacuum
m
e
d
e
c
c
(13) (13) ring that
m
is a logarithmic mean value for the angular frequency over the electromagnetic g
m
g
g
q
y
g
spectrum, roughly
12
~ 2 10
m
Hz
, we obtain
10
3
3 10
vacuum
J m
which is in good agreement
with the astrophysical observations. Thus, the energy density of the electromagnetic vacuum
fluctuations is quite plausible to represent the Dark Energy considered responsible for the cosmic
acceleration. spectrum, roughly
12
~ 2 10
m
Hz
, we obtain
10
3
3 10
vacuum
J m
which is in good agreement
with the astrophysical observations. Thus, the energy density of the electromagnetic vacuum
fluctuations is quite plausible to represent the Dark Energy considered responsible for the cosmic
acceleration. 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum. At the origin of Dark Matter? 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum. At the origin of Dark Matter? 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum. At the origin of Dark Matter? 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum.
At the origin of Dark Matter? As an example, muon mass is obtained for ni = 3, pion for ni = 4, kaon for ni =14, rho for ni = 22, nucleon
for ni = 27, lambda for ni = 32, sigma for ni = 34, tau for ni = 51…etc. According to the last equations, we draw that the electron/positron charge is related to photons and
derive from the electromagnetic vacuum while the particles masses are quantum states of the vacuum
originating from charges and their magnetic moment. The presence in equation (17) of an integer
in characterizing the particles rest masses implies that the
electromagnetic vacuum must have a complex structure of quantum levels yet to discover, which might
be related to string theory. Through this lens, all neutral elementary particles must be composed of
positive and negative charges and consequently, gravity should be an electromagnetic effect. The presence in equation (17) of an integer
in characterizing the particles rest masses implies that the
electromagnetic vacuum must have a complex structure of quantum levels yet to discover, which might
be related to string theory. Through this lens, all neutral elementary particles must be composed of
positive and negative charges and consequently, gravity should be an electromagnetic effect. Indeed, considering Planck’s length Pl = 1.616 10-35 m, which is the shorter possible wavelength for a
single photon beyond which the electromagnetic energy density transforms to a black hole, the
gravitational constant G is obtained exactly through the elementary charge e and the electromagnetic
vacuum constants ,
0and
0
[17] Indeed, considering Planck’s length Pl = 1.616 10-35 m, which is the shorter possible wavelength for a
single photon beyond which the electromagnetic energy density transforms to a black hole, the
gravitational constant G is obtained exactly through the elementary charge e and the electromagnetic
vacuum constants ,
0and
0
[17] 2
0
0
4
pl
G
e
(18) (18) It is worthy investigating whether gravitation originates from the radiation pressure of the
electromagnetic quantum vacuum field (Push Gravity) felt by the bodies in their own frame depending
on their charge densities [16]. Under this condition, light rays should follow the paths in the
electromagnetic vacuum imposed by the charge densities in space modifying locally the vacuum
fluctuations and the refractive index. Now, we can draw a quite interesting feature concerning matter, antimatter and gravity. 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum.
At the origin of Dark Matter? On the other hand, the mass
i
m of any other elementary particle i, lepton, meson or baryon, can be
expressed with a precision of roughly 1%, through the vacuum constant and the elementary
charge e On the other hand, the mass
i
m of any other elementary particle i, lepton, meson or baryon, can be
expressed with a precision of roughly 1%, through the vacuum constant and the elementary
charge e 2
2
i
i
m
ce
(16) (16) where
2
i
B
i
µ
n
, is the fine structure constant and ni is simply a positive integer. where
2
i
B
i
µ
n
, is the fine structure constant and ni is simply a positive integer. where
2
i
B
i
µ
n
, is the fine structure constant and ni is simply a positive integer.
Obviously, the particles rest mass are purely of electromagnetic nature and the mass-charge equivalence
relation writes simply Obviously, the particles rest mass are purely of electromagnetic nature and the mass-charge equivalence
relation writes simply 3
3
,
;
e
i
i
p
e
e
m
e
m
n
e
(17) 3
3
,
;
e
i
i
p
e
e
m
e
m
n
e
(17) where e is the electron or positron charge. Using the relation (2) for , the obtained numerical values of the proportionality constants are
26
3
0 /8
2.21510
e
B
µ
c
kg C
and
2
28
3
0 / (4 )
1.51710
p
B
µ
c
kg C
. Using the relation (2) for , the obtained numerical values of the proportionality constants are
26
3
0 /8
2.21510
e
B
µ
c
kg C
and
2
28
3
0 / (4 )
1.51710
p
B
µ
c
kg C
. 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum.
At the origin of Dark Matter? It has been established that the electron/positron elementary charge e, a fundamental physical constant,
is obtained exactly from the electromagnetic vacuum quantized amplitude constant
2
0
4
e
(14) (14) It becomes evident that the photon vector potential amplitude
k
k
a
and the ele It becomes evident that the photon vector potential amplitude
k
k
a
and the elementary charge e
are directly related to the electromagnetic vacuum through the amplitude constant demonstrating that It becomes evident that the photon vector potential amplitude
k
k
a
and the elementary charge e
are directly related to the electromagnetic vacuum through the amplitude constant demonstrating that
photons and leptons/antileptons derive from this vacuum field, putting the basis for a physical It becomes evident that the photon vector potential amplitude
k
k
a
and the elementary charge e p
p
p
k
k
y
g
are directly related to the electromagnetic vacuum through the amplitude constant demonstrating that
photons and leptons/antileptons derive from this vacuum field, putting the basis for a physical
comprehension of their mutual transformation mechanism. are directly related to the electromagnetic vacuum through the amplitude constant demonstrating that
photons and leptons/antileptons derive from this vacuum field, putting the basis for a physical
comprehension of their mutual transformation mechanism. We recall that, from the historical experimental evidence, Planck’s constant
is intrinsically related
to the energy quantization of the electromagnetic field to a single photon state. Despite of this
characteristic physical origin Planck’s constant is used in quantum physics for the description of all
particles. Consequently, we may intuitively guess that the electromagnetic nature should be an inherent
property of any particle. 4 Indeed, the rest mass of the electron/positron
,
e
e
m
is expressed directly through the elementary charge
e and its magnetic moment Indeed, the rest mass of the electron/positron
,
e
e
m
is expressed directly through the elementary charge
e and its magnetic moment 2
,
2
e
e
B
m
ce
(15) (15) with
B
the Bohr magneton. 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum.
At the origin of Dark Matter? In fact, for
0
and
0
to be positive quantities in equations (10), e and should have the same sign. In this way, the
gravitational constant expressed by (18) is positive for both matter and antimatter. Thus, gravitation
forces are attractive between bodies of ordinary matter, as well as between bodies of antimatter, but they
should be repulsive between matter and antimatter since they have positive and negative masses
according to the relations (16) and (17). In fact, particles and antiparticles are attracted by Coulomb
forces overcoming naturally the weak gravitational repulsion and annihilate mutually giving generally
birth to photons. Conversely, matter and antimatter neutral structures must be pushed away due to 5 repulsive gravitational forces. This is in agreement with previous studies that have shown that CPT
symmetry (Charge conjugation, Parity and Time reversal) and General Relativity cannot be compatible
unless matter and antimatter are mutually repelled [21]. Note that, from equations (14) for the electron, is negative and consequently the ordinary masses in
equations (15), (16) and (17) appear as negative and those of antimatter obtained from the positron as
positive. This is because historically, a negative charge was conventionally attributed to the electron and
a positive one to the positron. Inverting, eventually, the traditional convention and attributing a positive
charge to the electron and a negative to the positron, which becomes a “negatron”, results to positive
mass for matter (positive ) and negative for antimatter (negative ). Also,
0
0
,
,
,
B G
as well as the photon frequencies and energies are positive and identical for matter
and antimatter. According to the established equations (2), (11) and (14), all the fundamental constants
1/2
2
2
3
1/2
3/2
0
0
0
0
0
(
)
,
(4 )
/
,
(4 )
c
e
and
2
3
2
0
/ (4 )
p
G
l
are expressed
uniquely through the vacuum constants
0
0
,
,
and entailing that electromagnetism and gravitation
are natural manifestations of the electromagnetic quantum vacuum field. It is of high importance to mention that we have made no hypothesis and advanced no postulates in order
to obtain the equations (2), (10) and (14) to (18). 2.2 Transient charges (particles and antiparticles) from the electromagnetic quantum vacuum.
At the origin of Dark Matter? Everything derives directly from Maxwell’s theory
once the vector potential amplitude is normalized at a single photon level. Finally, we deduce that fluctuations of the electromagnetic vacuum may also give birth to transient states
of positive and negative charges of matter and antimatter, corresponding to known and unknown
particles. Halos of transient particles concentrations could be strongly favored near important real
charges (mass) distributions and consequently might contribute to the Dark Matter. Conclusions We have visited the basic cosmological features that derive naturally from the electromagnetic quantum
vacuum, the Dark Light. Photons (electromagnetic waves) are oscillations of the vacuum field. The
vacuum electric permittivity and magnetic permeability,
0 and
0
respectively, are intrinsic properties
of the electromagnetic quantum vacuum and fix the speed of light c. Elementary charges appear to be states of the same vacuum field. Particles masses originate from
charges and their magnetic moment witnessing a complex quantum structure for the electromagnetic
vacuum that may be related to the string theory. Gravitation is also an intrinsic property of the
electromagnetic quantum vacuum. The gravitation constant G derives from the vacuum properties and
is the same for matter and antimatter. Conversely, the masses of particles and antiparticles bear opposite
signs entailing a mutual gravitational repulsion. Matter-antimatter antigravity needs to be explored
experimentally. The electromagnetic quantum vacuum may constitute the physical basis for the development of a
coherent unified field theory. Dark Energy and Dark Matter might both originate from transient states of Dark Light fluctuations. The
first due to transient photons and the second to transient known and unknown particles. Under these
conditions, free space is not Lorentz invariant and an observer with uniform velocity, in absence of any
other reference frame, would be able to detect his motion from the Doppler shift of the electromagnetic
vacuum fluctuations spectrum. Next, we advanced the hypothesis that photons as well as matter and antimatter emerge spontaneously
from the quantum vacuum as residues of its associated fluctuations. Thus, Dark Energy, Dark Matter,
photons, matter and antimatter all derive from the electromagnetic quantum vacuum, the Dark Light. Energy-mass annihilation mechanisms, such as black holes, appear naturally in the later stage of the
evolution of a local finite universe transforming the initially generated energy-mass back to the vacuum
state. This also provides a satisfactory explanation to what the tremendous quantities of energy-mass
swallowed by black holes become in the singularity. Energy-mass and charges are conserved between the initial and final states of the overall cosmic
creation-annihilation processes. A large number of finite universes, as ours, as well as anti-universes
might be born, extend, live and die in an infinite and eternal space. 2.3 The electromagnetic quantum vacuum cosmic source of photons (energy) and charges (mass).
Quantum vacuum cosmological model. We have seen that transient states of the Dark Light fluctuations might be at the origin of Dark Energy
and Dark Matter. Now, we may assume that a small part of the vacuum fluctuations can indeed remain
in space as residual real states. Certainly, this conflicts with the mass-energy and charge conservation
laws but, as we will see later, these laws are satisfied between the initial and final states of the overall
cosmic process. In this way, real photons and charges (particles and antiparticles), can be created
continuously in space as a result of vacuum fluctuation residues. Thus, the electromagnetic vacuum
turns to be a cosmic source of energy (photons) and charges (matter and antimatter). The vacuum
fluctuation particles residues might be in thermal equilibrium at ~3 K, which could explain the
homogeneous and isotropic Cosmic Microwave Background (CMB). In the first stage of the universe, the energy-mass creation process dominates. Photons and charges
(particles and antiparticles) are created continuously everywhere in an infinite space entailing that
universe is homogeneous and flat. Some particle-antiparticles pairs annihilate producing photons, others
combine progressively to electrons-protons and positrons-antiprotons forming respectively hydrogen
and antihydrogen atoms, then molecules and gas which are progressively separated by gravitational
forces to form distant accumulations, the first with ordinary matter and the second with antimatter. The
presence of matter favors particles generation and that of antimatter antiparticles generation. Under the
effect of gravitation, following the well-known mechanisms, the increasing concentrations of hydrogen
(antihydrogen) give birth to stars (antimatter stars, ‘anti-stars’). Next, heavier elements (anti-elements)
are produced in stars (anti-stars) by the also well-known baryon genesis process. Galaxies (antimatter
galaxies, ‘anti-galaxies’) and clusters of galaxies (anti-galaxies) are formed progressively. Vacuum
fluctuation residues are enhanced mostly near already generated massive structures entailing the
formation of a local finite universe. If matter and antimatter structures are not separated completely since
the beginning due to gravitation repulsion to form a local universe and a distant anti-universe, then
remnants of antimatter (matter) might persist in the universe (antimatter universe, ‘anti-universe’). Recent works have shown that antihydrogen atoms have the same properties with those of ordinary
hydrogen atoms and particularly the same energy levels [22, 23]. We may reasonably assume that 6 antimatter stars and galaxies should have the same birth, life and death as the matter ones, as well as
similar radiation properties yielding a particular difficulty for their detection. 2.3 The electromagnetic quantum vacuum cosmic source of photons (energy) and charges (mass).
Quantum vacuum cosmological model. antimatter stars and galaxies should have the same birth, life and death as the matter ones, as well as
similar radiation properties yielding a particular difficulty for their detection. Vacuum transient photons fluctuations are mostly enhanced near charge (mass) concentrations and
consequently are higher within a local universe system than in the space outside contributing by this
way to a smooth accelerated expansion [19]. The presence of antimatter structures in an ordinary mass
universe would also contribute to this effect and they might probably play a quite important role, which
is worthy to be further investigated. In a second stage, energy-mass annihilation mechanisms in the universe (anti-universe), like black holes
(antimatter black holes, ‘anti black holes’) and probably other yet unknown cosmic structures, start
appearing following the death of massive stars (anti-stars), mostly in the center of galaxies (anti-
galaxies). Obviously, such annihilation mechanisms appear to older galaxies, which could explain why
quasars are found in big distances. A period of equilibrium might be established in the local universe (anti-universe) during which the
energy-mass annihilation and creation rates balance. In a later stage, the annihilation processes might
overcome the creation ones and the generated mass-energy returns progressively to the vacuum state. In
the overall energy-mass creation and annihilation processes in the universe (anti-universe) the energy-
mass and charge conservation laws are respected between the initial and final state. Conclusions A new cosmological model could be developed on this basis overcoming the well-known Big Bang
inconveniences such as, initial state, faster than the speed of light inflation, as well as the horizon,
flatness and antimatter issues. 7 7 References References 1. Frieman, J., Turner, M. S., Huterer, D. Dark energy and the accelerating universe. Ann. Rev. Astron. Astrophys. 46, 385-432 (2008). 2. Hey, T. The New Quantum Universe. (Cambridge University Press, 2003). 3. Jim Baggott, Origins : The Scientific Story of Creation. (Oxford Universit gg
,
g
f
y f
(
y
,
)
4. Linda S. Sparke and John S. Gallagher III, Galaxies in the Universe. An introduction. 2nd
Edition, (Cambridge University Press, 2007). 5. Gibbons, Gary W.; Hawking, Stephen W.; Siklos, S.T.C., eds., "Natural Inflation," in the Very
Early Universe, (Cambridge University Press, 1983). y
g
y
6. Weinberg, S. The cosmological constant problem, Rev. Mod. Phys. 61, 1 (1989) y
g
y
6. Weinberg, S. The cosmological constant problem, Rev. Mod. Phys. 61, 1 (1989). 7. Hobson, M.P., Efstathiou, G.P. and Lasenby, A.N. General Relativity: An introduction for
physicists (Cambridge University Press, 2006). 7. Hobson, M.P., Efstathiou, G.P. and Lasenby, A.N. General Relativity: An introduction for
physicists (Cambridge University Press, 2006). 8. Meis, C. Photon wave-particle duality and virtual electromagnetic waves. Foundations of Physics, 27, 865 (1997). y
9. Meis, C. and Dahoo P.R., Vector potential quantization and the photon wave-particle
representation. Jour. Phys. Conference Series, 738 012099 (2016). 10. Meis, C. Light and Vacuum, 2nd Edition (World Scientific, Singapore, 2017) 10. Meis, C. Light and Vacuum, 2nd Edition (World Scientific, Singapore, 2017) 11. Meis, C. Vector potential quantization and the quantum vacuum. Physics Research International, Vol. 2014, ID 187432 (2014). 12. Meis, C. and Dahoo, P.R., Vector potential quantization and the photon intrinsic
electromagnetic properties: Towards nondestructive photon detection. International Journal of Quantum Information 15, N°8, (2017) 1740003. 12. Meis, C. and Dahoo, P.R., Vector potential quantization and the photon
electromagnetic properties: Towards nondestructive photon detection. I t
ti
l J
l f Q
t
I f
ti
15 N°8 (2017) 1740003 International Journal of Quantum Information 15, N°8, (2017) 1740003. 13. Meis, C. and Dahoo, P.R., Vector potential quantization and the photon wave function. Jour. Phys. Conference Series, 936, 012004 (2017). 14. Meis, C. Quantized field of single photons
https://www.intechopen.com/online-first/quantized-field-of-single-photons (2019)
DOI: 10.5772/intechopen.88378 14. Meis, C. Quantized field of single photons
https://www.intechopen.com/online-first/quantized-field-of-single-photons (2019)
DOI: 10.5772/intechopen.88378 15. Meis, C. Electric potential of the quantum vacuum. Physics Essays, 22, 1, (2009) 15. Meis, C. Electric potential of the quantum vacuum. Physics Essays, 22, 1, (2009) 16. References Meis, C., The Electromagnetic Field Ground State and the Cosmological Evolution
Jour. Phys. Conference Series, 1141, 012072 (2018) 16. Meis, C., The Electromagnetic Field Ground State and the Cosmological Evolution
Jour. Phys. Conference Series, 1141, 012072 (2018) 16. Meis, C., The Electromagnetic Field Ground State and the
Jour. Phys. Conference Series, 1141, 012072 (2018) 17. Meis, C., Primary role of the quantum electromagnetic vacuum in Gravitation and Cosmology,
in Cosmology 2020 – The Current State, (IntechOpen, London, 2020), Ed. Michael L.Smith,
DOI: 10.5772/intechopen.87805. ISBN: 978-1-83968-267-4 17. Meis, C., Primary role of the quantum electromagnetic vacuum in Gravitation and Cosmology,
in Cosmology 2020 – The Current State, (IntechOpen, London, 2020), Ed. Michael L.Smith,
DOI: 10.5772/intechopen.87805. ISBN: 978-1-83968-267-4 p
8. Garrison, J. C. and Chiao, R. Y., Quantum Optics, Oxford University Press, New York, 2008. 18. Garrison, J. C. and Chiao, R. Y., Quantum Optics, Oxford University Press, New York, 2008. 19. Meis, C. The vacuum electromagnetic energy density - Towards a physical comprehension of
the vacuum energy scale problem. International Journal of Modern Physics A Vol 35 No 25 (2020) 2050153 Q
p
y
19. Meis, C. The vacuum electromagnetic energy density - Towards a physical comprehension of
the vacuum energy scale problem. International Journal of Modern Physics A. Vol.35, No. 25 (2020) 2050153 19. Meis, C. The vacuum electromagnetic energy density - Towards a physical comprehension of
the vacuum energy scale problem. International Journal of Modern Physics A. Vol.35, No. 25 (2020) 2050153 20. Meis, C. and Dahoo, P.R., The single photon state, the quantum vacuum and the elementary
electron/positron charge. American Institute of Physics Publications, Conference Proceedings 2040, 020011 (2018). 20. Meis, C. and Dahoo, P.R., The single photon state, the quantum vacuum and the elementary
electron/positron charge. A
i
I
tit t
f Ph
i
P bli
ti
C
f
P
di
2040 020011 (2018) p
g
American Institute of Physics Publications, Conference Proceedings 2040, 020011 (2018). 21. Villata, M., Europhysics Letters, 94, 20001 (2011). 22. Ahmadi, M., Alves, B. X. R., Wurtele, J. S., Observation of the hyperfine spectrum of
antihydrogen
Nature, Vol. 548, 66–69 (2017) 23. Andresen, G. B. (ALPHA Collaboration); et al. "Confinement of antihydrogen for 1,000
seconds". Nature Physics. 7 (7): 558–564, (2011). 8
|
https://openalex.org/W1988193962
|
https://europepmc.org/articles/pmc3796558?pdf=render
|
English
| null |
An Interactive Association of Advanced Glycation End-Product Receptor Gene Four Common Polymorphisms with Coronary Artery Disease in Northeastern Han Chinese
|
PloS one
| 2,013
|
cc-by
| 5,893
|
Xiaohong Yu1, Jun Liu1, Hao Zhu1, Yunlong Xia1, Lianjun Gao1, Zhen Li1, Nan Jia2, Weifeng Shen3,
Yanzong Yang1*, Wenquan Niu4,5* Xiaohong Yu1, Jun Liu1, Hao Zhu1, Yunlong Xia1, Lianjun Gao1, Zhen Li1, Nan Jia2, Weifeng Shen3,
Yanzong Yang1*, Wenquan Niu4,5* 1 Department of Cardiology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China, 2 Department of Hypertension, Ruijin Hospital, School of
Medicine, Shanghai Jiao Tong University, Shanghai, China, 3 Department of Cardiology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai,
China, 4 State Key Laboratory of Medical Genomics, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China, 5 Shanghai Institute of
Hypertension, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Abstract This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Grant support: Liaoning Provincial Department of Education Scientific Research Project (Grant No. L2011155 and L2012321). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Funding: Grant support: Liaoning Provincial Department of Education Scientific Research Project (Grant No. L2011155 and L2012321)
study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yyzheart@126.com (YY); niuwenquan_shcn@163.com (WN) Abstract Background: Growing evidence indicates that advanced glycation end-product receptor (RAGE) might play a contributory
role in the pathogenesis of coronary artery disease (CAD). To shed some light from a genetic perspective, we sought to
investigate the interactive association of RAGE gene four common polymorphisms (rs1800625 or T-429C, rs1800624 or T-
374A, rs2070600 or Gly82Ser, and rs184003 or G1704A) with the risk of developing CAD in a large northeastern Han Chinese
population. Methodology/Principal Findings: This was a hospital-based case-control study incorporating 1142 patients diagnosed with
CAD and 1106 age- and gender-matched controls. All individuals were angiographically confirmed. Risk estimates were
expressed as odds ratio (OR) and 95% confidence interval (CI). Overall there were significant differences in the genotype and
allele distributions of rs1800625 and rs184003, even after the Bonferroni correction. Logistic regression analyses indicated
that rs1800625 and rs184003 were associated with significant risk of CAD under both additive (OR = 1.20 and 1.23; 95% CI:
1.06–1.37 and 1.06–1.42; P = 0.006 and 0.008) and recessive (OR = 1.75 and 2.39; 95% CI: 1.28–2.40 and 1.47–3.87; P,0.001
and ,0.001) models after adjusting for confounders. In haplotype analyses, haplotypes C-T-G-G and T-A-G-T (alleles in order
of rs1800625, rs1800624, rs2070600 and rs184003), overrepresented in patients, were associated with 52% (95% CI: 1.19–
1.87; P = 0.0052) and 63% (95% CI: 1.14–2.34; P = 0.0075) significant increases in adjusted risk for CAD. Further interactive
analyses identified an overall best multifactor dimensionality reduction (MDR) model including rs1800625 and rs184003. This model had a maximal testing accuracy of 0.6856 and a cross-validation consistency of 10 out of 10 (P = 0.0016). The
validity of this model was substantiated by classical Logistic regression analysis. Conclusions: Our findings provided strong evidence for the potentially contributory roles of RAGE multiple genetic
polymorphisms, especially in the context of locus-to-locus interaction, in the pathogenesis of CAD among northeastern Han
Chinese. tation: Yu X, Liu J, Zhu H, Xia Y, Gao L, et al. (2013) An Interactive Association of Advanced Glycation End-Product Receptor
lymorphisms with Coronary Artery Disease in Northeastern Han Chinese. PLoS ONE 8(10): e76966. doi:10.1371/journal.pone.0076966 Editor: Barry I. Hudson, University of Miami, United States of America Editor: Barry I. Hudson, University of Miami, United States of America Received June 19, 2013; Accepted August 26, 2013; Published October 14, 2013 Received June 19, 2013; Accepted August 26, 2013; Published October 14, 2013 Copyright: 2013 Yu et al. Editor: Barry I. Hudson, University of Miami, United States of America
Received June 19, 2013; Accepted August 26, 2013; Published October 14, 2013
Copyright: 2013 Yu 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. Copyright: 2013 Yu 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. October 2013 | Volume 8 | Issue 10 | e76966 Citation: Yu X, Liu J, Zhu H, Xia Y, Gao L, et al. (2013) An Interactive Association of Advanced Glycation End-Product Receptor Gene Four Common
Polymorphisms with Coronary Artery Disease in Northeastern Han Chinese. PLoS ONE 8(10): e76966. doi:10.1371/journal.pone.0076966
Editor: Barry I. Hudson, University of Miami, United States of America
Received June 19, 2013; Accepted August 26, 2013; Published October 14, 2013
Copyright: 2013 Yu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Grant support: Liaoning Provincial Department of Education Scientific Research Project (Grant No. L2011155 and L2012321). The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: yyzheart@126.com (YY); niuwenquan_shcn@163.com (WN) Introduction or the genetic knockout of RAGE [4,5]. In humans, soluble forms
of RAGE or sRAGE in plasma can predict the development and
progression of heart failure, irrespective of the presence of diabetes
[6]. Likewise, plasma sRAGE levels were negatively associated
with the angiographically-confirmed CAD, and this association
was dose-dependent with patients in the lowest quartile of sRAGE
exhibiting the highest risk of CAD [7]. On the basis of these
observations, it is reasonable to speculate that RAGE gene might
play a contributory role in the pathogenesis of CAD. Advanced glycation end-product receptor (protein: RAGE;
gene: RAGE) is a member of the immunoglobulin superfamily of
cell surface receptors, and it interacts with advanced glycation
end-products and other molecules implicated in inflammation,
atherogenesis and vasoconstriction, eventually leading to coronary
dysfunction, atherosclerosis and thrombosis [1–3]. Evidence is
mounting from animal experiments suggesting the protection
against inflammatory conditions, heart failure, and coronary
artery disease (CAD) after the pharmacological blockade of RAGE October 2013 | Volume 8 | Issue 10 | e76966 1 PLOS ONE | www.plosone.org Yu X et al. RAGE and CAD in Han Chinese genotyping. Plasma was prepared for quantifying routine biolog-
ical profiles. The gene encoding RAGE is highly polymorphic, and more than
twenty polymorphisms so far have been validated. Best evaluated
with respect to the association with CAD or related intermediate
phenotypes in RAGE gene are four common polymorphisms, viz. rs1800625 (T-429C) and rs1800624 (T-374A) in the promoter
region, rs2070600 (Gly82Ser) in 3rd exon, and rs184003 (G1704A)
in 7th intron. Despite a large panel of the RAGE-CAD genetic
association
studies,
it
remains
unclear
whether
individuals
possessing the genetic defect (s) of these polymorphisms, in
isolation or in combination, are more susceptible to CAD than
those with the alternative one (s) [8–11]. To make definitive claims
about the involvement of RAGE gene in the development of CAD,
comprehensive genetic approaches such as replication studies with
other populations have attracted special attention. To generate
more information, we sought to investigate the interactive
association of these four common polymorphisms in RAGE gene
with the risk of developing CAD in a large northeastern Han
Chinese population. All polymorphisms were genotyped according to the polymerase
chain reaction-ligase detection reaction (PCR-LDR) method as
previously described [12]. The primers for PCR amplification and
the probes for LDR can be obtained by request. PCR reactions
were performed in the EDC-810 Amplifier. Study population This study was conducted on a hospital-based case-control
design involving 2248 unrelated individuals admitted to the
Department of Cardiology, the First Affiliated Hospital of Dalian
Medical University. All study individuals were Han Chinese and
resided in Dalian city, Liaoning province, and they were classified
into CAD group and control group according to the angiographic
results. Coronary angiography was undertaken by the standard
Judkins techniques or through the radial approach. The CAD
group enrolled was angiographically confirmed in the presence of
more than 50% stenosis in at least one of the three major coronary
arteries or major branches. Patients were excluded if they had
simple spasm of coronary arteries, myocardial bridge or other non-
coronary atherosclerotic lesions. The controls had no history of
any vascular event and had normal coronary arteries on
angiography. In total, there were 1142 patients diagnosed with
CAD and 1106 age- and gender-matched controls. Introduction For each polymorphism, two specific probes were synthesized to
discriminate specific bases, and additionally one common probe
was synthesized and labeled at the 39 end with 6-carboxy-
fluorescein (FAM) and phosphorylated at the 59 end. The
multiplex ligation reaction was carried out in a reaction volume
of 10 ml containing 2 ml of PCR product, 1 ml 106Taq DNA
ligase buffer, 1 mM of each discriminating probe, 5 U Taq DNA
ligase, and the ligation parameters were 30 cycles of 94uC for
30 seconds and 56uC for 3 minutes. After reaction, 1 ml LDR
reaction product was mixed with 1 ml ROX passive reference and
1 ml loading buffer, and then denatured at 95uC for 3 minutes,
chilled rapidly in ice water. The fluorescent products of LDR were
differentiated using ABI 3730XL sequencer (Applied Biosystems,
USA). Statistical analysis
2 Pearson x2 and unpaired Student’s t-test or Mann-Whitney U
test were adopted to examine the differences between CAD
patients and controls for categorical (including genotypes and
alleles of examined polymorphisms) and continuous variables,
respectively. Testing for deviations from Hardy-Weinberg equi-
librium was carried out using a Pearson goodness-of-fit test. Two-
tailed P,0.05 was accepted as statistical significance. Each genotype of examined polymorphisms was assessed by
Logistic regression analyses under the additive (major homozy-
gotes versus heterozygotes versus minor homozygotes), dominant
(major homozygotes versus heterozygotes plus minor homozy-
gotes) and recessive (major homozygotes plus heterozygotes versus
minor homozygotes) models of inheritance after adjusting for
confounding factors, respectively. The haplotype frequencies of four examined polymorphisms in
RAGE gene were estimated by haplo.em program, which computes
the maximum likelihood estimates of haplotype probabilities using
the progressive insertion algorithm which progressively inserts
batches of loci into haplotypes of growing lengths. Only haplotype
with frequency $3% was considered in haplotype analyses. The
haplo.cc and haplo.glm programs were employed to calculate the
adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for
each haplotype. These two programs are based on a generalized
linear model, and compute the regression of a trait on haplotypes
and other covariates [13]. Simulated P values were calculated
based on 1000 replicates. All mentioned haplo.* programs were
implemented in Haplo.Stats software (version 1.4.0) operated in
the R language (version 2.14, available at the website http://www. r-project.org). All individuals signed written informed consent prior to
enrollment. This study was reviewed and approved by the Ethics
Committee of Dalian Medical University, and was conducted in
agreement with the Declaration of Helsinki Principles. Study characteristics At enrollment, body weight and height were recorded, and body
mass index (BMI) was calculated as weight in kilograms divided by
height in meters squared. Systolic and diastolic blood pressures
(SBP and DBP) at sitting position were measured twice with a five-
minute interval by certified nurses. Venous blood was extracted from each individual after an
overnight fasting of at least 8 hours. Fasting glucose was measured
in fluoride plasma by an electrochemical glucose oxidase method. Plasma levels of triglyceride (TG), total cholesterol (TC), high-
density lipoprotein cholesterol (HDL-C), lipoprotein (a), blood
urea nitrogen (BUN), creatinine and urea acid (UA) were
determined enzymatically using available kits and auto analyzers. Plasma high sensitivity C-reactive protein (hsCRP) levels were
determined using the high-sensitivity enzyme-linked immunosor-
bent assay (ELISA) kit. Interactive
analyses
were
conducted
in
the
open-source
multifactor dimensionality reduction (MDR) software (version
2.0) (www.epistasis.org) [14,15]. All possible combinations of four
examined polymorphisms were constructed using MDR construc-
tive induction. The accuracy of each model was evaluated by a
Bayes classifier in the context of 10-fold cross-validation. In
general, a single best model simultaneously has the maximal
testing accuracy and cross-validation consistency. The cross-
validation consistency is a measure of the number of times of 10
divisions of the dataset that the best model is extracted. Statistical
significance was evaluated using a 1000-fold permutation test to
compare observed testing accuracies with those expected under October 2013 | Volume 8 | Issue 10 | e76966 Genotyping Genomic DNA was obtained from peripheral blood leukocytes
by TIANamp Blood DNA Kit (Tiangen Biotect (Beijing) Co.,
China) and was stored at 240uC until required for batch PLOS ONE | www.plosone.org 2 October 2013 | Volume 8 | Issue 10 | e76966 Yu X et al. RAGE and CAD in Han Chinese polymorphisms (P,0.0125). Correspondingly, the power to reject
the null hypothesis of no difference in genotype frequencies for
rs1800625 and rs184003 between patients and controls was 94.4%
and 99.6%, respectively. No significance was reached for the other
two polymorphisms under study. Moreover, considering the
absolute
linkage
disequilibrium
between
rs1800625
and
rs1800624 reported in Euro- and Afro-Brazilians [16], the relation
of these two polymorphisms was checked in all individuals, and the
linkage disequilibrium was only moderate (D’ = 0.67), indicating
the potential existence of genetic heterogeneity across ethnicities. polymorphisms (P,0.0125). Correspondingly, the power to reject
the null hypothesis of no difference in genotype frequencies for
rs1800625 and rs184003 between patients and controls was 94.4%
and 99.6%, respectively. No significance was reached for the other
two polymorphisms under study. Moreover, considering the
absolute
linkage
disequilibrium
between
rs1800625
and
rs1800624 reported in Euro- and Afro-Brazilians [16], the relation
of these two polymorphisms was checked in all individuals, and the
linkage disequilibrium was only moderate (D’ = 0.67), indicating
the potential existence of genetic heterogeneity across ethnicities. the null hypothesis of null association. Permutation testing corrects
for multiple testing by repeating the entire analysis on 1000
datasets that are consistent with the null hypothesis. Further to
validate the soundness of MDR method, a classical Logistic
regression analysis was undertaken to check the derived best
model. Statistical analyses were conducted by STATA software v11.0
for Windows (StataCorp LP, College Station, TX, USA). Study
power was estimated by adopting the Power and Sample Size
Calculations (PS) software (v3.0.7) [6]. The linkage disequilibrium
was performed by Haploview (v.4.0), and the linkage disequilib-
rium coefficient was expressed as D’. Three models of inheritance including additive, dominant and
recessive models were explored for each polymorphism. Genotyping Results
from Logistic regression analyses indicated that rs1800625 and
rs184003 were significantly associated with the risk of having CAD
under both additive (OR = 1.20 and 1.23; 95% CI: 1.06–1.37 and
1.06–1.42; P = 0.006 and 0.008, respectively) and recessive
(OR = 1.75
and
2.39;
95%
CI:
1.28–2.40
and
1.47–3.87;
P,0.001 and ,0.001, respectively) models after adjusting for
age, gender, BMI, SBP and fasting glucose. Baseline characteristics Differences of study characteristics between CAD group and
control group are compared in Table 1. Age and gender
distributed similarly between the two groups. CAD patients had
relatively higher BMI than controls (P = 0.0637). Blood pressures
and fasting glucose levels were strikingly higher in patients than in
controls (P,0.0005). Plasma total cholesterol and HDL-C levels
were significantly lower in patients than in controls (P,0.0005). In
contrast,
plasma
lipoprotein
(a)
(P,0.0005),
creatinine
(P = 0.0006) and hsCRP (P,0.0005) levels were significantly
higher in patients than in controls. There were no significant
differences for BUN and uric acid. Haplotype analyses p
yp
y
Table 3 presents the haplotype frequencies ($3%) of four
examined polymorphisms in patients and controls with the
cumulative frequencies reaching 92.89% and 88.83% respectively. The most common haplotype T-T-A-G (alleles in order of
rs1800625, rs1800624, rs2070600 and rs184003) was comparable
in frequencies between patients and controls (PSim = 0.1026), and
was assigned as the reference group in risk estimates. Haplotypes
C-T-G-G and T-A-G-T, which were remarkably overrepresented
in patients, were respectively associated with a 52% (95% CI:
1.19–1.87; P = 0.0052) and 63% (95% CI: 1.14–2.34; P = 0.0075)
increased risk of developing CAD after adjusting for age, gender,
BMI, SBP and fasting glucose. Accordingly for these two
haplotypes, the power to reject the null hypothesis of no difference
between patients and controls was 98.6% and 99.1%, respectively. Single-locus analyses The genotype distributions and allele frequencies of four
examined polymorphisms in RAGE gene and their risk prediction
for CAD are summarized in Table 2. There was no detectable
deviation from the Hardy-Weinberg equilibrium for all polymor-
phisms in both patients and controls (P.0.05). Overall there were
statistically significant differences in the genotypes and alleles of
rs1800625 and rs184003, even after applying a Bonferroni
correction to account for multiple testing with respect to the four Table 1. Baseline characteristics of study population. Table 1. Baseline characteristics of study population. Table 1. Baseline characteristics of study population. Characteristics
CAD patients (n = 1142)
Controls (n = 1106)
P
Age, years
62.0769.07
62.4269.85
0.3749
Gender (Males)
46.58%
49.10%
0.2334
BMI, kg/m2
26.19615.32
24.963.64
0.0637
SBP, mmHg
141.44616.82
137.31620.52
,0.0005
DBP, mmHg
84.86610.63
81.09611.92
,0.0005
Fasting glucose, mmol/L
6.1462.15
5.4761.26
,0.0005
Triglycerides, mmol/L
1.961.04
1.9261.45
0.7240
Total cholesterol, mmol/L
4.5961.18
4.8161.0
,0.0005
HDL-C, mmol/L
1.1260.32
1.3560.4
,0.0005
LDL-C, mmol/L
2.7560.95
2.7560.77
0.8986
Lipoprotein (a), mmol/L
0.360.45
0.2160.19
,0.0005
BUN, mmol/L
5.9263.89
5.7663.71
0.3794
Creatinine, mmol/L
87.49636.81
81.35635.96
0.0006
Uric acid, mmol/L
329.066100.37
328.85692.52
0.9644
hsCRP, mmol/L
12.37611.42
2.2163.71
,0.0005
Abbreviations: CAD, coronary artery disease; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; BUN, blood
urea nitrogen; hsCRP, high sensitivity C-reactive protein. Data are expressed as mean 6 standard deviation unless otherwise indicated. doi:10.1371/journal.pone.0076966.t001 Abbreviations: CAD, coronary artery disease; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; BUN, blood
urea nitrogen; hsCRP, high sensitivity C-reactive protein. Data are expressed as mean 6 standard deviation unless otherwise indicated. doi:10.1371/journal.pone.0076966.t001 October 2013 | Volume 8 | Issue 10 | e76966 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 3 3 Yu X et al. RAGE and CAD in Han Chinese Table 2. Genotype distributions and allele frequencies of four examined polymorphisms in RAGE gene between patients and
healthy controls and their risk prediction for coronary artery disease. Single-locus analyses Polymorphisms
CAD patients (n = 1142)
Controls (n = 1106)
Px2
OR; 95% CI; P*
rs1800625
TT
557
577
1.20; 1.06–1.37; 0.006
1.14; 0.97–1.35; 0.114
1.75; 1.28–2.40; ,0.001
Genotype (n):
TC
468
461
0.002
CC
117
68
Allele (%):
C
30.74
26.99
0.006
rs1800624
TT
608
604
1.04; 0.92–1.19; 0.515
1.06; 0.9–1.25; 0.501
1.05; 0.78–1.41; 0.759
Genotype (n):
TA
436
410
0.808
AA
98
92
Allele (%):
A
27.67
26.85
0.538
rs2070600
GG
482
496
1.11; 0.98–1.26; 0.086
1.11; 0.94–1.31; 0.235
1.26; 0.97–1.62; 0.079
Genotype (n):
GA
507
489
0.158
AA
153
121
Allele (%):
A
35.6
33.05
0.072
rs184003
GG
729
742
1.23; 1.06–1.42; 0.008
1.16; 0.98–1.38; 0.091
2.39; 1.47–3.87; ,0.001
Genotype (n):
GT
355
339
0.001
TT
58
25
Allele (%):
T
20.62
17.59
0.011
Abbreviations: CAD, coronary artery disease; OR, odds ratio; 95% CI, 95% confidence interval. * OR, 95% CI, and P values were calculated under the additive (the upper),
dominant (the middle), and recessive (the lower) models of inheritance after adjusting for age, gender, body mass index, systolic blood pressure, and fasting glucose. doi:10.1371/journal.pone.0076966.t002 Table 2. Genotype distributions and allele frequencies of four examined polymorphisms in RAGE gene between patients and
healthy controls and their risk prediction for coronary artery disease. Table 2. Genotype distributions and allele frequencies of four examined polymorphisms in RAGE gene between patients and
healthy controls and their risk prediction for coronary artery disease. Table 2. Genotype distributions and allele frequencies of four examined polymorphisms in RAGE gene between patients and
healthy controls and their risk prediction for coronary artery disease. Abbreviations: CAD, coronary artery disease; OR, odds ratio; 95% CI, 95% confidence interval. * OR, 95% CI, and P values were calculated under the additive (the upper),
dominant (the middle), and recessive (the lower) models of inheritance after adjusting for age, gender, body mass index, systolic blood pressure, and fasting glucose. doi:10.1371/journal.pone.0076966.t002 of four examined polymorphisms in RAGE gene is shown in
Table 4. Specifically, each best model was accompanied with the
testing accuracy, cross-validation consistency and significant level
as determined by permutation testing. The overall best MDR
model included rs1800625 and rs184003, and this model had a
maximal testing accuracy of 0.6856 and a cross-validation
consistency of 10 out of 10. Interactive analyses To shed some light on the potential genetic interactions, an
exhaustive MDR analysis that evaluates all possible combinations Table 3. Haplotype frequencies of four polymorphisms
examined in RAGE gene between patients and controls and
their risk prediction for coronary artery disease. Table 3. Haplotype frequencies of four polymorphisms
examined in RAGE gene between patients and controls and
their risk prediction for coronary artery disease. Haplotype*
CAD patients
Controls PSim
OR; 95% CI; P{
T-T-G-G
25.68
26.36
0.1026
Reference group
T-T-A-G
16.44
17.94
0.1225
0.97; 0.76–1.21; 0.6274
C-T-G-G
13.95
9.92
0.0038
1.52; 1.19–1.87; 0.0052
T-A-G-G
9.08
11.9
0.009
0.79; 0.52–1.06; 0.1115
C-T-A-G
7.72
6.17
0.0387
1.33; 0.91–1.82; 0.1397
T-T-G-T
6.64
4.41
0.0184
1.53; 0.97–2.11; 0.0806
T-A-G-T
5.75
3.1
0.0091
1.63; 1.14–2.34; 0.0075
T-A-A-G
4.75
5.48
0.2145
0.98; 0.66–1.46; 0.8347
C-A-G-G
2.88
3.55
0.2798
0.91; 0.53–1.37; 0.7904
Abbreviations: CAD, coronary artery disease, PSim, simulated P value; OR, odds
ratio; 95% CI, 95% confidence interval. PSim was calculated based on randomly
permuting the trait and covariates and then computing the haplotype score
statistics. *Alleles in haplotype were presented in order of polymorphisms
rs1800625, rs1800624, rs2070600 and rs184003. {OR, 95% CI, and P values were
calculated after considering age, gender, body mass index, systolic blood
pressure, and fasting glucose as covariates. doi:10.1371/journal.pone.0076966.t003 To further validate the predictive value of MDR model,
classical Logistic regression analysis was employed accordingly. The interaction of rs1800625 and rs184003 (rs1800625*rs184003)
was associated with 1.12-fold (95% CI: 1.05–1.2; P = 0.001), 1.09-
fold (95% CI: 1.01–1.9; P = 0.031) and 1.83-fold (95% CI: 1.42–
2.36; P,0.0005) increased risk of having CAD under additive,
dominant and recessive models of inheritance after adjusting for
age, gender, BMI, SBP and fasting glucose. Single-locus analyses Moreover, this model was significant
at the level of 0.0016, indicating that a model this good or better
was observed only by less than 2 out of 1000 permutations and was
thus unlikely under the null hypothesis of null association. October 2013 | Volume 8 | Issue 10 | e76966 Discussion They are characterized by genetic
homogeneity and geographic stability, and are probably more
uniform in their environmental exposures, including the habitual
dietary intake of high salt and high fat. All these characteristics
render
this
population
more
appropriate
to
enhance
our
understanding of genetic architecture of CAD and related
intermediate phenotypes such as blood pressure. Moreover, it
cannot be totally ruled out that the evolutionary history of linkage
disequilibrium patterns will vary significantly in different ethnic
populations. For example, the degrees of linkage disequilibrium
between rs1800625 and rs1800624 were differentiated between
Euro- and Afro-Brazilians [16] and Han Chinese in this study. Further in this study all examined polymorphisms respected the
Hardy-Weinberg equilibrium in both patients and controls,
lowering the likelihood of being biased by faulty genotyping or
population stratification. Importantly it is worth noting that our
sample size of 2248 individuals is large enough to ensure a high
level of study power (.94%) to detect the small-to-moderate
impact of common polymorphisms. Despite the clear strengths of our study, including the relatively
large sample size, the angiographically-confirmed CAD patients
and controls, and the selection of candidate gene and polymor-
phisms with strong biological plausibility, the interpretation of our
results, however, should be viewed in light of several limitations. First, the retrospective design of this study has inherent drawbacks
and precludes causal inferences [26]. Second, we only focused on
four common polymorphisms of RAGE gene, and it is encouraged
to examine more polymorphisms, especially the low-penetrance
polymorphisms from other promising CAD-susceptibility genes,
such as interleukin-6 gene [27]. More importantly, because CAD
is a multifactorial disease, characterizing the interaction of
multiple polymorphisms from different chromosomes is deemed
as an effective approach to elucidate final genetic architecture of
complex disease [28]. Third, the MDR method used in this study
has some underling drawbacks including computational intensive-
ness, indistinct interpretation, lack of sensitivity, and heterogene-
ity-free assumption [22,29]. Fourth, we recruited study individuals
aged more than 50 years, and future larger association studies in a
young population of CAD patients are of specific interest, because
genetic factors may have greater contribution to those suffering
premature CAD and in the absence of strong environmental risk
factors [30]. Last but not the least, the fact that our study
population was of Han Chinese descent limited the generalizability
of our findings, calling for further confirmation in other ethnic
groups. Discussion In the present study, we sought to investigate the association of
RAGE gene four common polymorphisms with the risk of
developing CAD in a large northeastern Han Chinese population
involving 2248 individuals. The principal finding was the potential
interactive roles of RAGE gene rs1800625 (T-429C) and rs184003
(G1704A) in the development of CAD. To the best of our
knowledge, this report so far is the largest case-control association October 2013 | Volume 8 | Issue 10 | e76966 October 2013 | Volume 8 | Issue 10 | e76966 4 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org Yu X et al. RAGE and CAD in Han Chinese Table 4. MDR analysis summary. Best combination of each model
Cross-validation consistency
Testing accuracy
P
rs1800625
8
0.6243
0.0637
rs1800625, rs184003
10
0.6856
0.0016*
rs1800625, rs1800624, rs184003
9
0.6637
0.0039
rs1800625, rs1800624, rs2070600, rs184003
10
0.6709
0.0021
*The overall best MDR model. doi:10.1371/journal.pone.0076966.t004 Best combination of each model informative nature of haplotype approach on the premise of the
synergistic effects within polymorphisms [23]. Although residual
confounding by incompletely measured or unmeasured physio-
logic covariates might exist, it seems unlikely that our results could
be explained by confounding. In addition, from a biological
standpoint, besides the potential impact of promoter rs1800625 on
transcriptional regulation [24], it cannot be overlooked that the
intronic rs184003 might be functional given the potential
regulatory effect of intronic loci on the stability of DNA molecule
[25], or alternatively this polymorphism might act as a surrogate
marker in linkage disequilibrium with other functional loci in
regulatory regions of RAGE gene. It is therefore reasonable to
hypothesize that the interaction of multiple genetic polymorphisms
in RAGE gene might play a contributory role in the pathogenesis of
CAD in Han Chinese. study examining the susceptibility of RAGE multiple genetic
polymorphisms to CAD in Chinese. More recently, Wang and colleagues have conducted a meta-
analysis by synthesizing data from 17 studies on RAGE gene three
polymorphisms (T-429C, T-374A, Gly82Ser) and the risk of CAD,
but unfortunately they failed to detect any suggestive association
[17]. This negative finding is possibly due to genetic heterogeneity
that is not uncommon in any disease identification strategy [18],
where
this
heterogeneity
can
be
somewhat
avoided
when
homogeneous populations are used [19]. Factually in this study,
all study individuals are of Han descent and local residents of
northeastern regions of China. October 2013 | Volume 8 | Issue 10 | e76966 References (1992) Cloning and
expression of a cell surface receptor for advanced glycosylation end products of
proteins. J Biol Chem 267: 14998–15004. 7. Falcone C, Emanuele E, D’Angelo A, Buzzi MP, Belvito C, et al. (2005) Plasma
levels of soluble receptor for advanced glycation end products and coronary
artery disease in nondiabetic men. Arterioscler Thromb Vasc Biol 25: 1032–
1037. nA1C levels in diabetic patients. Mol Immunol 44: 648–655. 22. Gui J, Andrew AS, Andrews P, Nelson HM, Kelsey KT, et al. (2010) A simple
and computationally efficient sampling approach to covariate adjustment for
multifactor dimensionality reduction analysis of epistasis. Hum Hered 70: 219–
225. 8. Selejan SR, Poss J, Hewera L, Kazakov A, Bohm M, et al. (2012) Role of
receptor for advanced glycation end products in cardiogenic shock. Crit Care
Med 40: 1513–1522. 23. Niu WQ, Zhao HY, Zhou L, Dai XX, Wang DY, et al. (2009) Interacting effect
of genetic variants of angiotensin II type 1 receptor on susceptibility to essential
hypertension in Northern Han Chinese. J Hum Hypertens 23: 68–71. 9. Park HJ, Baek JY, Shin WS, Kim DB, Jang SW, et al. (2011) Soluble receptor of
advanced glycated endproducts is associated with plaque vulnerability in patients
with acute myocardial infarction. Circ J 75: 1685–1690. 24. Hudson BI, Stickland MH, Futers TS, Grant PJ (2001) Effects of novel
polymorphisms in the RAGE gene on transcriptional regulation and their
association with diabetic retinopathy. Diabetes 50: 1505–1511. 10. Cai XY, Lu L, Wang YN, Jin C, Zhang RY, et al. (2011) Association of
increased S100B, S100A6 and S100P in serum levels with acute coronary
syndrome and also with the severity of myocardial infarction in cardiac tissue of
rat models with ischemia-reperfusion injury. Atherosclerosis 217: 536–542. p
y
25. Roy SW, Irimia M (2008) Intron mis-splicing: no alternative? Genome Biol 9:
208. 26. Gu M, Dong X, Zhang X, Wang X, Qi Y, et al. (2012) Strong association
between two polymorphisms on 15q25.1 and lung cancer risk: a meta-analysis. PLoS One 7: e37970. 11. Gao J, Shao Y, Lai W, Ren H, Xu D (2010) Association of polymorphisms in the
RAGE gene with serum CRP levels and coronary artery disease in the Chinese
Han population. J Hum Genet 55: 668–675. 27. Niu W, Liu Y, Qi Y, Wu Z, Zhu D, et al. (2012) Association of interleukin-6
circulating levels with coronary artery disease: a meta-analysis implementing
mendelian randomization approach. References 15. Hahn LW, Ritchie MD, Moore JH (2003) Multifactor dimensionality reduction
software for detecting gene-gene and gene-environment interactions. Bioinfor-
matics 19: 376–382. 1. Hegab Z, Gibbons S, Neyses L, Mamas MA (2012) Role of advanced glycation
end products in cardiovascular disease. World J Cardiol 4: 90–102. 1. Hegab Z, Gibbons S, Neyses L, Mamas MA (2012) Role of advanced glycation
end products in cardiovascular disease. World J Cardiol 4: 90–102. 2. Shrikhande GV, Scali ST, da Silva CG, Damrauer SM, Csizmadia E, et al. (2010) O-glycosylation regulates ubiquitination and degradation of the anti-
inflammatory protein A20 to accelerate atherosclerosis in diabetic ApoE-null
mice. PLoS One 5: e14240. 16. Torres MC, Beltrame MH, Santos IC, Picheth G, Petzl-Erler ML, et al. (2012)
Polymorphisms of the promoter and exon 3 of the receptor for advanced
glycation end products (RAGE) in Euro- and Afro-Brazilians. Int J Immunogenet
39: 155–160. 3. Pollreisz A, Hudson BI, Chang JS, Qu W, Cheng B, et al. (2010) Receptor for
advanced glycation endproducts mediates pro-atherogenic responses to
periodontal infection in vascular endothelial cells. Atherosclerosis 212: 451–456. 17. Wang J, Zou L, Song Z, Lang X, Huang S, et al. (2012) Meta-analysis of RAGE
gene polymorphism and coronary heart disease risk. PLoS One 7: e50790. 18. Evangelou E, Ioannidis JP (2013) Meta-analysis methods for genome-wide
association studies and beyond. Nat Rev Genet 14: 379–389. 4. Ramasamy R, Yan SF, Schmidt AM (2009) RAGE: therapeutic target and
biomarker of the inflammatory response – the evidence mounts. J Leukoc Biol
86: 505–512. y
19. Pineda-Krch M, Lehtila K (2004) Costs and benefits of genetic heterogeneity
within organisms. J Evol Biol 17: 1167–1177. 5. Volz HC, Laohachewin D, Seidel C, Lasitschka F, Keilbach K, et al. (2012)
S100A8/A9 aggravates post-ischemic heart failure through activation of RAGE-
dependent NF-kappaB signaling. Basic Res Cardiol 107: 250. 20. Kalea AZ, Schmidt AM, Hudson BI (2009) RAGE: a novel biological and
genetic marker for vascular disease. Clin Sci (Lond) 116: 621–637. 21. Laki J, Kiszel P, Vatay A, Blasko B, Kovacs M, et al. (2007) The HLA 8.1
ancestral haplotype is strongly linked to the C allele of 2429T.C promoter
polymorphism of receptor of the advanced glycation endproduct (RAGE) gene. Haplotype-independent association of the 2429C allele with high hemoglobi-
nA1C levels in diabetic patients. Mol Immunol 44: 648–655. 6. Neeper M, Schmidt AM, Brett J, Yan SD, Wang F, et al. Author Contributions Conceived and designed the experiments: YY WN. Performed the
experiments: XY JL. Analyzed the data: XY WN. Contributed reagents/ Conceived and designed the experiments: YY WN. Performed the
experiments: XY JL. Analyzed the data: XY WN. Contributed reagents/ Discussion Selection of RAGE gene as a CAD-susceptibility candidate is
founded on strong biological and genetic bases [3,17,20]. The
RAGE gene is located in the crowded major histocompatibility
complex (MHC) class III region, and there is strong evidence
supporting a tight linkage between RAGE gene rs1800625 and
tumor necrosis factor-a gene G-308A polymorphism [21]. Also
worth mentioning in the present study is the potential interactions
of RAGE gene two identified polymorphisms, rs1800625 and
rs184003, in susceptibility to CAD. As demonstrated in our single-
locus analyses, these two polymorphisms by itself were significantly
associated with the risk of developing CAD, especially under the
recessive
model. Further
in
haplotype
analyses,
nearly
all
haplotypes harboring either risk-conferring allele of two identified
polymorphisms had an increased risk for CAD, suggesting the
potential existence of locus-to-locus interaction. To shed some
light, a promising data-mining analytical approach MDR, which is
nonparametric and genetic model-free nature in design [22], was
employed, and as expected the aforementioned two polymor-
phisms constituted the overall best interactive model, reinforcing
the results of both single-locus and haplotype analyses. These
findings further confirmed our previous claims regarding the Taken together, our findings provided strong evidence for the
potentially contributory roles of RAGE genetic polymorphisms,
especially in the context of locus-to-locus interaction, in the
pathogenesis of CAD among 2248 northeastern Han Chinese. Moreover, corrections from statistical and practical points of view
established the robustness of our findings. For practical reasons,
large, well-designed longitudinal studies attempting to account for
gene-gene and gene-environment interactions, as well as studies
seeking to provide biological or clinical implications, are warrant-
ed in the future investigation. October 2013 | Volume 8 | Issue 10 | e76966 PLOS ONE | www.plosone.org 5 Yu X et al. RAGE and CAD in Han Chinese Yu X et al. RAGE and CAD in Han Chinese materials/analysis tools: HZ YX LG ZL NJ WS. Wrote the paper: WN
YY. References Int J Cardiol 157: 243–252. 12. Khanna M, Park P, Zirvi M, Cao W, Picon A, et al. (1999) Multiplex PCR/
LDR for detection of K-ras mutations in primary colon tumors. Oncogene 18:
27–38. 28. Niu W, Qi Y (2012) Matrix metalloproteinase family gene polymorphisms and
risk for coronary artery disease: systematic review and meta-analysis. Heart 98:
1483–1491. 13. Stram DO, Leigh Pearce C, Bretsky P, Freedman M, Hirschhorn JN, et al. (2003) Modeling and E-M estimation of haplotype-specific relative risks from
genotype data for a case-control study of unrelated individuals. Hum Hered 55:
179–190. 29. Moore JH, Ritchie MD (2004) STUDENTJAMA. The challenges of whole-
genome approaches to common diseases. JAMA 291: 1642–1643. 14. Pattin KA, White BC, Barney N, Gui J, Nelson HH, et al. (2009) A
computationally efficient hypothesis testing method for epistasis analysis using
multifactor dimensionality reduction. Genet Epidemiol 33: 87–94. 30. Zintzaras E, Raman G, Kitsios G, Lau J (2008) Angiotensin-converting enzyme
insertion/deletion gene polymorphic variant as a marker of coronary artery
disease: a meta-analysis. Arch Intern Med 168: 1077–1089. October 2013 | Volume 8 | Issue 10 | e76966 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 6
|
https://openalex.org/W2085817212
|
https://hal-brgm.archives-ouvertes.fr/hal-03652262/document
|
English
| null |
In Salah gas CO2 storage JIP: Surface gas and biological monitoring
|
Energy procedia
| 2,011
|
cc-by
| 6,459
|
To cite this version: D.G. Jones, T.R. Lister, D.J. Smith, J.M. West, P. Coombs, et al.. In Salah gas CO2
storage JIP: Surface gas and biological monitoring. Energy Procedia, 2011, 4, pp.3566-3573. 10.1016/j.egypro.2011.02.285. hal-03652262 In Salah gas CO2 storage JIP: Surface gas and biological
monitoring
D.G. Jones, T.R. Lister, D.J. Smith, J.M. West, P. Coombs, Alain Gadalia,
M. Brach, A. Annunziatellis, S. Lombardi, J Smith, et al. HAL Id: hal-03652262
https://brgm.hal.science/hal-03652262v1
Submitted on 26 Apr 2022 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. * Corresponding author. Tel.: +44-115-936-3576; fax: +44-115-936-3446.
E-mail address: dgj@bgs.ac.uk. Energy
Procedia www.elsevier.com/locate/procedia Abstract Surface gas and biological monitoring were carried out in 2009 at the In Salah Gas project (Krechba, Algeria), where geological
storage of CO2 has been underway since mid-2004. The CO2 is removed from produced natural gas and re-injected below the
gas-water contact on the flanks of the reservoir. The biological work was the first such study undertaken at the site. Observations
were made in: uplifted areas around the three CO2 injection wells, around the KB-5 well where breakthrough of CO2 from the
KB-502 injector had occurred, around the KB-4 well and in a background area away from CO2 injection and gas production. Near ground atmospheric measurements were made with a mobile open path laser system, with soil gas and flux measurements in
support of these and of a botanical and microbiological survey. Longer term monitoring was initiated for radon and other gases
using buried probes and activated charcoal integrative collectors. Laser measurements appeared to show only natural variations,
but interference from the vehicle exhaust, windblown dust and rain was apparent. Modifications are needed to overcome these
problems. Natural variation of atmospheric CO2 needs to be better constrained to identify anomalous values. Soil gas
concentrations and fluxes were very low but slightly higher values over the KB-5 well could indicate low-level leakage. This is
likely to be a legacy of breakthrough prior to the abandonment of the well. A variety of monocotyledonous and dicotyledenous
plants was present, particularly in dry wadis or shallow depressions. The xerophytic flora and the microbial numbers were typical
of such desert environments and the data provide baseline values since there were no indications of elevated CO2. There were
analytical problems with the microbial activity determinations but it can be concluded that activities were low. © 2010 Natural Environment Research Council. Published by Elsevier LTD. All rights reserved. c⃝2011 Published by Elsevier Ltd. Open access under CC BY-NC-ND license. Keywords: In Salah; CO2; gas monitoring; biological monitoring; Algeria 1. Introduction The In Salah Gas Project is a joint venture between Sonatrach, BP and Statoil. At the Krechba site, CO2 stripped
from natural gas is injected at a rate of 0.5–1 Mt per annum. Injection has been carried out since mid-2004 with
almost 3 Mt emplaced to date [1]. The CO2 is injected into a 20 m thick sandstone reservoir at approximately doi:10.1016/j.egypro.2011.02.285 Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3567 1800 m depth. The reservoir is an anticlinal structure with injection down dip of the natural gas reserves and the
production wells. It is sealed by a 950 m thick succession of Carboniferous mudstones,which is overlain by about
900 m of Cretaceous sandstone and mudstones. Cretaceous carbonates outcrop at surface. Injection is through three
deviated wells KB501, KB502 and KB503 (Figure 1). Satellite imaging (InSAR) indicates ground deformation has
taken place around the injection wells [2-4] as a result of pressure-induced poro-elastic expansion of the storage
reservoir in the area surrounding the injection points [5]. CO2 injection at the In Salah site is on-going and a range of monitoring technologies has been or is being
deployed. Other techniques are under consideration or have been considered and rejected [1]. The present study
outlines the second deployment of near surface gas monitoring methods at the site, following an initial appraisal in
August 2004 [6], and the first use of biological monitoring methods. The storage of CO2 in underground geological formations poses a very small risk of leakage to the surface. Monitoring strategies need to include a range of techniques to cover different monitoring requirements. Monitoring
of the surface of a storage site is one method for the detection and mitigation of any leakage. The nature of CO2
storage poses some challenges to surface monitoring: 1. The site must be monitored over its full life cycle, throughout the injection phase and after closure and well
abandonment. The slow dissolution and reaction of CO2 to form stable aqueous or mineral phases means that
buoyant and mobile CO2 can exist in the subsurface for tens to hundreds of years after injection has ceased [7]. Monitoring strategies need to be designed to cover this long time scale. 2. 1. Introduction The mobility and migration of supercritical CO2 (the preferred storage phase) and the large masses being
injected in full scale storage projects mean that leakage could occur in a wide area (i.e. the risk footprint of the site
may be greater than the actual area of the storage reservoir). Therefore, monitoring may need to cover large areas of
ground at the surface. g
3. Carbon dioxide is relatively abundant in the atmosphere (~380 ppm), groundwater, and soil, and detection of
small leaks above background is problematic, particularly since the background itself is variable. g
3. Carbon dioxide is relatively abundant in the atmosphere (~380 ppm), groundwater, and soil, and detection of
small leaks above background is problematic, particularly since the background itself is variable. When considering the challenges above, surface monitoring surveys need to be deployed over wide areas, and be
carried out relatively quickly and cheaply. However, they need to be sensitive enough to detect leakage, which may
occur at different rates giving rise to variable near surface gas concentrations and fluxes. It is crucial to assess
baseline conditions and their variability so that changes can be identified and quantified. When considering the challenges above, surface monitoring surveys need to be deployed over wide areas, and be
carried out relatively quickly and cheaply. However, they need to be sensitive enough to detect leakage, which may
occur at different rates giving rise to variable near surface gas concentrations and fluxes. It is crucial to assess
baseline conditions and their variability so that changes can be identified and quantified. Surface gas measurements at Krechba were made in March 2009 by BGS with BRGM and Sapienza Università
di Roma. The objectives of the short field visit were: Surface gas measurements at Krechba were made in March 2009 by BGS with BRGM and Sapienza Università
di Roma. The objectives of the short field visit were: make near ground atmospheric measurements of CO2 using a mobile open path laser system
To make near ground atmospheric measurements of CO2 using a mobile open path
To obtain supporting observations of CO2 and other gases in the very shallow subsurface (<1 m) by field
and laboratory measurements and to obtain CO2 flux data from the ground into the atmosphere
To deploy buried probes for continuous monitoring of Rn and passive samplers (activated charcoal) for
integrated monitoring to look at longer term gas migration effects. Gas Measurements A Boreal Laser open-path laser CO2 detector
CO2 of around 5-10 ppm),
mounted on the front of a Toyota Landcruiser 105, was linked to a GasFinder FC analyser. Data were collected
every second with positions from either a Trimble ProXT or a Pharos GPS receiver. This system allows the rapid
coverage of relatively large areas. The laser probe was mounted at a height of 38 cm (above flat ground), which was
as low as could be achieved whilst maintaining safe ground clearance. The probe used to collect soil gas samples consisted of an 8 mm diameter (4 mm ID), stainless-steel tube onto
which two solid steel cylinders were welded to act as pounding surfaces when installing and removing the probe
with a co-axial hammer. Prior to insertion, a sacrificial tip was fitted to the bottom of the probe in order to prevent
blockage. Because of the hardness of much of the ground, most sites sampled in March 2009 were predrilled using a
portable hammer drill before the probe was inserted. This does lead to potential problems with the effective
sampling depth as a perfect seal between the probe and the predrilled hole cannot be guaranteed and gas may,
therefore, come from around the probe annulus. All sites on the later visit were sampled by hammering in the probe. In-situ soil gas measurements were mostly made with a Draeger X-am 7000, although some used a Geotechnical
Instruments GA2000 or an LFG. All these instruments use an infrared analyser for CO2 and CH4 and
electrochemical detection for O2, H2S (X-am 7000 and GA2000) and CO (GA2000). Soil gas was drawn to the
instruments using their in-built pumps until a stable CO2 reading was seen. The analyser was purged with
atmospheric air between readings. The Draeger is limited to a 0-5% CO2 concentration range, compared with 0-
100% for the GA2000 and LFG20 and therefore should give better precision and accuracy at the low levels
encountered. In order to collect samples for laboratory analysis, at a number of sites a septum holder was attached to the upper
end of the probe tube. The needle of a 60ml plastic syringe was inserted through the septum and gas was drawn up
through the syringe and discarded to purge the probe. A 60 ml gas sample was then drawn up and injected into a
previously evacuated stainless steel container. Gas Measurements These containers were then transported back to the laboratory and
analysed for hydrocarbon species (C1-C3 alkanes and C2H4) and permanent gases (N2, O2 and CO2) using two
Fisons 8000-series bench gas chromatographs. The resolution of the gas chromatographs is 0.1 ppm with an
accuracy of ±5%. CO2 flux measurements were taken using a West Systems portable flux meter with a LICOR LI-820 IR detector
connected via Bluetooth to an Acer n300 palm-top computer (PDA) with built in GPS positioning. Barasol probes measure Rn gas concentrations continuously along with soil temperature and pressure The probes
are autonomous and can operate for several months on their internal battery. Data is stored to built-in memory and
downloaded when the instrument is retrieved. Radon can be carried by other gases and therefore may act as a natural
tracer. It also provides indications of the fluctuations of gas e.g. diurnally, seasonally and in response to changing
meteorological or ground conditions. The Barasol probes were buried vertically at a depth of 1 m in large excavated
pits, which were then backfilled. Ground gas measurements were made in the base of each pit during Barasol
deployment. The passive samplers used comprise activated charcoal cloth, buried at a depth of 0.3 m in the same plastic pipe
used for the Barasol or buried on their own in a similar way. They provide a cumulative record of the flux of gas and
accompanying elements, which can be liberated from the sampler on return to the laboratory and analysed. 1. Introduction
To make shallow groundwater measurements in existing water wells
To make shallow groundwater measurements in existing water wells The particular area of interest was between KB-502 and KB-5 (Figure 1) owing to the breakthrough of CO2 from
KB-502 into the latter well [1]. A secondary target was around KB-4. A background site to the west of the gas
reservoir was also measured to provide contrasting data in an area well away from the producing zone and the CO2
injectors. The particular area of interest was between KB-502 and KB-5 (Figure 1) owing to the breakthrough of CO2 from
KB-502 into the latter well [1]. A secondary target was around KB-4. A background site to the west of the gas
reservoir was also measured to provide contrasting data in an area well away from the producing zone and the CO2
injectors. Two Barasol probes were buried in excavated pits, one to the east of KB-502, close to the planned KB-602
aquifer monitoring well and KB-601 microseismic well, the second far away from CO2 injection to the south near
KB-7 (Figure 1). Passive sensors were deployed close to the three injection wells, KB-501-503, that near KB-502
being placed at the Barasol probe location (Figure 1). Weather conditions were mixed, with fine weather being followed by unsettled conditions with appreciable
amounts of rain. There had also been rainfall in the preceding weeks and significant amounts of surface vegetation
were apparent, particularly in the wadis. A second visit was made by BGS in November–December 2009. This was primarily to undertake biological
baseline measurements for which supporting soil gas and flux measurements were made [8] The aims were to
conduct a botanical survey and to assess microbial numbers and activity. The biological work centred on the three
injection wells. A traverse was made from KB-5 south-eastwards towards KB-502, observations were made around
KB-503, and on a traverse westwards from that well, and a traverse was undertaken north-eastwards from KB-501 Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3568 (Figure 1). The opportunity was also taken to make further atmospheric CO2 measurements in the areas of the
injectors and measurements at the background site. (Figure 1). The opportunity was also taken to make further atmospheric CO2 measurements in the areas of the
injectors and measurements at the background site. Gas Measurements The mobile laser data were plotted using Jenks’ Natural Breaks in ArcMap v9.2 to define the different classes. This identifies break points by picking the class breaks that best group similar values and maximise the differences
between classes by iteratively comparing sums of the squared difference between observed values within each class
and class means. Traverses with the mobile laser were first made on the plateau around KB-502. They were then extended to the
area between KB-502 and KB-5 to cover the area of uplift and look for any surface manifestation of the known
migration of CO2 between the two wells [1]. The majority of data values were close to a ‘normal’ atmospheric level of around 380 ppm CO2 with the
differences between classes being only small (a few tens of ppm). The highest values, and only marked changes in
CO2 concentration, to in excess of 500 ppm, were seen around the KB-502 well itself and were clearly related to the
vehicle exhaust being blown forward into the laser probe (Figure 2). Thus, with the wind coming from the south to
south west, higher values were seen on the NE side of the well on a clockwise circuit and on the west side on an
anticlockwise circuit. Similar effects were seen elsewhere and could not always be avoided despite careful planning
of the direction of survey lines. Mobile laser measurements were made near KB-5 and then in the accessible parts of the wadi floor between KB-
5 and KB-502. A number of circuits were made around the outer perimeter of the KB-5 well pad. At certain points
rapid increases in CO2 concentration were observed, but these were almost certainly from the vehicle exhaust. However, more subtle variations (several tens of ppm) were also seen and these appeared, from examination of the
data in the field, to occur consistently in an area to the SE of the well on different circuits. Static measurements were
therefore made at this location with the vehicle engine switched off (Figure 3). Initially there were slightly higher
values (400–420 ppm) but they tailled off over time to 380–400 ppm. Soil gas and flux measurements were made at
this location and around the rest of the well pad, but showed no signs of higher CO2. Biological studies In order to cover the large areas needed for the surveys, widely spaced (100–200 m) sample points were used on
each biological transect. Given the sparse vegetation in the desert setting, sampling points generally targeted
vegetated areas in order to carry out a baseline botanical and microbiological survey. As such, the sampling points
are not representative of the wider area, but hopefully reflect the flora present. A handheld GPS receiver was used to
locate the sampling points. Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3569 At each sample location, a 0.5 x 0.5 m quadrat survey was taken on a 2.5 m cross from a central point (where the
soil samples and gas measurements were normally taken). For each quadrat the percentage area covered by each
plant was estimated and photographs and samples taken to aid species identification. Soils were sampled with a Dutch auger, at 10–20 cm, 30–40 cm and, where possible, 40–50 cm depth. The auger
head was cleaned with disinfectant wipes to minimise contamination. Sub-samples were taken for immediate
analysis to evaluate microbial biomass by adenosine tri phosphate (ATP) assay using a Deltatox analyser and also
preserved for determination of microbial numbers using epifluorescence microscopy in BGS laboratories. Further
details of the methods can be found in Smith et al. 2010 [8]. Gas Measurements Similar apparently higher values were also observed over the area of uplift to the east of KB-5 but once again the
values fell when observed over a longer time period. It seems likely that such fluctuations of 30–40 ppm are within
the natural diurnal variation and this needs to be considered when trying to detect a leakage signal above
background. Soil gas and flux measurements at this location gave low values (0.04 % CO2 and negative flux). Traverses with the open path laser around the KB-4 well showed changes that could be attributed to rainfall
affecting the measurements both directly and through splash-up of dirt onto the laser optics. Other than differences
seen during and after rainfall no obvious elevated atmospheric CO2 values were seen. However, soil gas CO2
readings, although low did appear to be somewhat higher than elsewhere ranging from 0.16–0.27 % for lab results
and 0.06–0.19 % for the most sensitive field instrument. This contrasts with the majority of sites where most of the
data were close to atmospheric levels (0.03–0.05 %). A series of lines were run with the open path laser in a background area to the west of the gas field. These
covered the same area where point measurements were made in 2004 [6]. Surface gas/flux measurements were
repeated at similar locations to 2004. It also rained during these traverses, which had some impact on the laser
results. The field soil gas and flux measurements were all very low at the background sites, with CO2 concentrations
at or only slightly higher than atmospheric levels and flux levels no higher than 5 g m2 d1. Some additional gas data were obtained during the November-December visit, although this was focussed
primarily on biological observations and associated gas measurements. The atmospheric data obtained with the laser
system were affected by the very dry, dusty and rather breezy conditions. Some vehicle exhaust effects were seen, Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3570 but a more serious problem was the varying amounts of airborne dust both within the laser beam and settling on the
external optical interfaces. It was hard to separate the effect of dust from real variations in atmospheric CO2 content. Cleaning the probe windows had only a relatively minor effect, suggesting that dust in the air had more of an
influence on the data. Biological studies All the plants observed were typical desert flora, identified using Ozenda (2004) [9] and Sahara-Nature.com, and
all were seed-formers (Spermatophyta). A variety of angiosperm (flowering plant) families were detected (e.g. Compositaceae, Graminaceae, Brassicaceae) with both monocotyledonous (e.g. Graminaceae) and dicotyledonous
(e.g. Compositaceae) groupings present. One gymnosperm family was also observed (Ephedra spp). All exhibit
typical xerophytic adaptations (reduced leaves, thick stems) necessary for life in an arid environment. The Krechba
area was sparsely vegetated at the time of this visit, with large expanses of ground completely free from visible
flora. In any given sampling area, vegetation cover (Figure 4) was never higher than 50%, and was commonly
around 10%. Vegetation was more plentiful in dry wadi channels, where there may be water at depth and in shallow
depressions where water would collect temporarily, as seen on the transects between KB-5 and KB-502 and to the
east of KB-503. The KB-501 transect, on the plateau, provided conditions more representative of background for the
region. The dicotyledenous angiosperm (Hammada spp) dominated here with other unidentified plants . The ATP results appear to have been affected by a contaminated blank giving rise to negative values. This
implies the samples had a disinfecting quality and/or the change in pH caused by adding sample to the de-ionised
water produced conditions outside the tolerance range of the microbial population. The ATP results appear to have been affected by a contaminated blank giving rise to negative values. This
implies the samples had a disinfecting quality and/or the change in pH caused by adding sample to the de-ionised
water produced conditions outside the tolerance range of the microbial population. 5
1
6
1 In general, microbial counts averaged ~105 g1 at 10–20 cm depth although ~106 g1 were observed in two
samples (Figure 4). However, some samples had so few organisms present that it was not possible to obtain
meaningful results. At 30–40 cm depth numbers vary between ~104 g1 to ~105 g1 although fewer samples were
taken because of sampling difficulties. Even fewer samples were taken from 40–50 cm but numbers remain at ~105
g1 where counts were possible. Two samples had zero counts meaning that the samples from these sites were
devoid of microbial life. Soil gas concentrations on the transects at 90 cm depth were very low; most values were at or below atmospheric
levels (Figure 4). Biological studies The exceptions were 2 sites on the KB-5 to KB-502 line and 2 on the KB-503 transect, which
reached 0.09 to 0.15% CO2. Fluxes were also low, mostly less than 2 g m1 d1 (Figure 4). Higher values (5–9 g m2
d1) were seen at a few points on the KB-5 to 502 and KB-503 transects. Gas Measurements However, certain lines where windblown dust should have been less of a factor, owing to the
strength and direction of the wind, suggest that higher CO2 concentrations (in excess of 500 ppm) are possible in this
kind of environment. The only soil gas CO2 values above 1.0 % were seen directly over the KB-5 well, in unconsolidated sand in a
trench at the wellhead. It is most likely that they are a relict of breakthrough prior to the full abandonment of the
well. Some slightly higher values (0.5–0.8 % CO2) was also seen in the bottom of the 2 m deep pit dug for the
Barasol probe to the SE of KB-502. These may indicate true gas concentrations, free from atmospheric dilution, as
they are from holes drilled into bedrock rather than in highly permeable sand and gravel. Data were only obtained from the Barasol radon probe near KB-502; the probe near KB-7, intended to measure
background values was faulty. Results were obtained from 27/03/2009 until 01/03/2010. The raw data show a
narrow pressure range (942 to 967 mBar) and wider temperature variation (12.1 to 31.3°C). The average Rn value
was 1.45 kBq.m-3 with small peaks in the range 6-14 kBq.m-3. Preliminary analysis does not suggest CO2 leakage. The passive sampler (activated charcoal) leachates display slight contamination either by carbonate (Ca, Mg),
clay and/ or sand (Si, Al). There were no indications of deep transport by CO2 on an initial appraisal. The passive sampler (activated charcoal) leachates display slight contamination either by carbonate (Ca, Mg),
clay and/ or sand (Si, Al). There were no indications of deep transport by CO2 on an initial appraisal. 4. Discussion It is clear from experiences with the mobile open path laser system that the readings can be affected by vehicle
exhaust gases, rain, dust in the atmosphere and dust or dirt on the windows of the laser probe. The impact of exhaust gases can be minimised by running lines at right angles to the wind direction and moving
upwind. However, the terrain, or the need to cover a particular feature (e.g. the area of uplift) may constrain the line
direction. The only way to totally eliminate exhaust problems would be to use an electric vehicle, but this is not a Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3571 viable option at Krechba. An alternative is to extend the tail pipe of the vehicle exhaust to increase the likelihood
that the gases are mixed and dispersed away from the laser probe. Dust in the atmosphere and on the windows of the laser probe can cause higher apparent readings. Alternatively,
in wet conditions, the lenses can get muddy because of splash-up from the ground and this can cause reduced values
and noisy data, which are only rectified by cleaning. A dust cover for the probe is being considered although this
might reduce the response time. Natural background atmospheric CO2 concentrations vary during the day and this needs to be considered. Measurements of CO2 fluxes have been made at different sites around the world, normally with eddy covariance
techniques. However, although these studies involve measuring the CO2 concentration and flux at high frequencies
(e.g. 10 Hz) the data are more often reported in terms of net long term fluxes. The short term variability is not
therefore readily obtainable from publications. Measurements are usually made at a height of 2 m or greater and so
may not be indicative of values closer to the ground. Also it is more usual for the readings to be made in vegetated
areas, such as grasslands or over forest canopies. There appear to be few data for desert areas with very sparse
vegetation cover. g
A better understanding of natural variability in near ground CO2 values would help greatly in identifying gas
anomalies. It could then be used to determine methods for identifying anomalies. 4. Discussion The indications at present, from
the data collected so far at Krechba, would suggest that atmospheric CO2 varies on the timescale of a few minutes
and longer. Thus a strategy to identify anomalies could involve looking for shorter term changes in CO2
concentration (seconds to tens of seconds) and ignore longer term trends. This would, however, require the
elimination of the other effects of vehicle exhaust, rain and dust. Shallow soil gas and flux measurements are problematical at Krechba because of the hard ground, although it is
possible, with patience, to get measurements in most places. The highly permeable dry gravel and sand adds a real
risk of atmospheric gas being drawn down through the substrate into the analyser. A solution to this problem would be increasing the depth of sampling. A network of shallow boreholes (5–10 m
depth) was proposed after an initial visit to Krechba [6]. However, this approach is not readily amenable to the rapid
investigation of an atmospheric CO2 anomaly. Soil gas and flux data from the two visits show clearly the higher CO2 concentrations in March in one of the
Barasol pits. These were taken in bedrock and thus less susceptible to atmospheric dilution. They may better reflect
the true ground CO2 concentrations. Laboratory data were somewhat higher and this may be because the sampling
method (involving withdrawal of a much smaller volume of gas than the in situ measurements) minimised
atmospheric dilution. Other field data show CO2 concentrations at or below atmospheric levels except for sites near
KB-4, measured under wet conditions that might have reduced atmospheric ingress. Fluxes in March were mostly
below 5 g m1 d1. Data from the later visit show the anomalous nature of the CO2 concentrations measured at the KB-5 wellhead,
which are also reflected by some of the higher flux readings. These suggest that the KB-5 data merit further
investigation as they could indicate leakage from the well. Other sites showed somewhat higher CO2 concentrations
and fluxes than seen in March (albeit still very low values) and these may reflect biological production as a bias
towards vegetated areas was necessary for the botanical work. In comparison with more vegetated sites from temperate regions the soil gas values are lower by at least an order
of magnitude. 4. Discussion The flux rates are also lower in general by an order of magnitude although the highest values (> 1 g
m1 d1) overlap with typical autumnal temperate flux rates. All the results from this study provide baseline data
after more than 2 months without rain (TuTiempo.net, 2010). The botanical study revealed a wide range of plants at Krechba, all of which exhibited xerophytic characteristics
(e.g. reduced leaves, thick stems) necessary to survive in the arid conditions. All those observed were seed-formers
(Spermatophyta). A variety of angiosperm (flowering plant) families were detected with both monocotyledonous
and dicotyledonous groupings present. Sensitivities to exposure between monocotyledonous and dicotyledeonous
plants to different CO2 soil gas concentrations have been demonstrated in temperate climates [10, 11] so there is the
potential for these same plant groupings to have similar sensitivities in this desert environment. It is likely that, after rain, short-lived annuals would appear which would rapidly complete their cycle, producing
seeds to survive through the drought until the next rain event [9]. Characterisation of these plant families and species
would only be possible during and immediately after rain which often falls in March/April. 3572 Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 In general, microbial numbers observed are typical of desert aerobic microbial populations [12] where numbers
vary from <10 (Atacama Desert) to 1.6 x 107 g1 in soils of Nevada. Microbial activity in this environment is highly
dependent on the availability of moisture but other factors, such as temperature and nutrient availability, will also
play a role. It would be useful to carry out the same evaluation after a period of rain to ascertain the microbiological
background under these conditions, whilst also observing the change in the flora. In general, microbial numbers observed are typical of desert aerobic microbial populations [12] where numbers
vary from <10 (Atacama Desert) to 1.6 x 107 g1 in soils of Nevada. Microbial activity in this environment is highly
dependent on the availability of moisture but other factors, such as temperature and nutrient availability, will also
play a role. It would be useful to carry out the same evaluation after a period of rain to ascertain the microbiological
background under these conditions, whilst also observing the change in the flora. This study did not establish the types of microbes present – only the numbers present. 4. Discussion It is extremely likely
further analysis of the composition of the population will reveal a complex ecosystem as suggested in other studies
[e.g. 12–14]. A qualitative conclusion from the problematical ATP measurements was that the microbial ATP in
Krechba samples is generally low (below the contaminated blank in all cases). The data presented in this study represent a baseline survey of plant life, plant coverage and microbial activity in
the soils of the Krechba site. Soil gas data do not indicate any CO2 leakage except possibly at KB-5. However, the
higher values there are not likely to reflect continuing leakage as the well has now been completely sealed and
abandoned. This could be verified by further measurements. As expected for a hot, dry, low nutrient ecosystem,
microbial populations and total vegetative cover were low. Plant life in particular exploits topographic lows,
presumably to make better use of the little water available. In spite of the environmental factors and scarcity of
vegetation, plant diversity is relatively high, with monocotyledonous and dicotyledonous angiosperms, and
gymnosperms represented by a number of species. References [1] Mathieson, A, Midgley, J, Dodds, K, Wright, I, Ringrose, P and Saoul, N.. CO2 sequestration monitoring and
verification technologies applied at Krechba, Algeria. The Leading Edge, 2010,. 29, 216-22. [1] Mathieson, A, Midgley, J, Dodds, K, Wright, I, Ringrose, P and Saoul, N.. CO2 sequestr
verification technologies applied at Krechba, Algeria. The Leading Edge, 2010,. 29, 216-22 verification technologies applied at Krechba, Algeria. The Leading Edge, 2010,. 29, 216-22. [2] Mathieson, A, Wright, I, Roberts, D and Ringrose, P.. Satellite imaging to monitor CO2 movement at Krechba,
Algeria. Energy Procedia, 2009, 1, 2201-9. [2] Mathieson, A, Wright, I, Roberts, D and Ringrose, P.. Satellite imaging to monitor CO2
Algeria. Energy Procedia, 2009, 1, 2201-9. [3] Onuma, T and Ohkawa, S.. Detection of surface deformation related with CO2 injection by DInSAR at In Salah,
Algeria. Energy Procedia, 2009, 1, 2177-84. [4] Vasco, DW, Alessandro, F And Fabrizio, N.. Reservoir monitoring and characterization using satellite geodetic
data: Interferometric synthetic aperture radar observations from the Krechba field, Algeria. Geophysics, 2008, 73,
WA113-22. [5] Rutqvist, J, Vasco, DW and Myer, L.. Coupled reservoir-geomechanical analysis of CO2 injection and ground
deformations at In Salah, Algeria. International Journal of Greenhouse Gas Control, 2010, 4, 225-30. [6] Jones, DG and Annunziatellis, A. Soil gas feasibility study at In Salah, Algeria. British Geological Survey
Commissioned Report, CR/04/260, 2004, pp. 30. [7] World Resources Institute. CCS Guidelines: Guidelines for Carbon Dioxide Capture, Transport, and Storage. Washington, DC: World Resources Institute, 2008. [8] Smith, DJ, West, JM, Jones, DG, Coombs, P, And Lister, TR. Baseline botany and microbiology survey results
from the Krechba area (In Salah Gas Project). British Geological Survey Internal Report, CR/10/051, 2010, pp. 44. [9] Ozenda, P. Flore et vegetation du Sahara. 3rd Edition. Paris: CNRS, 2004 [10] Beaubien, SE, Ciotoli, G, Coombs, P, Dictor, MC, Krüger, M, Lombardi, S, Pearce, JM, and West, JM. The
impact of a naturally occurring CO2 gas vent on the shallow ecosystem and soil chemistry of a Mediterranean
pasture (Latera, Italy). International Journal of Greenhouse Gas Control, 2008, 2, 373-87 [11] West, JM, Pearce, JM, Coombs, P, Ford, Jr, Scheib, C, Colls, JJ, Smith, KL, and Steven,MD.. The impact of
controlled injection of CO2 on the soil ecosystem and chemistry of an English lowland pasture. Energy Procedia,
2009, 1, 1863-70 [12] Bhatnagar, A, and Bhatnagar, M.. Microbial diversity in desert ecosystems. Acknowledgements g
This study was carried out under the EC FP6 project CO2ReMoVe. We are grateful for the support of In Salah
Gas, BP and Statoil and in particular, John Midgley, Nabil Saoul and Allan Mathieson. g
This study was carried out under the EC FP6 project CO2ReMoVe. We are grateful for the support of In Salah
Gas, BP and Statoil and in particular, John Midgley, Nabil Saoul and Allan Mathieson. [14] Thomas, AD, and Hoon, SR.. Carbon dioxide fluxes from biologically-crusted Kalahari Sands after simulated
wetting. Journal of Arid Environments, 2010,. 74, 131-9 References Current Science, 2005, 89, 91-10
[13] Shamir, I, and Steinberger, Y.. Vertical distribution and activity of soil microbial population in a sandy desert
ecosystem. Microbial Ecology, 2007, 53, 340-7 [14] Thomas, AD, and Hoon, SR.. Carbon dioxide fluxes from biologically-crusted Kalahari Sands after simulated
wetting. Journal of Arid Environments, 2010,. 74, 131-9 Author name / Energy Procedia 00 (2010) 000–000
D.G. Jones et al. / Energy Procedia 4 (2011) 3566–3573 3573 8
Author name / Energy Procedia 00 (2010) 000 000
Figure 1. Location map of Krechba field on Quickbird satellite
image with main study areas. Bar-A and Bar-B are Barasol
locations. Solid red dots are passive sensor locations. Inset map
shows location of Krechba in Algeria
Figure 2. Data for near ground atmospheric CO2 (concentrations in
ppm) from mobile open path laser measurements around KB-502. Note higher values to the east and NW of the well due to vehicle
exhaust affecting measurements with the laser probe
Figure 3. Static measurement of near ground atmospheric CO2 SE
of KB-5 in area of apparently higher CO2. Note decline with time
Figure 4. Comparison of microbial counts, soil CO2 concentration
and plant coverage datasets. Kb-503
Bar-A
Kb-502
Kb-501
Bar-B
Background
Kb-4
250
300
350
400
450
500
83524
84844
90204
CO2 (ppm)
Time (hmmss)
KB-5_4: CO2
gy
(
) Figure 3. Static measurement of near ground atmospheric CO2 SE
250
300
350
400
450
500
83524
84844
90204
CO2 (ppm)
Time (hmmss)
KB-5_4: CO2 Figure 1. Location map of Krechba field on Quickbird satellite
image with main study areas. Bar-A and Bar-B are Barasol
locations. Solid red dots are passive sensor locations. Inset map
shows location of Krechba in Algeria
Kb-503
Bar-A
Kb-502
Kb-501
Bar-B
Background
Kb-4 Kb-503
Bar-A
Kb-502
Kb-501
Bar-B
Background
Kb-4 Figure 3. Static measurement of near ground atmospheric CO2 SE
of KB-5 in area of apparently higher CO2. Note decline with time Figure 3. Static measurement of near ground atmospheric CO2 SE
of KB-5 in area of apparently higher CO2. Note decline with time Figure 4. Comparison of microbial counts, soil CO2 concentration
and plant coverage datasets. Figure 4. Comparison of microbial counts, soil CO2 concentration
and plant coverage datasets. Figure 1. Location map of Krechba field on Quickbird satellite
image with main study areas. Bar-A and Bar-B are Barasol
locations. Solid red dots are passive sensor locations. Inset map
shows location of Krechba in Algeria
Figure 2. References Data for near ground atmospheric CO2 (concentrations in
ppm) from mobile open path laser measurements around KB-502. Note higher values to the east and NW of the well due to vehicle
exhaust affecting measurements with the laser probe
Figure 4. Comparison of microbial counts, soil CO2 concentration
and plant coverage datasets. Figure 1. Location map of Krechba field on Quickbird satellite
image with main study areas. Bar-A and Bar-B are Barasol
locations. Solid red dots are passive sensor locations. Inset map
shows location of Krechba in Algeria Figure 2. Data for near ground atmospheric CO2 (concentrations in
ppm) from mobile open path laser measurements around KB-502. Note higher values to the east and NW of the well due to vehicle
exhaust affecting measurements with the laser probe Figure 4. Comparison of microbial counts, soil CO2 concentration
and plant coverage datasets. Figure 2. Data for near ground atmospheric CO2 (concentrations in
ppm) from mobile open path laser measurements around KB-502. Note higher values to the east and NW of the well due to vehicle
exhaust affecting measurements with the laser probe
|
https://openalex.org/W4388302591
|
https://imn.ac.id/pastabiq/index.php/pastabiq/article/download/40/17
|
Indonesian
| null |
MERDEKA DARI SAMPAH, SEBAGAI PRAKTIK SOSIAL DALAM KEHIDUPAN SOSIAL MASYARAKAT PEDULI SAMPAH
|
Pastabiq
| 2,023
|
cc-by-sa
| 2,930
|
Pastabiq : Jurnal Pengabdian kepada Masyarakat
ISSN 2828-8556 (print) | ISSN 2829-2685 (online)
Vol. 2, No. 1, April, 2023, pp. 19-25
https://doi.org/10.56223/pastabiq.v2i1.40 Pastabiq : Jurnal Pengabdian kepada Masyarakat
ISSN 2828-8556 (print) | ISSN 2829-2685 (online)
Vol. 2, No. 1, April, 2023, pp. 19-25
https://doi.org/10.56223/pastabiq.v2i1.40 Abstrak Sampah plastik berkontribusi terhadap kerusakan lingkungan dan
merupakan salah satu masalah utama di Indonesia. Hal ini disebabkan
pertumbuhan penduduk dan kurangnya kesadaran masyarakat, serta daur
ulang sampah plastik. Salah satunya terjadi di Kecamatan Cijeruk di Desa
Cipelang, karena minimnya lahan untuk pembuangan sampah, banyak
masyarakat yang mulai membuang sampah di sungai atau di dekat
pemukiman penduduk. Artikel ini bertujuan untuk memberikan informasi
pembuangan sampah yang meningkatkan kreativitas masyarakat di desa
Cipelang yaitu cara mengolah sampah plastik. Cara ini dilakukan dengan
membuat kerajinan dan karya dari botol bekas berupa Vas Bunga Cantik. Oleh karena itu, cara ini efektif membantu mengurangi sampah plastik. Artikel ini bertujuan untuk memberikan informasi untuk membantu
mengurangi sampah plastik di desa Cipelang dan mendorong kreativitas
masyarakat untuk memanfaatkan sampah dengan lebih baik dan menjaga
kelestarian lingkungan. Abstract Plastic waste contributes to environmental damage and is one of the main
problems in Indonesia. This is due to population growth and lack of public
awareness, as well as the recycling of plastic waste. One of them occurred in
Cijeruk District in Cipelang Village, due to the lack of land for garbage
disposal, many communities have begun dumping garbage in rivers or near
residential areas. This article aims to provide information on waste disposal
that increases the creativity of the community in Cipelang village, namely
how to process plastic waste. This method is done by making crafts and
works from used bottles in the form of Beautiful Flower Vases. Therefore, this
method effectively helps to reduce plastic waste. This article aims to provide
information to help reduce plastic waste in Cipelang village and encourage
community creativity to make better use of waste and preserve the
environment. Diajukan:
15 September 2022
Diterima:
30 April 2023
Diterbitkan:
30 April 2023 MERDEKA DARI SAMPAH, SEBAGAI PRAKTIK SOSIAL DALAM
KEHIDUPAN SOSIAL MASYARAKAT PEDULI SAMPAH Muhamad Rahman Halim*, Asep Maulana
Institut Madani Nusantara, Sukabumi, Indonesia
*e-mail : rahmanhlm061@gmail.com Info Artikel
Abstract
Diajukan:
15 September 2022
Diterima:
30 April 2023
Diterbitkan:
30 April 2023
Keywords:
KKN,
Plastic Waste,
Bottle, Trash
Kata Kunci:
KKN,
limbah Plastik,
Botol Plastik. Plastic waste contributes to environmental damage and is one of the main
problems in Indonesia. This is due to population growth and lack of public
awareness, as well as the recycling of plastic waste. One of them occurred in
Cijeruk District in Cipelang Village, due to the lack of land for garbage
disposal, many communities have begun dumping garbage in rivers or near
residential areas. This article aims to provide information on waste disposal
that increases the creativity of the community in Cipelang village, namely
how to process plastic waste. This method is done by making crafts and
works from used bottles in the form of Beautiful Flower Vases. Therefore, this
method effectively helps to reduce plastic waste. This article aims to provide
information to help reduce plastic waste in Cipelang village and encourage
community creativity to make better use of waste and preserve the
environment. Abstrak
Sampah plastik berkontribusi terhadap kerusakan lingkungan dan
merupakan salah satu masalah utama di Indonesia. Hal ini disebabkan
pertumbuhan penduduk dan kurangnya kesadaran masyarakat, serta daur
ulang sampah plastik. Salah satunya terjadi di Kecamatan Cijeruk di Desa
Cipelang, karena minimnya lahan untuk pembuangan sampah, banyak
masyarakat yang mulai membuang sampah di sungai atau di dekat
pemukiman penduduk. Artikel ini bertujuan untuk memberikan informasi
pembuangan sampah yang meningkatkan kreativitas masyarakat di desa
Cipelang yaitu cara mengolah sampah plastik. Cara ini dilakukan dengan
membuat kerajinan dan karya dari botol bekas berupa Vas Bunga Cantik. Oleh karena itu, cara ini efektif membantu mengurangi sampah plastik. Artikel ini bertujuan untuk memberikan informasi untuk membantu
mengurangi sampah plastik di desa Cipelang dan mendorong kreativitas
masyarakat untuk memanfaatkan sampah dengan lebih baik dan menjaga
kelestarian lingkungan. Pendahuluan Sampah padat adalah sisa padat kegiatan manusia sehari-hari dan/atau proses
alam (Suyoto, 2008). Tingkat Timbunan Sampah Tidak Hanya Terus Meningkat Tidak
hanya sejalan dengan pertumbuhan penduduk, tetapi juga sejalan dengan pola
konsumsi masyarakat yang semakin meningkat. Di sisi lain, kapasitas pembuangan
sampah masyarakat dan kota belum optimal. Sampah yang tidak dibuang dengan benar
berdampak pada kesehatan lingkungan dan masyarakat sekitar (Riswan et al., 2015)
Masalah sampah merupakan masalah yang cukup rumit diakibatkan karena kurangnya
pengertian masyarakat terhadap akibat – akibat yang dapat ditimbulkan oleh sampah. Oleh karena itu bila tidak ditangani secara benar, maka akan menimbulkan dampak
seperti pencemaran air, udara, dan tanah yang mengakibatkan sumber penyakit. Sampah sebagai barang yang amsih bisa dimanfaatkan tidak seharusnya diperlakukan Pastabiq : Junal Pengabdian kepada Masyarakat I 19 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana sebagai barang yang menjijikan, melainkan harus dapat dimanfaatkan sebagai bahan
mentah atau bahan lainnya (Sarlia, 2020) Sampah adalah hasil buangan kegiatan manusia yang sudah tidak berguna lagi. Sampah bukan hanya benda padat yang dibuang begitu saja tanpa ada manfaatnya. Sampah melibatkan tiga prinsip yang harus dipenuhi: keberadaan suatu benda atau
benda padat, hubungan langsung dan tidak langsungnya dengan aktivitas manusia, dan
benda tersebut sudah tidak digunakan lagi.(Nababan, 2007). Sampah yang tidak
dikelola dengan baik dapat memiliki konsekuensi kesehatan yang serius. Efek langsung
disebabkan oleh kontak langsung orang ke orang karena adanya vektor yang
menularkan patogen penghasil sampah ke manusia (Muhammad, 2017). Pengelolaan sampah rumah tangga dan sampah rumah tangga sejenis
berdasarkan Undang-Undang Nomor 18 Tahun 2008 tentang Pengelolaan Sampah. Terdiri dari pengurangan sampah dan pengelolaan sampah. Pengurangan sampah
meliputi kegiatan pengurangan timbulan sampah, daur ulang sampah, dan pemanfaatan
kembali sampah. Salah satu kegiatan pengelolaan sampah adalah pemilahan, dimana
sampah dikelompokkan dan dipilah berdasarkan jenis, jumlah dan jenisnya. Pelaksanaan pengelolaan sampah menurut Peraturan Pemerintah Nomor 81 Tahun
2012 tentang Pengelolaan Sampah Rumah Tangga dan Sampah Sejenis Sampah Rumah
Tangga meliputi pengurangan sampah dan pengelolaan sampah yang harus dilakukan
oleh semua pihak (Brunner, 2016). Menurut Lawrence Green yang dikutip dalam Notoatmodjo (2015), perilaku
kesehatan seseorang dipengaruhi oleh tiga faktor yaitu predisposisi, dukungan dan
penguatan. Faktor
predisposisi
meliputi
tingkat
pengetahuan,
sikap,
tradisi
kepercayaan, tingkat pendidikan, dan tingkat sosial ekonomi. Faktor pendukungnya
adalah ketersediaan sarana dan prasarana kesehatan, dan kinerja ekonomi. Pastabiq : Junal Pengabdian kepada Masyarakat I 20 Pendahuluan Faktor
penguat terdiri dari sikap tokoh masyarakat, peran tenaga kesehatan, dan kebijakan
kesehatan.Pengetahuan masyarakat tentang pengelolaan sampah sebagian besar baik,
namun tidak semua orang yang memiliki pengetahuan yang cukup melakukan hal yang
benar dalam pengelolaan sampah. Perubahan perilaku atau penerimaan perilaku baru
melewati tiga fase proses: pengetahuan, sikap, dan perubahan praktik. Berdasarkan
pengalaman dan penelitian, perilaku berbasis pengetahuan terbukti lebih persisten
dibandingkan perilaku yang tidak berbasis pengetahuan (Notoatmodjo, 2015). Upaya pengelolaan sampah yang dapat mempengaruhi sikap masyarakat tentang
pengolahan dan pengelolaan sampah dipantau, ditegur bila ada sikap yang salah, dan
bertanggung jawab untuk meninjau ulang agar sampah tidak menumpuk diharapkan
dapat ditugaskan.Sikap mempengaruhi perilaku masyarakat. Saya berharap bahwa
sikap yang baik mengarah pada perilaku yang baik, meskipun tidak selalu. Kurangnya
fasilitas dapat mempengaruhi pembuangan sampah yang tidak tepat. Hal ini dibuktikan
dengan penelitian yang menunjukkan bahwa mayoritas masyarakat yang memiliki sikap
negatif cenderung lebih banyak melakukan tindakan membuang sampah dengan cara
yang tidak baik (Notoatmodjo, 2015). g
Dengan produksi kemasan skala besar, pengelolaan sampah menjadi semakin
penting.dasar plastik. Namun, saat membuang sampah plastik, diperlukan metode yang
tepat dan rasional agar aman bagi lingkungan. Jumlah sampah plastik semakin
meningkat dari tahun ke tahun, dan sampah plastik bersifat anorganik dan sulit terurai. Sampah plastik yang terurai juga tidak sempurna karena berubah bentuk menjadi
mikroplastik yang nantinya dapat merusak sumber daya alam. Pengolahan sampah
plastik dapat dilakukan dengan berbagai cara.Nilai 3R (Reuse, Reduce, Recycle). Ada
banyak hal yang bisa kita manfaatkan atau atasi dengan mengurangi ketergantungan Pastabiq : Junal Pengabdian kepada Masyarakat I 2 I 20 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana kita pada plastik. Contoh penerapan proses daur ulang atau daur ulang adalah
pengolahan botol plastik. Botol plastik yang sulit terurai bisa menjadi ladang kreatif,
termasuk membuat kreasi yang bermanfaat(Muis et al., 2022). kita pada plastik. Contoh penerapan proses daur ulang atau daur ulang adalah
pengolahan botol plastik. Botol plastik yang sulit terurai bisa menjadi ladang kreatif,
termasuk membuat kreasi yang bermanfaat(Muis et al., 2022). g
Masalah sampah yang kami dapat saat pengabdian kepada masyarakat di desa
yang kami tinggali sebagai peserta KKN IMN Sukabumi tahun 2022. Kami mendapatkan
informasi yang sangat penting terkait dengan Judul Artikel yang kami buat tentang
masalah sampah di daerah yang kami tinggali. Pendahuluan Masih banyak warga atau masyarakat
yang belum tau dampak dari membuang sampah kesungai dapat mengakibatkan
terjadinya berbagai dampak yang terjadi pada dirinya nantinya entah itu pencemaran
penyakit yang menyebar ke lingkungan masyarakat setempat. Juga menjadi dampak
bagi rumah yang dekat dengan sungai yang mengakibatkan longsor dan lain – lain. Dan
kami datang bukan hanya sekedar untuk bertamu dan berbincang ria. kami datang
dengan tujuan yang akan membawa kegiatan bermanfaat bagi masyarakat setempat dan
juga bagi warga desa yang kami kunjungi sebagai tempat pengamdian kepada
masyarakat mahasiswa/i IMN Sukabumi tahun 2022 yang membawa perubahan bagi
masyarakat setempat. Berdasarkan uraian di atas maka peneliti merumuskan masalah
yaitu faktor – faktor apa saja yang berhubungan dengan perilaku pengelolaan sampah
rumah tangga dan lingkungan sekolah berdasarkan penelusuran artikel ilmiah ?. Kami mendapatkan ide untuk mengelola sampah untuk dikelola dan
dikembangkan Bersama masyarakat Kp. Babakan Jampang membuat kreasi seni yang
indah untuk dipandang dan untuk dirawat dengan sebaik – baiknya. Dan mengahsilkan
karya seni yang berkelanjutan dan mambawa perubahan yang tadinya banyak sampah
menjadi kurangnya sampah yang ada di lingkungan setempat. Tujuan umum penelitian
ini adalah untuk mengetahui faktor – faktor yang berhubungan dengan perilaku
pengelolaan sampah rumah tangga dan lingkungan sekolah berdasarkan penelusuran
artikel ilmiah. Tujuan khusus yaitu gambaran tingkat pengetahuan, gambaran sikap,
gambaran sarana dan prasarana, dan juga hubungan antara tingkat pengetahuan, sikap
dan hubungan sarana dan prasarana dengan perilaku pengolahan sampah rumah
tangga dan lingkungan sekolah di daerah setempat. Metode Kegiatan pembuatan Vas Bunga botol dari sampah plastik berupa botol
Berkreasilah dengan mengurangi sampah plastik dan memanfaatkan sampah di
sekitar kita, terutama sampah plastik yang ada di sekitar kita. Misalnya, mendaur
ulang botol plastik menjadi tempat vas bunga yang lebih bermanfaat dan lebih
indah dipandang. Kerajinan ini memiliki beberapa tahap: 1. Tahap persiapan Tahap persiapan yang harus dilakukan pada tahap ini adalah
menyiapkan alat dan bahan untuk membuat tempat sampah dari limbah botol
plastik. Silakan kumpulkan sampah botol plastik terlebih dahulu lalu bersihkan. Alat dan bahan tambahan seperti gunting, tang, kawat besi, , lilin, korek api untuk
merakit botol plastik, serta alat dan bahan yang mempercantik penampilan seperti
cat, kuas, sikat. 2. Tahap Pelaksanaan Pada tahap ini terdapat tahap pembuatan tempat vas bunga
dari sampah plastik. Produk yang dihasilkan digunakan sebagai tempat vas bunga. Kami memilih produk ini karena mengurangi limbah lingkungan dan meningkatkan
kreativitas. 3. Tahap Akhir Tahap akhir ini meliputi penyusunan laporan akhir berdasarkan
kegiatan yang dilakukan selama tahap implementasi. Karya ini akan 3. Tahap Akhir Tahap akhir ini meliputi penyusunan laporan akhir berdasarkan
kegiatan yang dilakukan selama tahap implementasi. Karya ini akan Pastabiq : Junal Pengabdian kepada Masyarakat I 21 Pastabiq : Junal Pengabdian kepada Masyarakat I 21 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana dipublikasikan melalui distribusi ke SDN Cipelang 2, selain laporan akhir. Has dipublikasikan melalui distribusi ke SDN Cipelang 2, selain laporan akhir. Hasil Hasil dan Pembahasan Masalah sosial yang berkembang dari limbah plastik berlebih memiliki
konsekuensi yang menghancurkan di planet kita, merenggut banyak nyawa. Akibat
sanitasi yang buruk, banyak orang, terutama di Indonesia, menderita penyakit seperti
penyakit kulit, malaria, dan demam berdarah. Upaya pemusnahan sampah plastik
seperti pembakaran plastik juga berdampak buruk bagi kesehatan, dan tidak semua
sampah plastik terurai dan hilang dengan baik. Asap yang dihasilkan selama proses
pembakaran mengandung karbon dan dapat menyebabkan penyakit kronis seperti
kanker dan gangguan internal lainnya. Oleh karena itu, masyarakat ditantang untuk
mencari solusi permasalahan sampah plastik. Salah satu inisiatif untuk mengurangi
sampah plastik adalah program 3R (Reduce or Reduce, Reuse or Reuse, Recycle or
Recycle). Pada Program KKN tahun 2022 Institut Madanu Nusantara Sukabumi
Kelompok 2 mencanangkan program daur ulang dengan harapan dapat mengurangi
sampah plastik di sekitar Kecamatan Cujeruk khususnya di sekitar Desa Cipelang,
memacu kreativitas mahasiswa dan memajukan masyarakat. Atau memilih untuk
mendaur ulang. Tahapan pembuatannya adalah: Persiapan yang perlu dilakukan pada tahap ini adalah menyiapkan alat dan
bahan untuk membuat tempat sampah dari sampah plastik, terlebih dahulu
mengumpulkan sampah botol plastik, kemudian gunting, tang, kawat besi, tali benang
dan lilin. dan bahan untuk botol plastik, seperti korek api, serta alat dan bahan untuk
memperbaiki penampilannya seperti sikat dan koas. Gambar 1. Tahap Pengumpulan Botol Plastik
Gambar 2. Botol plastik yang sudah dibersihkan
Selanjutnya lubangi botol dengan alat yang sudah dipersiapkan, gunakan pisau
t k
b
t l b
d b
i
t
h b t l
t k dit
i t
b h
Al t
t k Gambar 1. Tahap Pengumpulan Botol Plastik Gambar 2. Botol plastik yang sudah dibersihkan Gambar 1. Tahap Pengumpulan Botol Plastik Gambar 1. Tahap Pengumpulan Botol Plastik Gambar 2. Botol plastik yang sudah dibersihkan Selanjutnya lubangi botol dengan alat yang sudah dipersiapkan, gunakan pisau
untuk membuat lubang pada bagian tengah botol untuk ditanami tumbuhan. Alat untuk
membuat lubang kecil pada bagian untuk penyangga botol saat di gantung di pagar
gunakan lilin untuk mengahsilkan api kemudian siapkan paku untuk melubangi botol,
gunakan paku yang sudah dipanaskan menggunakan api dari lilin untuk melubangi
bagian bawah botol agar ada oksigen masuk ke dalam botol untuk menyerap air pada
saat tanaman sisiram. Pastabiq : Junal Pengabdian kepada Masyarakat I 22 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana Jika sudah selesai proses melubangi selanjutnya tinggal rangkai botol yang sudah
dilubangi dan diikat menggunakan tali benang yang sudah disiapkan lalu rangkai
dengan yang sudah diberi tanah, tanaman, pupuk dan juga air yang sudah disiapkan
untuk menyiram bunga saat sudah di ikat oleh tali benang. Susunlah botol yang sudah
disiapkan lalu sejajarkan biar terlihat rapih dan jangan lupa untuk selalu siram
tanamannya. Gambar 3. Tahap Merangkai Botol ke dingding atau pagar yang sudah sisiapkan Gambar 3. Tahap Merangkai Botol ke dingding atau pagar yang sudah sisiapkan Setelah selesai merangkai lalu kita lihat hasil dari proses pengolahan daur ulang
botol bekas menjadi kreasi seni yang menarik dan juga bermanfaat bagi lingkungan
sekitar dan lingkungan sekolah. Guna mengahsilkan taman yang indah saat di lihat dan
juga pemandangan yang sangat hijau di lingkungan sekitar masyarakat rumah tangga
menjadikan penghijauan yang berguna bagi pernapasan kita, juga bisa menghirup udara
segar dari tanaman yang kita tanam menggunakan botol bekas yang di jadikan vas
bunga yang indah. Gambar 4. Hasil vas bunga dari limbah Botol plastik Gambar 4. Hasil vas bunga dari limbah Botol plastik Pastabiq : Junal Pengabdian kepada Masyarakat I 23 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana Inilah hasil dari pengolahan limbah plastik dari botol bekas diubah menjadi
kreasi seni yang menarik dan bermanfaat bagi lingkungan masyarakat rumah tangga
dan juga lingkungan sekolah yang ada di desa cipelang. Acknowledgements Ucapan terima kasih kami sampaikan kepada dosen pembimbing lapangan yang
telah membimbing selama kegiatan KKN berlangsung, dan juga kepada seluruh warga
masyarakat desa Cipelang, serta aparatur Desa yang mendukung penuh kegiatan kami
dan juga kepada Pihak Institut Madani Nusantara Sukabumi. Simpulan Sampah plastik merupakan salah satu masalah utama di Indonesia yang
berkontribusi terhadap kerusakan lingkungan. Hal ini disebabkan meningkatnya
permintaan plastik akibat pertumbuhan penduduk dan kurangnya kesadaran
masyarakat untuk mendaur ulang sampah plastik. Proses ini dilakukan dengan
mendaur ulang atau mengolah kembali sampah yang sudah ada yaitu botol plastik,
dengan cara membersihkan sampah botol plastik, melubangi bagian atas dan bawahnya
serta menyambung botol dengan tali benang hingga membentuk Vas Bunga yang indah. Oleh karena itu cara ini sangat efektif untuk sedikit mengurangi sampah plastik
khususnya Desa Cipelang karena untuk membuat Vas Bunga membutuhkan banyak
botol, semoga dapat membantu mengurangi sampah dan memotivasi masyarakat
khususnya pelajar dan anak-anak. Untuk mendistribusikan. Daftar Referensi Ariani, A. (2018). Pemanfaatan Botol Plastik Bekas Menjadi Media Tanam (POT) Di
Lahan
Sempit.Abdimas
Pedagogi:
Jurnal
Ilmiah
Pengabdian
kepada
Masyarakat,2(1), 1-7. Asropah, Asropah, et al. "Pemanfaatan Barang Bekas Botol Plastik dalam Pembuatan
Vertical Garden."E-Dimas: Jurnal Pengabdian Kepada Masyarakat, vol. 7, no. 2,
2016, pp. 9-16. Brunner, P. H., & Rechberger, H. (2016). Handbook of material flow analysis: For
environmental, resource, and waste engineers. CRC press. Choirunnisa, F., & Hestiana, R. A. Solusi Cerdas Dan Praktis Pengolahan Sampah Plastik
Dengan Ecobriks Di Kelurahan Kemijen Rw 03. Muis, A. A., Mursalim, N., Nacjmi, N. Y., Setiawan, I., S, N., Aris, M. R., Asdar, M.,
Ramadhani, S., Afdal, A., & Aziza, N. (2022). Pemanfaatan Sampah Plastik Dalam
Upaya Merawat Lingkungan Guna Menumbuhkan Kreativitas Masyarakat. Community Development Journal : Jurnal Pengabdian Masyarakat, 2(3), 611–617. https://doi.org/10.31004/cdj.v2i3.2484 Nababan, B. B. R. (2007). Bab I Pendahuluan ٰ ﻟ ٰ ﻟ ٰ ﻟﻟ ﻟ ﻟ ﻟ ٰ ٰ ﻟ ﻟ ٰ ﻟ. 7, 1–
11. Notoatmodjo, S. (2015). Promosi Kesehatan dan Perilaku Kesehatan (Cetakan V). Jakarta:
Rineka Cipta. Riswan, Sunoko, H. R., & Hadiyanto, A. (2015). Kesadaran Lingkungan. Jurnal Ilmu
Lingkungan,
9(1),
31–39. https://ejournal.undip.ac.id/index.php/ilmulingkungan/article/view/2085 Wulandari, S., Nurmala, D., & Dewi, R. S. (2021). Pemanfaatan Limbah Plastik Menjadi
Barang Bernilai Ekonomi Untuk Meningkatkan Pendapatan Masyarakat Di Desa
Tanjung Rejo Percut Sei Tuan. Amaliah: Jurnal Pengabdian Kepada Masyarakat, 5
(1), 44 -47. Sunarsih, E. (2014). Konsep pengolahan limbah rumah tangga dalam upaya pencegahan
pencemaran lingkungan. Jurnal Ilmu Kesehatan Masyarakat, 5 (3). Wahyudin. 2016. Analisis Teknik Operasional Pengelolaan Sampah Perkotaan di Kota
Bima. Jurnal Kesehatan Masyarakat UNTB. Pastabiq : Junal Pengabdian kepada Masyarakat I 24 Merdeka dari Sampah, Sebagai Praktik Sosial... Halim, Maulana Wahyudin. 2016. Analisis Teknik Operasional Pengelolaan Sampah Perkotaan di Kota
Bima. Jurnal Kesehatan Masyarakat UNTB. Merdeka dari Sampah, Sebagai Praktik Sosial...
Halim, Maulana
|
https://openalex.org/W2254999921
|
https://ueaeprints.uea.ac.uk/id/eprint/67953/1/1_s2.0_S0161642015014256_main.pdf
|
English
| null |
Associations with Intraocular Pressure in a Large Cohort
|
Ophthalmology
| 2,016
|
cc-by
| 10,764
|
Results from the UK Biobank Michelle P.Y. Chan, FRCOphth,1 Carlota M. Grossi, PhD,1 Anthony P. Khawaja, PhD, FRCOphth,2
Jennifer L.Y. Yip, PhD, MRCOphth,2 Kay-Tee Khaw, MBBChir, FRCP,2 Praveen J. Patel, FRCOphth, MD(Res),1
Peng T. Khaw, PhD, FRCOphth,1 James E. Morgan, DPhil, FRCOphth,3 Stephen A. Vernon, DM, FRCOphth,4
Paul J. Foster, PhD, FRCOphth,1 on behalf of the UK Biobank Eye and Vision Consortium* Purpose:
To describe the associations of physical and demographic factors with Goldmann-correlated
intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) in a British cohort. Design:
Cross-sectional study within the UK Biobank, a large-scale multisite cohort study in the United
Kingdom. Purpose:
To describe the associations of physical and demographic factors with Goldmann-correlated
intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) in a British cohort. g
Participants:
We included 110 573 participants from the UK Biobank with intraocular pressure (IOP) mea-
surements available. Their mean age was 57 years (range, 40e69 years); 54% were women, and 90% were white. g
Participants:
We included 110 573 participants from the UK Biobank with intraocular pressure (IOP) mea-
surements available. Their mean age was 57 years (range, 40e69 years); 54% were women, and 90% were white. Methods:
Participants had 1 IOP measurement made on each eye using the Ocular Response Analyzer
noncontact tonometer Linear regression models were used to assess the associations of IOP with physical and Participants:
We included 110 573 participants from the UK Biobank with intraocular pressure (IOP) mea-
surements available. Their mean age was 57 years (range, 40e69 years); 54% were women, and 90% were white. Methods:
Participants had 1 IOP measurement made on each eye using the Ocular Response Analyzer
noncontact tonometer. Linear regression models were used to assess the associations of IOP with physical and
demographic factors. g
y
(
g ,
y
);
,
Methods:
Participants had 1 IOP measurement made on each eye using the Ocular Response Analyzer
noncontact tonometer. Linear regression models were used to assess the associations of IOP with physical and
demographic factors. g
p
Main Outcome Measures:
The IOPg and IOPcc. g
Results:
The mean IOPg was 15.72 mmHg (95% confidence interval [CI], 15.70e15.74 mmHg), and the mean
IOPcc was 15.95 mmHg (15.92e15.97 mmHg). After adjusting for covariates, IOPg and IOPcc were both
significantly associated with older age, male sex, higher systolic blood pressure (SBP), faster heart rate, greater
myopia, self-reported glaucoma, and colder season (all P < 0.001). Results from the UK Biobank The strongest determinants of both IOPg and
IOPcc were SBP (partial R2: IOPg 2.30%, IOPcc 2.26%), followed by refractive error (IOPg 0.60%, IOPcc 1.04%). The following variables had different directions of association with IOPg and IOPcc: height (0.77 mmHg/m IOPg;
1.03 mmHg/m IOPcc), smoking (0.19 mmHg IOPg, 0.35 mmHg IOPcc), self-reported diabetes (0.41 mmHg
IOPg, 0.05 mmHg IOPcc), and black ethnicity (0.80 mmHg IOPg, 0.77 mmHg IOPcc). This suggests that
height, smoking, diabetes, and ethnicity are related to corneal biomechanical properties. The increase in both
IOPg and IOPcc with age was greatest among those of mixed ethnicities, followed by blacks and whites. The
same set of covariates explained 7.4% of the variability of IOPcc but only 5.3% of the variability of IOPg. Conclusions:
This analysis of associations with IOP in a large cohort demonstrated that some variables
clearly have different associations with IOPg and IOPcc, and that these 2 measurements may reflect different
biological characteristics. Ophthalmology 2016;123:771-782 ª 2016 by the American Academy of Ophthal-
mology. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). *Supplemental material is available at www.aaojournal.org. Elevated intraocular pressure (IOP) is one of the most sig-
nificant risk factors for the development1 and progression2 of
open-angle glaucoma. Intraocular pressure is a multifactorial
trait with a heritability of 29% to 62%.3,4 Many epidemio-
logic studies have examined the association of IOP with
physical and sociodemographic factors across different
populations, and these factors have been shown to account for
approximately 10% of IOP variability.5e8 Although some
associations with IOP have been demonstrated consistently,
such as systolic blood pressure (SBP),7e10 other factors such
as age7,8,11,12 and sex7e9,11,13 have a less consistent effect. There is also growing evidence that corneal biomechanics
influence IOP measurements.14e16 The UK Biobank is one of the largest prospective cohort studies with ocular data
globally and will lend statistical power to detecting
weaker associations of IOP. In this study, we explore the
associations of both Goldmann-correlated IOP (IOPg) and
corneal-compensated IOP (IOPcc) measured by the Ocular
Response Analyzer noncontact tonometer (ORA). Associations with Intraocular Pressure in a
Large Cohort Results from the UK Biobank 2016 by the American Academy of Ophthalmology
This is an open access article under the CC BY license (http://creativecommons.org/
licenses/by/4.0/). Published by Elsevier Inc. Statistical Analysis protocol (http://www.ukbiobank.ac.uk/resources/) and protocols
for individual tests (http://biobank.ctsu.ox.ac.uk/crystal/docs.cgi)
are available online. In brief, an extensive baseline questionnaire,
physical measurements, and biological samples were undertaken
in 22 assessment centers between 2006 and 2010. All UK resi-
dents aged 40 to 69 years who were registered with the National
Health Service and living up to 25 miles from 1 of the 22 study
assessment centers were invited to participate. The work was
carried out with the approval of the North West Research Ethics
Committee (reference number 06/MRE08/65), in accordance
with the principles of the Declaration of Helsinki. Left eye IOP values were chosen for the main analyses because
they were measured after the right eye and were possibly less prone
to artifacts with the participant more familiar with the test. Par-
ticipants who reported having had laser refractive surgery or
corneal graft surgery in the left eye were excluded from the anal-
ysis because corneal surgery would bias the relationship between
IOPg and IOPcc. Body mass index was examined between 20 and
40 kg/m2 (95% of the study population), because BMI outside this
range showed a nonlinear relationship with IOP. Smoking status
was dichotomized to regular (smokes on most or all days) and
current nonsmokers (ex-smokers and never smokers) to maximize
the potential to detect an effect. Season of IOP measurement was
categorized into spring (March to May), summer (June to August),
autumn (September to November), and winter (December to
February). p
p
Ophthalmic data were collected in late 2009 in 6 assessment
centers as an additional enhancement to the initial baseline
assessment. These 6 centers are distributed widely across the
United Kingdom, including Croydon and Hounslow in Greater
London, Liverpool and Sheffield in Northern England, Birming-
ham in the Midlands, and Swansea in Wales. Participants
completed a touch-screen self-administered questionnaire on their
general health and socioeconomic status. The Townsend depriva-
tion index was determined according to the participants’ postcodes
at recruitment and the corresponding output areas from the pre-
ceding national census. The index was calculated on the basis of
the output area’s employment status, home and car ownership, and
household condition; the higher and more positive the index, the
more deprived an area. Methods The UK Biobank is a large-scale multisite cohort study estab-
lished by the Wellcome Trust medical charity, Medical Research
Council,
Department
of
Health,
Scottish
Government,
and
Northwest Regional Development Agency. The overall study 771 http://dx.doi.org/10.1016/j.ophtha.2015.11.031
ISSN 0161-6420/15 Ophthalmology
Volume 123, Number 4, April 2016 Ophthalmology
Volume 123, Number 4, April 2016 Statistical Analysis The choices for ethnicity include white
(English/Irish or other white background), Asian or British Asian
(Indian/Pakistani/Bangladeshi or other Asian background), black
or black British (Caribbean, African, or other black background),
Chinese, mixed (white and black Caribbean or African, white and
Asian, or other mixed background), or other ethnic group (not
defined). Smoking status was determined by the participant’s
answer to “Do you smoke tobacco now?,” from the selection of
yes, on most or all days/only occasionally/no/prefer not to answer. Diabetes status was determined as those who answered yes to “Has
a doctor ever told you that you have diabetes?” Glaucoma and
macular degeneration statuses were determined as those who
selected “glaucoma” or “macular degeneration” from a list of eye
disorders to the question, “Has a doctor told you that you have any
of the following problems with your eyes?” The variables to be examined for associations with IOP were
decided a priori on the basis of previous published studies. The
possibility of clustering of IOP within each center of assessment
was explored, but the intraclass correlation coefficients were very
low (0.004 for IOPcc, 0.0005 for IOPg), which indicated that
clustering accounted for a very small proportion of the variance in
IOP. Therefore, we elected to proceed with multiple regression
analysis using the center of assessment as a covariable to account
for the potential underlying small differences in associations with
IOP. Variations in characteristics between the centers were
explored using multiple 1-way analysis of variance with Bonfer-
roni correction for continuous variables and chi-square test for
categoric variables. Associations between IOP and continuous variables were first
explored graphically. The relationship with sex, age, Townsend
deprivation index, center of assessment, weight, height, waist
circumference, SBP and diastolic blood pressure (DBP), BMI,
refractive error, smoking status, diabetes, glaucoma, macular
degeneration, and season of IOP measurement were explored with
univariable linear regression. All examined variables were included
in a multivariable regression model. All statistical analyses were
performed using STATA (Stata/IC 12.0; StataCorp LP, College
Station, TX). A more robust statistical significance threshold of
P < 0.001 was used to avoid false-positives due to the large
number of tests carried out. Further details of the derivation of the
variables and missing data can be found on the UK Biobank online
data showcase (http://biobank.ctsu.ox.ac.uk/crystal/label.cgi). Measurements Blood pressure and heart rate were measured using the HEM-
70151T digital blood pressure monitor (Omron, Hoofddorp, The
Netherlands). Two measurements of each were taken, and the mean
was used in subsequent analysis. Weight was measured with the
BV-418 MA body composition analyzer (Tanita, Arlington
Heights, IL). Height was measured using a Seca 202 height
measure (Seca, Birmingham, UK). Body mass index (BMI) was
calculated as weight (kg)/height (m)2. Waist circumference at the
level of the umbilicus was measured using a Wessex non-
stretchable sprung tape measure. Autorefraction was performed
using an RC5000 Auto Refkeratometer (Tomey, Nagoya, Japan),
and refractive error (spherical equivalent) was calculated as sphere
power þ (cylinder power/2). The IOP was measured once for each
eye (right eye first) using the ORA (Reichert Corp., Philadelphia,
PA), and only 1 measurement per eye was taken. Participants who
had eye surgery within the previous 4 weeks or those with possible
eye infections were precluded from having IOP measured. The
ORA flattens the cornea with a jet of air and uses an electro-optical
system to measure the air pressures at which the cornea
flattens both inward and outward. The average of the 2 ORA
pressure values was calibrated against Goldmann applanation
tonometer measures to derive IOPg. The IOPcc was derived using
proprietary formulae to correct for the corneal biomechanical
properties.17 Results Of the 502 656 participants in the whole UK Biobank cohort,
112 690 underwent IOP measurements, and 112 285 had valid
measurements. Table 1 summarizes their mean IOP stratified by
age, sex, and laterality. Mean IOP was slightly higher in the
right eye than the left eye for both IOPg and IOPcc (mean
difference, 0.14 mmHg IOPg; 95% confidence interval [CI],
0.12e0.16 mmHg, paired t test P < 0.001; 0.07 mmHg IOPcc;
95% CI, 0.05e0.09 mmHg; P < 0.001). Therefore, left eye
values were used in all subsequent analyses because they were
measured after the right eye and were possibly less prone to
artifacts with the participant more familiar with the test. The
mean left IOPg was 15.72 mmHg (95% CI, 15.70e15.74
mmHg), and the mean left IOPcc was 15.95 mmHg (95% CI,
15.92e15.97 mmHg). The IOPg and IOPcc increased linearly
with age, SBP, DBP, pulse rate, and BMI (Fig 1AeD) and
decreased linearly with refractive error (Fig 1E). 772 Table 1. Intraocular Pressure Stratified By Age, Sex, and Eye
IOPg, mmHg (SD, 95% CI)
IOPcc, mmHg (SD, 95% CI)
Right (n¼111 434)
Left (n¼111 049)
Right (n¼111 434)
Left (n¼111 049)
Men
40e49 yrs
15.4 (3.7, 15.4e15.5)
15.4 (3.9, 15.3e15.4)
15.6 (3.6, 15.5e15.6)
15.6 (3.7, 15.5e15.6)
50e59 yrs
15.9 (4.0, 15.8e16.0)
15.7 (3.9, 15.7e15.8)
16.1 (3.9, 16.1e16.2)
16.1 (3.9, 16.0e16.1)
60e69 yrs
16.3 (4.0, 16.2e16.3)
16.2 (4.1, 16.1e16.2)
16.8 (4.0, 16.7e16.8)
16.7 (4.1, 16.7e16.8)
Women
40e49 yrs
15.3 (3.6, 15.2e15.3)
15.0 (3.5, 14.9e15.0)
15.0 (3.5, 15.0e15.1)
14.9 (3.4, 14.8e15.0)
50e59 yrs
15.6 (3.7, 15.6e15.7)
15.4 (3.8, 15.4e15.5)
15.6 (3.6, 15.5e15.6)
15.4 (3.7, 15.4e15.5)
60e69 yrs
16.2 (3.8, 16.1e16.2)
16.0 (3.9, 16.0e16.1)
16.3 (3.8, 16.3e16.4)
16.2 (3.9, 16.2e16.3)
Total
40e49 yrs
15.3 (3.7, 15.3e15.4)
15.2 (3.7, 15.1e15.2)
15.3 (3.5, 15.2e15.3)
15.2 (3.6, 15.1e15.2)
50e59 yrs
15.8 (3.9, 15.7e15.8)
15.6 (3.8, 15.5e15.6)
15.8 (3.8, 15.77e15.85)
15.7 (3.8, 15.7e15.8)
60e69 yrs
16.2 (3.9, 16.2e16.2)
16.1 (4.0, 16.1e16.1)
16.5 (3.9, 16.5e16.6)
16.5 (4.0, 16.45e16.52)
All
15.86 (3.8, 15.84e15.88)
15.72 (3.9, 15.70e15.74)
16.02 (3.8, 16.00e16.04)
15.95 (3.9, 15.92e15.97)
Difference (right-left)
0.14 (0.12e0.16), P < 0.001
0.07 (0.05e0.09), P < 0.001
CI ¼ confidence interval; IOPcc ¼ corneal-compensated intraocular pressure; IOPg ¼ Goldmann-correlated intraocular pressure; SD ¼ standard deviation. Data shown are for 112 690 participants in the whole UK Biobank cohort with valid IOP measurements. The IOP values shown are the mean for each age
group. Results The t test compares the difference between left and right eye values. Chan et al
Associations with IOP in the UK Biobank Chan et al
Associations with IOP in the UK Biobank Table 1. Intraocular Pressure Stratified By Age, Sex, and Eye CI ¼ confidence interval; IOPcc ¼ corneal-compensated intraocular pressure; IOPg ¼ Goldmann-correlated intraocular pressure; SD ¼ standard deviation. Data shown are for 112 690 participants in the whole UK Biobank cohort with valid IOP measurements. The IOP values shown are the mean for each age
group. The t test compares the difference between left and right eye values. Table 2 summarizes the characteristics of the 110 573 study
participants, which excluded those whose left eye has had laser
refractive surgery or corneal graft surgery. The completeness of
each variable is included in Table 2, which is generally high
(98.6%e100% complete). Their mean age was 57.3 years (range,
40e70 years), 54.1% were women, and the majority were white
(89.6%). Significant differences between men and women were
found for age, distribution of participants among the centers of
assessment, ethnicity, deprivation index, height, weight, BMI,
waist circumference, SBP, DBP, pulse rate, smoking status, and
the percentage with self-reported glaucoma and diabetes. season (baseline winter; IOPg 0.14 mmHg spring, 0.27 mmHg
summer; IOPcc 0.29 mmHg spring, 0.37 mmHg summer, P <
0.001). Systolic blood pressure was the most important determinant
of both IOPg and IOPcc, accounting for 2.30% and 2.26% (partial
R2) of their variations, respectively, followed by refractive error
(IOPg 0.60%, IOPcc 1.04%) (Table 5). Some examined factors had different relationships with IOPg
and IOPcc in the multivariable model. Self-reported diabetes was
significantly associated with IOPg (0.41 mmHg, P < 0.001) but
not with IOPcc (0.05 mmHg, P ¼ 0.38). The following cova-
riates had different directions of association with IOPg and IOPcc:
height (0.77 mmHg/m IOPg, P < 0.001; 1.03 mmHg/m IOPcc,
P < 0.001), smoking (0.19 mmHg IOPg, P < 0.001; 0.35 mmHg
IOPcc, P < 0.001), and ethnicity, where IOPg was highest among
whites (baseline) and lowest among blacks (0.80 mmHg, P <
0.001), but IOPcc was highest among blacks (0.77 mmHg, P <
0.001) and lowest among the Chinese (0.74 mmHg, P < 0.001)
(Fig 2). This suggests that height, smoking, and ethnicity are
strongly related to corneal biomechanical properties. Results The same
set of covariates explained 7.4% of the variability of IOPcc, but
only 5.3% of the variability of IOPg. Among the 6 centers of assessment, mean IOPcc was signifi-
cantly different (P < 0.001, analysis of variance) but not mean
IOPg (P ¼ 0.046). Specifically, IOPcc was significantly lower in
Birmingham than every center except Swansea by 0.05 to 0.41
mmHg (P < 0.001). The centers were also different in ethnicity,
deprivation index, and season of test (P < 0.0001). As a result, the
center of assessment was included as a variable in the regression
models. Croydon was selected as the baseline center because it
contributed the largest number of participants. The associations of IOP with physio-demographic factors were
tested using univariable linear regression stratified by sex (Tables 3
and 4) and multivariable regression (Table 5). All covariates in the
univariable model were included in the multiple regression model
to allow direct comparisons between IOPg and IOPcc. The DBP
and waist circumference were excluded because of collinearity
between DBP and SBP, and waist circumference with BMI. After adjusting for covariates, the following were significantly
associated with both IOPg and IOPcc: older age (0.18 mmHg
IOPg/decade, P < 0.001; 0.49 mmHg IOPcc/decade, P <
0.001), male sex (0.18 mmHg IOPg, P < 0.001; 0.35 mmHg
IOPcc P < 0.001), SBP (0.035 mmHg IOPg, P < 0.001; 0.033
mmHg IOPcc, P < 0.001), pulse rate (0.023 mmHg IOPg,
P < 0.001; 0.018 mmHg IOPcc, P < 0.001), myopic refractive
error (0.11 mmHg IOPg/diopter, P < 0.001; 0.14 mmHg
IOPcc/diopter, P < 0.001), self-reported glaucoma (1.97 mmHg
IOPg, P < 0.001; 2.30 mmHg IOPcc, P < 0.001), and colder The association of IOP and age was examined for each ethnic
group. Figure 3 demonstrates how changes in mean IOP with age
varies across the ethnic groups, showing a linear increase among
whites, Asians, blacks, and those of mixed ethnicities, and the
trends are similar between IOPg and IOPcc. The increase was
greatest among those of mixed ethnicities after adjusting for
covariates (mixed 0.55 mmHg IOPg/decade, 0.64 mmHg IOPcc/
decade), followed by black participants (0.42 mmHg IOPg/
decade, 0.54 mmHg IOPcc/decade) (Table 6). There was no
statistically
significant
trend
among
Chinese
and
“other”
ethnicities for IOP and age. Sensitivity analysis using right eye IOP values and right
eyeespecific variables (e.g., refraction) was performed for the
regression analysis. Chan et al
Associations with IOP in the UK Biobank *P values are from the t test or chi-square test comparing characteristics between men and women in the study cohort. P < 0.001 shown in bold. BMI ¼ body mass index; D ¼ diopters; DBP ¼ diastolic blood pressure; SBP ¼ systolic blood pressure. The study participants excluded those who had undergone laser refractive surgery or corneal graft surgery in their left eye. For continuous variables, the
values shown are mean (standard deviation). *P values are from the t test or chi-square test comparing characteristics between men and women in the study cohort. P < 0.001 shown in bold. Goldmann-Correlated Intraocular Pressure
versus Corneal-Compensated Intraocular
Pressure was no longer significant with IOPcc (0.005 mmHg, 95%
CI, 0.011 to 0.001 mmHg; P ¼ 0.11). was no longer significant with IOPcc (0.005 mmHg, 95%
CI, 0.011 to 0.001 mmHg; P ¼ 0.11). Results The only different results were for sex, which
was no longer significantly associated with IOPg (0.09 mmHg;
95% CI, 0.03e0.16 mmHg; P ¼ 0.007), and with BMI, which 773 Figure 1. Graphs showing that Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) increase linearly
with (A) age, (B) systolic blood pressure, (C) pulse rate, (D) body mass index (BMI). (E) The IOPg and IOPcc show an inverse relationship with refractive
error. (F) Height has an insignficant relationship with IOPg and IOPcc in univariable regression, but a differential relationship with IOPg and IOPcc in
multivariable regression. The error bars represent the 95% confidence intervals (CIs). Ophthalmology
Volume 123, Number 4, April 2016 Ophthalmology
Volume 123, Number 4, April 2016 Figure 1. Graphs showing that Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) increase linearly
with (A) age, (B) systolic blood pressure, (C) pulse rate, (D) body mass index (BMI). (E) The IOPg and IOPcc show an inverse relationship with refractive
error. (F) Height has an insignficant relationship with IOPg and IOPcc in univariable regression, but a differential relationship with IOPg and IOPcc in
multivariable regression. The error bars represent the 95% confidence intervals (CIs). Figure 1. Graphs showing that Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) increase linearly
with (A) age, (B) systolic blood pressure, (C) pulse rate, (D) body mass index (BMI). (E) The IOPg and IOPcc show an inverse relationship with refractive
error. (F) Height has an insignficant relationship with IOPg and IOPcc in univariable regression, but a differential relationship with IOPg and IOPcc in
multivariable regression. The error bars represent the 95% confidence intervals (CIs). 774 Chan et al
Associations with IOP in the UK Biobank Chan et al
Associations with IOP in the UK Biobank Table 2. Characteristics of the 110 573 Study Participants in the UK Biobank, by Sex
Total
Women
Men
P Value*
Sex, % women
54.1
Age, yrs (n¼110 573)
57.3 (8.1)
57.1 (8.0)
57.6 (8.2)
<0.001
Ethnicity, % (n¼110 573)
White
89.6
89.4
89.9
<0.001
Asian
3.9
3.4
4.5
Black
3.6
3.9
3.2
Chinese
0.46
0.55
0.36
Mixed
0.90
1.0
0.74
Others
1.6
1.7
1.3
Assessment center, % (n¼ 110 573)
Croydon
22.9
23.8
21.8
<0.001
Sheffield
22.4
22.0
22.8
Birmingham
21.6
21.0
22.2
Hounslow
18.6
18.9
18.3
Liverpool
14.2
14.0
14.4
Swansea
0.39
0.36
0.43
Townsend deprivation index (n¼110 438)
0.94 (3.0)
0.96 (3.0)
0.91 (3.1)
0.004
Height, m (n¼110 127)
1.69 (0.09)
1.63 (0.06)
1.76 (0.07)
<0.001
Weight, kg (n¼110 102)
78.2 (16.1)
71.6 (14.2)
86.0 (14.5)
<0.001
BMI, kg/m2 (n¼105 113)
27.4 (4.8)
27.1 (5.2)
27.8 (4.3)
<0.001
Waist circumference, cm (n¼110 268)
90.6 (13.6)
85.1 (12.7)
97.1 (11.5)
<0.001
SBP, mmHg (n¼110 510)
137.4 (18.4)
135.1 (19.0)
140.0 (17.2)
<0.001
DBP, mmHg (n¼110 510)
81.9 (10.0)
80.5 (9.9)
83.6 (9.9)
<0.001
Pulse rate, min1 (n¼110 510)
68.6 (11.1)
69.4 (10.4)
67.7 (11.8)
<0.001
Refractive error, D
Right eye (n¼1 109 376)
0.36 (2.8)
0.31 (2.8)
0.35 (2.7)
0.14
Left eye (n¼109 059)
0.31 (2.8)
0.31 (2.8)
0.30 (2.7)
0.42
Current smoking status, % (n¼107 115)
Regular smoker
7.2
6.2
8.5
<0.001
Occasional smoker
2.8
2.1
3.7
Nonsmoker
90.0
91.7
87.9
Self-reported diabetes, % (n¼109 832)
5.9
4.4
7.7
<0.001
Self-reported glaucoma, %
Right eye (n¼110 573)
1.46
1.17
1.80
<0.001
Left eye (n¼110 573)
1.45
1.14
1.82
<0.001
Self-reported macular degeneration, %
Right eye (n¼110 573)
0.85
0.92
0.77
0.006
Left eye (n¼110 573)
0.82
0.90
0.72
0.001
Season of test, % (n¼110 573)
Spring
35.0
34.9
35.25
0.040
Summer
19.9
20.1
19.6
Autumn
23.1
23.2
22.95
Winter
22.0
21.8
22.2
BMI ¼ body mass index; D ¼ diopters; DBP ¼ diastolic blood pressure; SBP ¼ systolic blood pressure
Chan et al
Associations with IOP in the UK Biobank Table 2. Characteristics of the 110 573 Study Participants in the UK Biobank, by Sex BMI ¼ body mass index; D ¼ diopters; DBP ¼ diastolic blood pressure; SBP ¼ systolic blood pressure. The study participants excluded those who had undergone laser refractive surgery or corneal graft surgery in their left eye. For continuous variables, the
values shown are mean (standard deviation). Discussion In this study, the associations of most variables with IOPg
and IOPcc were similar. However, after adjusting for con-
founders, there were clear differences in the association of
IOPg and IOPcc with self-reported diabetes (positively and
significantly associated with IOPg but not with IOPcc),
height (positively associated with IOPcc, negatively asso-
ciated with IOPg), smoking (positively associated with We examined the physical and demographic associations
with IOP in one of the largest cohort studies in recent years. This is also one of the few studies that examined and con-
trasted the associations of IOPg and IOPcc together in a
large cohort. 775 Ophthalmology
Volume 123, Number 4, April 2016 Table 3. Univariable Linear Regression with Goldmann-Correlated Intraocular Pressure (Left Eye) as the Dependent Variable Table 3. Univariable Linear Regression with Goldmann-Correlated Intraocular Pressure (Left Eye) as the Dependent Variable Table 3. Discussion Univariable Linear Regression with Goldmann-Correlated Intraocular Pressure (Left Eye) as the Dependent Variable
All
Women
Men
b (95% CI)
P
b (95% CI)
P
b (95% CI)
P
Age, decade
0.45 (0.43e0.48)
<0.001
0.50 (0.46e0.53)
<0.001
0.4 (0.36e0.44)
<0.001
Sex (baseline ¼ female)
0.25 (0.20e0.30)
<0.001
e
e
e
e
Ethnicity (baseline ¼ white)
Asian
0.52 (0.64 to 0.40)
<0.001
0.65 (0.82 to 0.48)
<0.001
0.44 (0.61 to 0.27)
0.001
Black
0.71 (0.83 to 0.58)
<0.001
0.83 (0.99 to 0.67)
<0.001
0.50 (0.70 to 0.30)
0.001
Chinese
0.55 (0.90 to 0.21)
0.001
0.72 (1.14 to 0.31)
0.001
0.19 (0.78 to 0.40)
0.53
Mixed
0.58 (0.82 to 0.34)
<0.001
0.58 (0.88 to 0.28)
<0.001
0.52 (0.93 to 0.11)
0.012
Others
0.58 (0.77 to 0.39)
<0.001
0.69 (0.93 to 0.46)
<0.001
0.37 (0.68 to 0.068)
0.017
Assessment center
(baseline ¼ Croydon)
Sheffield
0.088 (0.20e0.16)
0.012
0.13 (0.045e0.22)
0.003
0.018 (0.087 to 0.12)
0.73
Birmingham
0.011 (0.06 to 0.08)
0.76
0.008 (0.08 to 0.10)
0.87
0.052 (0.11 to 0.09)
0.79
Hounslow
0.044 (0.028 to 0.12)
0.23
0.015 (0.08 to 0.11)
0.76
0.072 (0.040 to 0.18)
0.21
Liverpool
0.14 (0.057e0.21)
0.001
0.081 (0.022 to 0.18)
0.12
0.18 (0.062e0.30)
0.003
Swansea
0.19 (0.18 to 0.57)
0.31
0.20 (0.31 to 0.71)
0.44
0.15 (0.39 to 0.70)
0.58
Deprivation index
0.010 (0.018 to 0.002)
0.011
0.024 (0.034 to 0.013)
<0.001
0.005 (0.007 to 0.016)
0.44
Weight, 10 kg
0.08 (0.062e0.091)
<0.001
0.07 (0.04e0.9)
<0.001
0.04 (0.01e0.06)
0.003
Height, m
0.09 (0.16 to 0.34)
0.47
1.84 (2.32 to 1.36)
0.001
0.15 (0.20 to 0.10)
<0.001
BMI, kg/m2
0.033 (0.027e0.038)
<0.001
0.031 (0.024e0.039)
<0.001
0.028 (0.019e0.038)
<0.001
Waist, cm
0.014 (0.013e0.016)
<0.001
0.014 (0.012e0.016)
<0.001
0.011 (0.008e0.015)
<0.001
SBP, mmHg
0.039 (0.038e0.040)
<0.001
0.037 (0.035e0.038)
<0.001
0.041 (0.039e0.043)
<0.001
DBP, mmHg
0.054 (0.052e0.056)
<0.001
0.053 (0.050e0.056)
<0.001
0.055 (0.051e0.058)
<0.001
Pulse, min1
0.030 (0.028e0.032)
<0.001
0.036 (0.033e0.039)
<0.001
0.027 (0.024e0.030)
<0.001
Refractive error, D
0.085 (0.093 to 0.077)
<0.001
0.073 (0.084 to 0.062)
<0.001
0.10 (0.11 to 0.088)
<0.001
Smoking
Nonsmoker ¼ 0
e
e
e
e
e
e
Regular smoker ¼ 1
0.12 (0.026e0.20)
0.011
0.049 (0.18 to 0.08)
0.45
0.22 (0.09e0.34)
0.001
Diabetes
0.69 (0.60e0.79)
<0.001
0.86 (0.71e1.00)
<0.001
0.52 (0.39e0.65)
<0.001
Glaucoma
2.34 (2.15e2.53)
<0.001
2.54 (2.25e2.83)
<0.001
2.15 (1.88e2.41)
<0.001
Macular degeneration
0.51 (0.26e0.80)
<0.001
0.34 (0.013e0.66)
0.041
0.81 (0.39e1.22)
0.001
Seasons (baseline ¼ winter)
Spring
0.20 (0.26 to 0.14)
<0.001
0.10 (0.19 to 0.021)
0.014
0.31 (0.40 to 0.21)
<0.001
Summer
0.43 (0.50 to 0.36)
<0.001
0.27 (0.36 to 0.17)
<0.001
0.62 (0.73 to 0.51)
<0.001
Autumn
0.13 (0.19 to 0.57)
<0.001
0.049 (0.14 to 0.042)
0.29
0.21 (0.31 to 0.10)
<0.001
BMI ¼ body mass index; CI ¼ confidence interval; D ¼ diopters; DBP ¼ diastolic blood pressure; IOPg ¼ Goldmann-correlated intraocular pressure; SBP ¼
systolic blood pressure. Discussion P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. BMI ¼ body mass index; CI ¼ confidence interval; D ¼ diopters; DBP ¼ diastolic blood pressure; IOPg ¼ Goldmann-correlated intraocular pressure; SBP ¼
systolic blood pressure. P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. IOPg but negatively associated with IOPcc), and black
ethnicity (negatively associated
with
IOPg, positively
associated with IOPcc). Previous studies using Goldmann
applanation tonometry found higher IOP to be associated
with self-reported diabetes,8,12 whereas no association had
been found with height, including 1 study that used
IOPg.9,12,18 A recent study comparing ORA data among 2
groups of diabetic patients (HbA1c <7%, HbA1c 7%) and
healthy controls did demonstrate similar differential asso-
ciations and found that IOPcc was not significantly different
among the 3 groups, whereas IOPg was significantly higher
in the diabetic patients than in the controls.19 For smoking,
findings have been variable, with some studies reporting no
association8,9,11,13,18 and other studies reporting higher IOP
in smokers.10,20 Among women, IOPg in this study was not
significantly associated with smoking in univariable (P ¼
0.004) or multiple regression (P ¼ 0.41, not shown in ta-
bles), a finding also seen in the Gutenberg Health Study
using noncontact tonometry.21 different biological features. The IOPg is calibrated against
the Goldmann applanation tonometer, whereas IOPcc is
derived by modeling IOP of patients who underwent laser-
assisted in situ keratomileusis to minimize the difference
in measured pressure before and after surgery,17 therefore
reflecting an IOP measure with minimal influence from
corneal
biomechanics.16
In
particular,
central
corneal
thickness (CCT) is correlated with IOPg but not IOPcc,
and
IOPcc
is
not
correlated
with
corneal
resistance
factor.17 However, it is not clear exactly which parameters
of corneal biomechanics best describe the difference
between IOPg and IOPcc. Height is related to a longer axial length, deeper anterior
chamber, and flatter cornea.22,23 Therefore, height is plau-
sibly related to determinants of collagen-related processes,
which may explain the different associations with IOPg and
IOPcc. There is a clear trend for men being taller than
women in our study, and that resulted in paradoxical
results in the univariable analysis when the sexes were
separated. This was resolved when sex was adjusted for in
the model. Chan et al
Associations with IOP in the UK Biobank Table 4. Chan et al
Associations with IOP in the UK Biobank Univariable Linear Regression with Corneal-Compensated Intraocular Pressure (Left Eye) as the Dependent Variable
All
Women
Men
b (95% CI)
P
b (95% CI)
P
b (95% CI)
P
Age, decade
0.67 (0.64e0.70)
<0.001
0.70 (0.67e0.73)
<0.001
0.61 (0.57e0.65)
<0.001
Sex (female ¼ 0, male ¼ 1)
0.61 (0.56e0.66)
<0.001
e
e
e
e
Ethnicity (baseline ¼ white)
Asian
0.16 (0.28 to 0.04)
0.009
0.25 (0.42 to 0.08)
0.004
0.15 (0.32 to 0.02)
0.08
Black
0.56 (0.44e0.69)
<0.001
0.49 (0.33e0.65)
<0.001
0.74 (0.54e0.94)
<0.001
Chinese
0.77 (1.11 to 0.44)
<0.001
0.80 (1.21 to 0.39)
<0.001
0.55 (1.13 to 0.03)
0.06
Mixed
0.31 (0.55 to 0.07)
0.013
0.18 (0.48 to 0.12)
0.23
0.38 (0.79 to 0.02)
0.06
Others
0.22 (0.41 to 0.035)
0.020
0.13 (0.36 to 1.02)
0.27
0.26 (0.57 to 0.04)
0.09
Center of assessment
(baseline ¼ Croydon)
Sheffield
0.053 (0.015 to 0.12)
0.13
0.028 (0.06 to 0.12)
0.53
0.042 (0.061 to 0.15)
0.42
Birmingham
0.37 (0.44 to 0.30)
<0.001
0.39 (0.48 to 0.30)
<0.001
0.40 (0.51 to 0.30)
<0.001
Hounslow
0.014 (0.086 to 0.057)
0.69
0.042 (0.13 to 0.051)
0.38
0.002 (0.11 to 0.11)
0.98
Liverpool
0.042 (0.036 to 0.12)
0.29
0.041 (0.14 to 0.061)
0.43
0.098 (0.02 to 0.22)
0.10
Swansea
0.065 (0.30 to 0.43)
0.73
0.19 (0.32 to 0.70)
0.46
0.13 (0.67 to 0.40)
0.63
Deprivation index
0.02 (0.03 to 0.01)
<0.001
0.02 (0.03 to 0.01)
<0.001
0.02 (0.04 to 0.01)
<0.001
Weight, 10 kg
0.12 (0.10e0.13)
<0.001
0.08 (0.06e0.10)
<0.001
0.0004 (0.02 to 0.02)
0.10
Height, m
2.02 (1.77e2.23)
<0.001
0.91 (1.38 to 0.43)
<0.001
0.18 (0.69 to 0.33)
0.48
BMI, kg/m2
0.025 (0.019e0.030)
<0.001
0.030 (0.023e0.037)
<0.001
0.0008 (0.01 to 0.009)
0.87
Waist, cm
0.018 (0.017e0.20)
<0.001
0.015 (0.013e0.017)
<0.001
0.004 (0.001e0.007)
0.007
SBP, mmHg
0.042 (0.040e0.043)
<0.001
0.040 (0.039e0.042)
<0.001
0.040 (0.038e0.042)
<0.001
DBP, mmHg
0.058 (0.056e0.061)
<0.001
0.057 (0.054e0.060)
<0.001
0.053 (0.049e0.056)
<0.001
Pulse, min1
0.021 (0.019e0.023)
<0.001
0.031 (0.028e0.033)
<0.001
0.016 (0.013e0.019)
<0.001
Refractive error, D
0.11 (0.12 to 0.11)
<0.001
0.63 (0.75 to 0.50)
<0.001
0.14 (0.15 to 0.13)
<0.001
Smoking
Nonsmoker ¼ 0
Regular smoker ¼ 1
0.51 (0.60 to 0.42)
<0.001
0.61 (0.74 to 0.49)
<0.001
0.51 (0.64 to 0.39)
<0.001
Diabetes
0.38 (0.28e0.48)
<0.001
0.53 (0.38e0.68)
<0.001
0.13 (0.03 to 0.26)
0.06
Glaucoma
2.34 (2.15e2.53)
<0.001
2.84 (2.55e3.13)
<0.001
2.74 (2.48e3.00)
<0.001
Macular degeneration
0.79 (0.53e1.04)
<0.001
0.63 (0.31e0.95)
<0.001
1.11 (0.69e1.51)
<0.001
Seasons (baseline ¼ winter)
Spring
0.36 (0.42 to 0.29)
<0.001
0.23 (0.31 to 0.14)
<0.001
0.50 (0.60 to 0.41)
<0.001
Summer
0.56 (0.62 to 0.48)
<0.001
0.40 (0.49 to 0.30)
<0.001
0.73 (0.83 to 0.62)
<0.001
Autumn
0.011 (0.06 to 0.08)
0.75
0.12 (0.028e0.21)
0.011
0.10 (0.21 to 0.0008)
0.05
BMI ¼ body mass index; CI ¼ confidence interval; D ¼ dipoters; DBP ¼ diastolic blood pressure; SBP ¼ systolic blood pressure. Chan et al
Associations with IOP in the UK Biobank P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. Table 4. Univariable Linear Regression with Corneal-Compensated Intraocular Pressure (Left Eye) as the Dependent Variable BMI ¼ body mass index; CI ¼ confidence interval; D ¼ dipoters; DBP ¼ diastolic blood pressure; SBP ¼ systolic blood pressure. P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. focusing on the magnitudes of association, self-reported
glaucoma has the greatest effect on IOP (b¼1.97 mmHg
IOPg, 2.30 mmHg IOPcc), which is equivalent to a 5- to
10-fold effect on IOP compared with a decade increase in
age (b¼0.18 mmHg IOPg, 0.49 mmHg IOPcc). It is also
notable that the effect of seasonal change in IOP between
winter and summer (b¼0.27 mmHg IOPg, 0.37 mmHg
IOPcc) is comparable to the difference in IOP between
women and men (b¼0.18 mmHg IOPg, 0.35 IOPcc), as well
as
the
difference
between
smokers
and
nonsmokers
(b¼0.19 mmHg IOPg, 0.35 mmHg IOPcc). toxicity from smoking could directly influence the cornea to
cause the differential associations with IOPg and IOPcc. Diabetes is known to cause corneal epithelial and endothe-
lial dysfunction and thickening of the basement membrane,
postulated to occur from advanced glycation end products
and changes in the polyol pathway.24 Although the damage
of cigarette smoke on the cornea is rarely examined,
smoking induces oxidative stress on lens protein and the
retina, thought to be related to cataract formation and
increased
risk
of
age-related
macular
degeneration.25
These tissue effects could be replicated in the cornea. Overall, the list of systemic and ocular factors examined
explained only a small proportion of IOPg and IOPcc
variation (adjusted R2: 5.3% IOPg, 7.4% IOPcc). Other
published studies reported similarly low explanatory power
in their models (R2 of 10.19%e11.0% using Goldmann
IOP),5e8 although the list of explanatory variables varies
greatly among studies, and therefore the R2 values cannot be
directly compared. Nevertheless, the power of large popu-
lation studies is to allow small effects to be detected, and
these small effects could be biologically important. By Discussion Chronic high serum glucose in diabetes and the The differential systemic associations of IOPg and IOPcc
demonstrated probably mean these 2 IOP measures reflect 776 Chan et al
Associations with IOP in the UK Biobank Age which supports our findings of significantly higher (by
0.80 mmHg) IOPg in whites than blacks, but the opposite
in IOPcc (by 0.77 mmHg) once the thinner cornea is
taken into account. Studies in the past have found inconsistent relationships of
IOP with age in regression analyses, ranging from a positive
association,7e10,18,28 an inverse relationship,11,12 and no
association.6,13,29 For subjects aged 40 to 69 years, this
study found a positive relationship with IOP, which per-
sisted after adjusting for confounders. The Beijing Eye
study found IOP increasing up to age 60 to 64 years and
decreasing thereafter to age 75 years.30 The EPIC-Norfolk
Eye Study also found the same trend among women,18
and there is a hint of the same “inverted U” trend in our
data as IOPg reaches a plateau at age 65 (Fig 1A),
although there were no data beyond age 69 years. Corneal-compensated IOP continues to increase at age 65
years or more, and this trend mirrors the increasing preva-
lence of glaucoma with age. Age is one of the most
important risk factors for open-angle glaucoma (OAG), and
the results of this study support the possibility that the effect Studies in the past have found inconsistent relationships of
IOP with age in regression analyses, ranging from a positive
association,7e10,18,28 an inverse relationship,11,12 and no
association.6,13,29 For subjects aged 40 to 69 years, this
study found a positive relationship with IOP, which per-
sisted after adjusting for confounders. The Beijing Eye
study found IOP increasing up to age 60 to 64 years and
decreasing thereafter to age 75 years.30 The EPIC-Norfolk
Eye Study also found the same trend among women,18
and there is a hint of the same “inverted U” trend in our
data as IOPg reaches a plateau at age 65 (Fig 1A),
although there were no data beyond age 69 years. Corneal-compensated IOP continues to increase at age 65
years or more, and this trend mirrors the increasing preva-
lence of glaucoma with age. Age is one of the most
important risk factors for open-angle glaucoma (OAG), and
the results of this study support the possibility that the effect Studies in the past have found inconsistent relationships of
IOP with age in regression analyses, ranging from a positive
association,7e10,18,28 an inverse relationship,11,12 and no
association 6,13,29 For subjects aged 40 to 69 years this Few studies directly compared IOP between ethnic
groups. Ethnicity For ethnicity, the differential associations with IOPg and
IOPcc could be related to ethnic differences in corneal
hysteresis or CCT. Thick or thin CCT is known to cause
overestimation or underestimation, respectively, of the
true IOP by Goldmann applanation tonometers and
corneal curvature.14 Studies have consistently found CCT
to be thinner in Africans than in white subjects,26,27 777 Ophthalmology
Volume 123, Number 4, April 2016 Table 5. Ethnicity Multivariable Linear Regression with Goldmann-Correlated Intraocular Pressure and Corneal-Compensated Intraocular Pressure
(Left Eye) as the Dependent Variables
IOPg
IOPcc
b (95% CI)
P
Standard
Coefficient*
Partial
R2 (%)
b (95% CI)
P
Standard
Coefficient*
Partial
R2 (%)
Age, decade
0.18 (0.15e0.21)
<0.001
0.15
0.12
0.49 (0.46e0.52)
<0.001
0.39
0.9
Sex (baseline ¼ female)
0.18 (0.11e0.25)
<0.001
n/a
0.03
0.35 (0.28e0.42)
<0.001
n/a
0.1
Ethnicity (baseline ¼ white)
n/a
n/a
Asian
0.61 (0.74 to 0.48)
<0.001
0.09
0.042 (0.09 to 0.17)
0.52
0
Black
0.80 (0.94 to 0.66)
<0.001
0.13
0.77 (0.63e0.90)
<0.001
0.13
Chinese
0.72 (1.08 to 0.36)
<0.001
0.02
0.74 (1.10 to 0.38)
<0.001
0.02
Mixed
0.55 (0.80 to 0.29)
<0.001
0.02
0.06 (0.30 to 0.19)
0.66
0
Others
0.50 (0.70 to 0.30)
<0.001
0.02
0.11 (0.088 to 0.30)
0.28
0
Center of assessment
(baseline ¼ Croydon)
Sheffield
0.07 (0.14 to 0.002)
0.058
0
0.012 (0.06 to 0.83)
0.74
0
Birmingham
0.056 (0.13 to 0.017)
0.13
n/a
0
0.32 (0.39 to 0.25)
<0.001
n/a
0.08
Hounslow
0.005 (0.07 to 0.08)
0.89
0
0.04 (0.11 to 0.04)
0.32
0
Liverpool
0.12 (0.21 to 0.04)
0.005
0.01
0.15 (0.23 to 0.06)
0.001
0.01
Swansea
0.24 (0.63 to 0.15)
0.23
0
0.014 (0.39 0.37)
0.94
0
Deprivation index
0.007 (0.001 to 0.016)
0.10
0.02
0
0.004 (0.013 to 0.004)
0.32
0.001
0
Height, m
0.77 (1.14 to 0.39)
<0.001
0.07
0.02
1.03 (0.65e1.40)
<0.001
0.095
0.03
BMI, kg/m2
0.008 (0.014 to 0.002)
0.009
0.03
0.01
0.016 (0.022 to 0.011) <0.001
0.067
0.03
SBP, mmHg
0.035 (0.033e0.036)
<0.001
0.63
2.29
0.033 (0.032e0.034)
<0.001
0.60
2.16
Pulse, min1
0.023 (0.021e0.025)
<0.001
0.26
0.43
0.018 (0.016e0.02)
<0.001
0.20
0.28
Refractive error, D
0.11 (0.12 to 0.10)
<0.001
0.30
0.58
0.14 (0.15 to 0.13)
<0.001
0.39
1.04
Smoking (baseline ¼ nonsmoker)
Regular smoker
0.19 (0.097e0.28)
<0.001
n/a
0.02
0.35 (0.44 to 0.26)
<0.001
n/a
0.06
Self-reported diabetes
0.41 (0.3e0.52)
<0.001
n/a
0.06
0.05 (0.15 to 0.06)
0.38
n/a
0
Self-reported glaucoma
1.97 (1.77e2.17)
<0.001
n/a
0.38
2.30 (2.11e2.50)
<0.001
n/a
0.54
Self-reported macular degeneration
0.21 (0.053 to 0.47)
0.12
n/a
0
0.34 (0.087e0.60)
0.009
n/a
0.01
Seasons (baseline ¼ winter)
Spring
0.14 (0.21 to 0.075)
<0.001
0.02
0.29 (0.35 to 0.22)
<0.001
0.08
Summer
0.27 (0.35 to 0.20)
<0.001
n/a
0.05
0.37 (0.44 to 0.30)
<0.001
n/a
0.1
Autumn
0.04 (0.11 to 0.03)
0.30
0
0.066 (0.003 to 0.14)
0.06
0
Adjusted R2 (%)
5.3
7.4
BMI ¼ body mass index; CI ¼ confidence interval; D ¼ diopters; DBP ¼ diastolic blood pressure; IOPcc ¼ corneal-compensated intraocular pressure;
IOPg ¼ Goldmann-correlated intraocular pressure; n/a ¼ not available; SBP ¼ systolic blood pressure. Ethnicity *For continuous covariates, standardized coefficient represents change in IOP (mmHg) per standard deviation of the covariate. P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. Table 5. Multivariable Linear Regression with Goldmann-Correlated Intraocular Pressure and Corneal-Compensated Intraocular Pressure
(Left Eye) as the Dependent Variables BMI ¼ body mass index; CI ¼ confidence interval; D ¼ diopters; DBP ¼ diastolic blood pressure; IOPcc ¼ corneal-compensated intraocular pressure;
IOPg ¼ Goldmann-correlated intraocular pressure; n/a ¼ not available; SBP ¼ systolic blood pressure. *For continuous covariates, standardized coefficient represents change in IOP (mmHg) per standard deviation of the covariate. P < 0.001 shown in bold. BMI between 20 and 40 kg/m2 was analyzed. BMI ¼ body mass index; CI ¼ confidence interval; D ¼ diopters; DBP ¼ diastolic blood pressure; IOPcc ¼ corneal-compensated intraocular pressure;
IOPg ¼ Goldmann-correlated intraocular pressure; n/a ¼ not available; SBP ¼ systolic blood pressure.
*For continuous covariates, standardized coefficient represents change in IOP (mmHg) per standard deviation of the covariate. P < 0.001 shown in bold.
BMI between 20 and 40 kg/m2 was analyzed. Body Mass Index In the univariable regression model, IOPg and IOPcc had a
positive relationship with BMI, but after adjusting for con-
founders, they were associated with lower BMI (IOPg P ¼
0.009, IOPcc P < 0.001). This contrasts with all previous
studies that found IOP to be associated with higher
BMI,7,8,10,11,21,35 even if not statistically significant.18 It was
the introduction of SBP into the model that switched the
direction of association, indicating that SBP was a major
confounder in the relationship between IOP and BMI in
this study. In the sensitivity analysis using right eye IOP
values, a similar attenuation effect was found, where BMI
was positively associated with both IOPg and IOPcc in
the univariable model (P < 0.0001), but the association
was no longer significant in the multivariable model
(IOPg 0.005 mmHg, 95% CI, 0.0005 to 0.011 mmHg,
P ¼ 0.073; IOPcc 0.005 mmHg, 95% CI, 0.02 to
0.001 mmHg, P ¼ 0.11). Again, it was the introduction of
SBP (IOPg) and SBP and pulse rate (IOPcc) into the
model that negated the association with BMI. In the univariable regression model, IOPg and IOPcc had a
positive relationship with BMI, but after adjusting for con-
founders, they were associated with lower BMI (IOPg P ¼
0.009, IOPcc P < 0.001). This contrasts with all previous
studies that found IOP to be associated with higher
BMI,7,8,10,11,21,35 even if not statistically significant.18 It was could be mediated partly by higher IOP in older people. In
sensitivity analysis using right eye IOP values, the associ-
ation with age was less significant with IOPg (P ¼ 0.007),
although the direction of association remains. It was the
introduction of SBP into the model that attenuated the effect
of age on right eye IOPg. the introduction of SBP into the model that switched the
direction of association, indicating that SBP was a major
confounder in the relationship between IOP and BMI in
this study. In the sensitivity analysis using right eye IOP
values, a similar attenuation effect was found, where BMI
was positively associated with both IOPg and IOPcc in
the univariable model (P < 0.0001), but the association
was no longer significant in the multivariable model
(IOPg 0.005 mmHg, 95% CI, 0.0005 to 0.011 mmHg,
P ¼ 0.073; IOPcc 0.005 mmHg, 95% CI, 0.02 to
0.001 mmHg, P ¼ 0.11). Chan et al
Associations with IOP in the UK Biobank Figure 2. Beta regression coefficients for each ethnic group in the
multivariable model, showing the differences between each group’s
Goldmann-correlated
intraocular
pressure
(IOPg)
and
corneal-
compensated intraocular pressure (IOPcc) compared with the baseline
group of white ethnicity. CI ¼ confidence interval. Blood Pressure Systolic blood pressure was the strongest determinant of IOP
in this study, which is in agreement with most other studies
reporting similar analyses.7e10,13,18,30 Other hemodynamic
factors such as DBP13,30 and pulse rate8,10,13 were also
associated with IOP in this study and previous publications. This reflects the dynamic role they have in aqueous produc-
tion, which is mediated by ciliary blood flow and ciliary ox-
ygen delivery, as well as in regulating aqueous outflow by
their effects on episcleral venous pressure and pulse-
dependent motion of the trabecular meshwork.34 Systolic blood pressure was the strongest determinant of IOP
in this study, which is in agreement with most other studies
reporting similar analyses.7e10,13,18,30 Other hemodynamic Figure 2. Beta regression coefficients for each ethnic group in the
multivariable model, showing the differences between each group’s
Goldmann-correlated
intraocular
pressure
(IOPg)
and
corneal-
compensated intraocular pressure (IOPcc) compared with the baseline
group of white ethnicity. CI ¼ confidence interval. Body Mass Index Again, it was the introduction of
SBP (IOPg) and SBP and pulse rate (IOPcc) into the
model that negated the association with BMI. g
g
y
g
The large size of this study allows us to further examine
the relationship of age with IOP in different ethnic groups. The increase in both IOPg and IOPcc with age was greatest
among those of mixed ethnicities, followed by blacks and
whites. The trend among Chinese and “other” ethnicities
was not clear, and this in part could be due to these 2 groups
having relatively smaller numbers, and the size of the
change in IOP per decade of age could inherently be small. The trend of increase in OAG prevalence with age has
been examined in 2 recently published meta-analyses, and
both studies found Hispanics to have the steepest rate of
increase in OAG cases with age.31,32 However, both studies
also confirmed that the prevalence of OAG was actually
highest among blacks, followed by Hispanics and Asians,
and lowest among white subjects. The large size of this study allows us to further examine
the relationship of age with IOP in different ethnic groups. The increase in both IOPg and IOPcc with age was greatest
among those of mixed ethnicities, followed by blacks and
whites. The trend among Chinese and “other” ethnicities
was not clear, and this in part could be due to these 2 groups
having relatively smaller numbers, and the size of the
change in IOP per decade of age could inherently be small. The trend of increase in OAG prevalence with age has
been examined in 2 recently published meta-analyses, and
both studies found Hispanics to have the steepest rate of
increase in OAG cases with age.31,32 However, both studies
also confirmed that the prevalence of OAG was actually
highest among blacks, followed by Hispanics and Asians,
and lowest among white subjects. Sex We found IOP to be higher in men than in women after
adjusting for confounders. This contrasts with several studies
that found IOP to be higher in women than in men7,8,10,18 or
found no difference,5,9,11,13,30 but it is supported by 1 study
that used noncontact tonometry.21 In addition, meta-analyses
of the prevalence of OAG have consistently shown men to be
1.36 to 1.37 times more likely than women to have OAG after
adjusting for age, race, and study design.32,33 A possible
reason other studies found different associations with IOP
could be their smaller sample sizes, which could be under-
powered to detect the difference, although there could be true
differences in the populations surveyed. Refractive Error Refractive error was the second most important predictor of
both IOPg and IOPcc. Higher IOP was associated with
increasing myopic refraction, which persisted even if
pseudophakic
participants
were
excluded
(results
not
shown). This corroborates other studies that reported
refractive error8,13,18,30 and studies that reported IOP in-
creases with longer axial length.7,12,18 Myopia is a well-
established risk factor for glaucoma,36e38 although the
exact mechanism is unknown. The current results support, at
least in part, an IOP-related mechanism. Although we cannot relate the observations of Hispanics to
our study because they were not identified as a separate group,
it seems that the trend in OAG prevalence found in recent
studies mirrors our findings in ethnic differences in IOPcc. Elevated IOPcc could be a useful indicator of OAG risk. Age With the large number of participants in the UK
Biobank, we were also able to demonstrate that white par-
ticipants had significantly higher IOPg than Asians, Chi-
nese, and those with mixed or other ancestries. However,
with IOPcc, the differences between whites and Asians, and
those with mixed and other ancestries, were no longer sig-
nificant, indicating that corneal biomechanics attenuated the
observed differences in IOPg. Of note, Chinese participants
had the lowest IOPcc and the second-lowest IOPg, indi-
cating little attenuation by corneal biomechanics. This cor-
roborates with the finding that the IOPg and IOPcc among
Chinese
participants
showed
no
statistical
difference
(P ¼ 0.52, paired t test), whereas all other ethnic groups
showed significant differences (all P < 0.001, paired t test). 778 Chan et al
Associations with IOP in the UK Biobank Season The effect of seasonality on IOP has been shown in longitu-
dinal studies in Sweden39 and Shanghai,40 and among ocular
hypertensives in Pakistan41 and the United States, as well as
cross-sectional population studies in the United States8 and
Barbados.10 These studies all demonstrated higher IOP in
the colder months than the warmer months. Our IOPg and
IOPcc data also corroborated these findings and showed
that the trend is not restricted to applanation tonometry. Temperature, hydration, and daylight hours41,42 all have
been suggested as possible explanations. 779 Figure 3. Variation of Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) with age for each ethnic
group. The error bars represent the 95% confidence intervals (CIs). Ophthalmology
Volume 123, Number 4, April 2016 Ophthalmology
Volume 123, Number 4, April 2016 Figure 3. Variation of Goldmann-correlated intraocular pressure (IOPg) and corneal-compensated intraocular pressure (IOPcc) with age for each ethnic
group. The error bars represent the 95% confidence intervals (CIs). Center of Assessment Swansea. The findings remain in multivariable regression, in
which IOPg was similar between the baseline center of
Croydon and all other centers, and IOPcc was significantly
lower in Birmingham than Croydon. Sensitivity analysis
was performed by excluding data from Birmingham, and the
main findings of differential associations of IOPg and IOPcc
with the physical characteristics remained. Swansea. The findings remain in multivariable regression, in
which IOPg was similar between the baseline center of
Croydon and all other centers, and IOPcc was significantly
lower in Birmingham than Croydon. Sensitivity analysis
was performed by excluding data from Birmingham, and the
main findings of differential associations of IOPg and IOPcc
with the physical characteristics remained. The 6 centers are widely distributed geographically within
the United Kingdom, covering the midlands (Birmingham),
northern England (Liverpool, Sheffield), Wales (Swansea),
and Greater London (Hounslow, Croydon). They each
contributed different proportions of subjects to the study
cohort, with Swansea accounting for only 0.4% of the study. Participants in these centers also differed in many physical
characteristics. Initial analysis of variance analysis showed
IOPg to be similar between the centers, but IOPcc was
significantly higher in Birmingham than all centers except 1. de Voogd S, Ikram MK, Wolfs RC, et al. Incidence of open-
angle glaucoma in a general elderly population: the Rotter-
dam Study. Ophthalmology 2005;112:1487–93. Chan et al
Associations with IOP in the UK Biobank Shown are regression coefficients for age (mmHg IOP/10 years of age) in
multivariable linear models for each ethnic group, with IOPg and IOPcc as
dependent variables. The following covariates were used in every model: sex,
center of assessment, deprivation index, height, body mass index, systolic
blood pressure, pulse, refractive error, smoking status, self-reported diabetes,
glaucoma and macular degeneration, and seasons. P < 0.001 shown in bold. 8. Klein BE, Klein R, Linton KL. Intraocular pressure in an
American community. The Beaver Dam Eye Study. Invest
Ophthalmol Vis Sci 1992;33:2224–8. 9. Foster PJ, Machin D, Wong T-Y, et al. Determinants of
intraocular pressure and its association with glaucomatous
optic neuropathy in Chinese Singaporeans: the Tanjong Pagar
Study. Invest Ophthalmol Vis Sci 2003;44:3885–91. population studies that reported associations with IOP.7,9
This allows weaker associations to be shown. However,
the price of achieving such a large sample size efficiently is
a low study response rate (5.5%). Together with the
volunteer nature and the relatively young age group of <70
years, the study participants are likely to be a healthier
sample of the UK population, and therefore are unrepre-
sentative of the general population. Nevertheless, a diverse
range of exposures and characteristics are likely to have
been captured in such a large study, such that the results
reported can still be applicable to other populations with a
different distribution of these exposures. The self-reported
nature of diabetes, glaucoma, and macular degeneration
could affect the observed associations because of recall and
misclassification errors. However, more advanced diseases
were likely to be included and to bias the outcome by
increasing the likelihood of an association being found. 10. Wu SY, Leske MC. Associations with intraocular pressure in
the Barbados Eye Study. Arch Ophthalmol 1997;115:1572–6. 11
id k
i
l O
l
d
i 11. Kawase K, Tomidokoro A, Araie M, et al. Ocular and systemic
factors related to intraocular pressure in Japanese adults: the
Tajimi study. Br J Ophthalmol 2008;92:1175–9. 12. Tomoyose E, Higa A, Sakai H, et al. Intraocular pressure and
related systemic and ocular biometric factors in a population-
based study in Japan: the Kumejima study. Am J Oph-
thalmol 2010;150:279–86. 13. Jonas JB, Nangia V, Matin A, et al. Intraocular pressure and
associated factors: the central India eye and medical study. J Glaucoma 2011;20:405–9. 14. Doughty MJ, Zaman ML. Chan et al
Associations with IOP in the UK Biobank Human corneal thickness and its
impact on intraocular pressure measures: a review and meta-
analysis approach. Surv Ophthalmol 2000;44:367–408. 15. Liu J, Roberts CJ. Influence of corneal biomechanical prop-
erties on intraocular pressure measurement: quantitative anal-
ysis. J Cataract Refract Surg 2005;31:146–55. Another limitation of this study is that only 1 IOP mea-
surement was made for each participant, rendering the data
more prone to measurement error than if multiple measure-
ments were taken. However, it is reassuring that the standard
deviation of 3.8 to 3.9 mmHg for IOP in this study is com-
parable to 3.7 mmHg reported in another population study
using the ORA, which used an average of 3 measurements.18 16. Medeiros FA, Weinreb RN. Evaluation of the influence of corneal
biomechanical properties on intraocular pressure measurements
using the ocular response analyzer. J Glaucoma 2006;15:364–70. 17. Luce D. Methodology for cornea compensated IOP and corneal
resistance factor for the Reichert Ocular Response Analyzer. Invest Ophthalmol Vis Sci 2006;47. E-Abstract 2266. In conclusion, this is the largest study of associations of
IOP with demographic and systemic factors to date. It has
confirmed many known associations and demonstrated
previously unknown differential associations with IOPg and
IOPcc. The findings provide insight into the relationship
between corneal biomechanics with systemic factors and
their effect on IOP measurements. 18. Foster PJ, Broadway DC, Garway-Heath DF, et al. Intraocular
pressure and corneal biomechanics in an adult British popu-
lation: the EPIC-Norfolk eye study. Invest Ophthalmol Vis Sci
2011;52:8179–85. 19. Yazgan S, Celik U, Kaldirim H, et al. Evaluation of the rela-
tionship between corneal biomechanic and HbA1C levels in
type 2 diabetes patients. Clin Ophthalmol 2014;8:1549–53. 20. Lee AJ, Rochtchina E, Wang JJ, et al. Does smoking affect
intraocular pressure? Findings from the Blue Mountains Eye
Study. J Glaucoma 2003;12:209–12. Study Strengths and Limitations The strength of this study is the large sample size of 110 573
participants, which is 19 to 55 times larger than to most 780 Chan et al
Associations with IOP in the UK Biobank Chan et al
Associations with IOP in the UK Biobank Table 6. Relationship of Age with Goldmann-Correlated Intra-
ocular Pressure and Corneal-Compensated Intraocular Pressure in
Different Ethnic Groups 2. Leske MC, Heijl A, Hyman L, et al. Predictors of long-term
progression in the early manifest glaucoma trial. Ophthal-
mology 2007;114:1965–72. Table 6. Relationship of Age with Goldmann-Correlated Intra-
ocular Pressure and Corneal-Compensated Intraocular Pressure in
Different Ethnic Groups
IOPg
IOPcc
b (95% CI)
P Value
b (95% CI)
P Value
White
0.18 (0.15e0.21)
<0.001
0.51 (0.47e0.54)
<0.001
Asian
0.07 (0.09 to 0.24)
0.39
0.25 (0.09e0.41)
<0.001
Black
0.42 (0.23e0.60)
<0.001
0.54 (0.35e0.73)
<0.001
Chinese 0.52 (1.04 to 0.01)
0.06
0.37 (0.84 to 0.11)
0.13
Mixed
0.55 (0.21e0.90)
<0.001
0.64 (0.30e0.98)
<0.001
Others
0.13 (0.16 to 0.41)
0.38
0.29 (0.02e0.56)
0.03
CI ¼ confidence interval; IOPcc ¼ corneal-compensated intraocular
pressure; IOPg ¼ Goldmann-correlated intraocular pressure. Shown are regression coefficients for age (mmHg IOP/10 years of age) in
multivariable linear models for each ethnic group, with IOPg and IOPcc as
dependent variables. The following covariates were used in every model: sex,
center of assessment, deprivation index, height, body mass index, systolic
blood pressure, pulse, refractive error, smoking status, self-reported diabetes,
glaucoma and macular degeneration, and seasons. P < 0.001 shown in bold. gy
3. Carbonaro F, Andrew T, Mackey DA, et al. Heritability of
intraocular pressure: a classical twin study. Br J Ophthalmol
2008;92:1125–8. IOPg
IOPcc
b (95% CI)
P Value
b (95% CI)
P Value
White
0.18 (0.15e0.21)
<0.001
0.51 (0.47e0.54)
<0.001
Asian
0.07 (0.09 to 0.24)
0.39
0.25 (0.09e0.41)
<0.001
Black
0.42 (0.23e0.60)
<0.001
0.54 (0.35e0.73)
<0.001
Chinese 0.52 (1.04 to 0.01)
0.06
0.37 (0.84 to 0.11)
0.13
Mixed
0.55 (0.21e0.90)
<0.001
0.64 (0.30e0.98)
<0.001
Others
0.13 (0.16 to 0.41)
0.38
0.29 (0.02e0.56)
0.03 4. Chang TC, Congdon NG, Wojciechowski R, et al. Determinants
and heritability of intraocular pressure and cup-to-disc ratio in a
defined older population. Ophthalmology 2005;112:1186–91. 5. Weih LM, Mukesh BN, McCarty CA, et al. Association of
demographic, familial, medical, and ocular factors with intra-
ocular pressure. Arch Ophthalmol 2001;119:875–80. 6. Wang D, Huang W, Li Y, et al. Intraocular pressure, central
corneal thickness, and glaucoma in Chinese adults: the Liwan
eye study. Am J Ophthalmol 2011;152:454–462 e1. 7. Memarzadeh F, Ying-Lai M, Azen SP, et al. Associations with
intraocular pressure in Latinos: the Los Angeles Latino Eye
Study. Am J Ophthalmol 2008;146:69–76. Ophthalmology
Volume 123, Number 4, April 2016 Ophthalmology
Volume 123, Number 4, April 2016 Central India Eye and Medical Study. Graefes Arch Clin Exp
Ophthalmol 2010;248:1657–66. systematic
review
and
meta-analysis. Ophthalmology
2014;121:2081–90. systematic
review
and
meta-analysis. Ophthalmology
2014;121:2081–90. Central India Eye and Medical Study. Graefes Arch Clin Exp
Ophthalmol 2010;248:1657–66. 23. Wong TY, Foster PJ, Johnson GJ, et al. The relationship between
ocular dimensions and refraction with adult stature: the Tanjong
Pagar Survey. Invest Ophthalmol Vis Sci 2001;42:1237–42. 33. Rudnicka AR, Mt-Isa S, Owen CG, et al. Variations in primary
open-angle glaucoma prevalence by age, gender, and race: a
Bayesian meta-analysis. Invest Ophthalmol Vis Sci 2006;47:
4254–61. 24. Kaji Y. Prevention of diabetic keratopathy. Br J Ophthalmol
2005;89:254–5. 34. Ramos RF, Sumida GM, Stamer WD. Cyclic mechanical stress
and trabecular meshwork cell contractility. Invest Ophthalmol
Vis Sci 2009;50:3826–32. 25. Galor A, Lee DJ. Effects of smoking on ocular health. Curr
Opin Ophthalmol 2011;22:477–82. 35. Mori K, Ando F, Nomura H, et al. Relationship between
intraocular pressure and obesity in Japan. Int J Epidemiol
2000;29:661–6. 26. Brandt JD, Beiser JA, Kass MA, et al. Central corneal thick-
ness in the Ocular Hypertension Treatment Study (OHTS). Ophthalmology 2001;108:1779–88. 36. Xu L, Wang Y, Wang S, et al. High myopia and glaucoma
susceptibility
the
Beijing
Eye
Study. Ophthalmology
2007;114:216–20. 27. Detry-Morel M, Jamart J, Hautenauven F, et al. Comparison of
the corneal biomechanical properties with the Ocular Response
Analyzer (ORA) in African and Caucasian normal subjects and
patients with glaucoma. Acta Ophthalmol 2012;90:e118–24. 37. Wong TY, Klein BE, Klein R, et al. Refractive errors, intra-
ocular
pressure,
and
glaucoma
in
a
white
population. Ophthalmology 2003;110:211–7. 28. Rochtchina E, Mitchell P, Wang JJ. Relationship between age
and intraocular pressure: the Blue Mountains Eye Study. Clin
Experiment Ophthalmol 2002;30:173–5. 38. Grodum K, Heijl A, Bengtsson B. Refractive error and glau-
coma. Acta Ophthalmol Scand 2001;79:560–6. 29. Wong TT, Wong TY, Foster PJ, et al. The relationship of
intraocular pressure with age, systolic blood pressure, and
central corneal thickness in an Asian population. Invest Oph-
thalmol Vis Sci 2009;50:4097–102. 39. Bengtsson B. Some factors affecting the distribution of intra-
ocular pressures in a population. Acta Ophthalmol (Copenh)
1972;50:33–46. 40. Qureshi IA, Xi XR, Lu HJ, et al. Effect of seasons upon
intraocular pressure in healthy population of China. Korean J
Ophthalmol 1996;10:29–33. 30. Xu L, Li J, Zheng Y, et al. Ophthalmology
Volume 123, Number 4, April 2016 Intraocular pressure in Northern
China in an urban and rural population: the Beijing eye study. Am J Ophthalmol 2005;140:913–5. 31. Kapetanakis VV, Chan MPY, Foster PJ, et al. Global varia-
tions and time trends in the prevalence of primary open angle
glaucoma (POAG): a systematic review and meta-analysis. Br J Ophthalmol 2016;100:86–93. 41. Qureshi IA, Xiao RX, Yang BH, et al. Seasonal and diurnal
variations of ocular pressure in ocular hypertensive subjects in
Pakistan. Singapore Med J 1999;40:345–8. 42. Stoupel E, Goldenfeld M, Shimshoni M, et al. Intraocular
pressure (IOP) in relation to four levels of daily geomagnetic and
extreme yearly solar activity. Int J Biometeorol 1993;37:42–5. p
32. Tham YC, Li X, Wong TY, et al. Global prevalence of glau-
coma and projections of glaucoma burden through 2040: a References 21. Hoehn R, Mirshahi A, Hoffmann EM, et al. Distribution of
intraocular pressure and its association with ocular features
and cardiovascular risk factors: the Gutenberg Health Study. Ophthalmology 2013;120:961–8. 22. Nangia V, Jonas JB, Matin A, et al. Body height and ocular
dimensions in the adult population in rural Central India. The 781 Abbreviations and Acronyms: BMI
¼
body
mass
index;
CCT
¼
central
corneal
thickness;
CI
¼
confidence
interval;
DBP
¼
diastolic
blood
pressure;
IOP ¼ intraocular pressure; IOPcc ¼ corneal-compensated intraocular
pressure; IOPg ¼ Goldmann-correlated intraocular pressure; OAG ¼ open-
angle glaucoma; ORA ¼ Ocular Response Analyzer; SBP ¼ systolic blood
pressure. BMI
¼
body
mass
index;
CCT
¼
central
corneal
thickness;
CI
¼
confidence
interval;
DBP
¼
diastolic
blood
pressure;
IOP ¼ intraocular pressure; IOPcc ¼ corneal-compensated intraocular
pressure; IOPg ¼ Goldmann-correlated intraocular pressure; OAG ¼ open-
angle glaucoma; ORA ¼ Ocular Response Analyzer; SBP ¼ systolic blood
pressure. BMI
¼
body
mass
index;
CCT
¼
central
corneal
thickness;
CI
¼
confidence
interval;
DBP
¼
diastolic
blood
pressure;
IOP ¼ intraocular pressure; IOPcc ¼ corneal-compensated intraocular
pressure; IOPg ¼ Goldmann-correlated intraocular pressure; OAG ¼ open-
angle glaucoma; ORA ¼ Ocular Response Analyzer; SBP ¼ systolic blood
pressure. Financial Disclosure(s): The author(s) have made the following disclosure(s): P.J.F.: Grant e Alcon;
Personal fees Allergan and Zeiss; Nonfinancial support Heidelberg
during the conduct of the study. The author(s) have made the following disclosure(s): P.J.F.: Grant e Alcon; Personal fees Allergan and Zeiss; Nonfinancial support Heidelberg
during the conduct of the study. UK Biobank was established by the Wellcome Trust medical charity,
Medical Research Council, Department of Health, Scottish Government,
and Northwest Regional Development Agency. It also had funding from the
Welsh Assembly Government, British Heart Foundation, and Diabetes UK. Footnotes and Financial Disclosures Originally received: March 17, 2015. Originally received: March 17, 2015. A.P.K.: Wellcome Trustefunded Clinical Research Fellow (094791/Z/10/Z). A.P.K.: Wellcome Trustefunded Clinical Research Fellow (094791/Z/10/Z). Final revision: October 3, 2015. P.J.F.: Supported by the Richard Desmond Charitable Trust via Fight for
Sight (1956), the Special Trustees of Moorfields Eye Hospital (ST 12 09),
and the Department for Health through an award made by the NIHR
Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation
Trust (BRC2_009). The funding organizations had no role in the design or
conduct of this research. P.J.F.: Supported by the Richard Desmond Charitable Trust via Fight for
Sight (1956), the Special Trustees of Moorfields Eye Hospital (ST 12 09),
and the Department for Health through an award made by the NIHR
Biomedical Research Centre at Moorfields Eye Hospital NHS Foundation
Trust (BRC2_009). The funding organizations had no role in the design or
conduct of this research. Accepted: November 15, 2015. Available online: January 12, 2016. 1 NIHR Biomedical Research Centre, Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, London, United
Kingdom. 2 Department of Public Health & Primary Care, University of Cambridge,
Cambridge, United Kingdom. Author Contributions: Author Contributions: Conception and design: Chan, Grossi, Khawaja, Yip, Foster 3 School of Optometry & Vision Sciences, Cardiff University, Cardiff,
United Kingdom. Data collection: Chan, Grossi, Khawaja, Yip, Foster Analysis and interpretation: Chan, Grossi, Khawaja, Yip, K-T Khaw, Patel,
Morgan Vernon, Foster 4 Department of Ophthalmology, Nottingham University Hospital NHS
Trust, Nottingham, United Kingdom. Morgan Vernon, Foster Obtained funding: Not applicable Presented at: the Association for Research in Vision and Ophthalmology
Annual Meeting, May 3e7, 2015, Denver, Colorado. Overall responsibility: Chan, Grossi, Khawaja, Yip, K-T Khaw, Patel, PT
Khaw, Morgan, Vernon, Foster Overall responsibility: Chan, Grossi, Khawaja, Yip, K-T Khaw, Patel, PT
Khaw, Morgan, Vernon, Foster *A list of the UK Biobank Eye and Vision Consortium members is avail-
able at www.aaojournal.org. Correspondence: Correspondence: Paul J. Foster, PhD, FRCOphth, Division of Genetics and Epidemiology,
UCL Institute of Ophthalmology, 11-43 Bath St., London EC1V 9EL, UK. E-mail: p.foster@ucl.ac.uk. M.P.Y.C.: Funded by an MRC/RCOphth Clinical Training Fellowship
(G1001939/1) and the International Glaucoma Association. 782
|
https://openalex.org/W4298623089
|
https://hal-insu.archives-ouvertes.fr/insu-03836300/file/s11053-022-10116-w.pdf
|
English
| null |
Tectonic Regime as a Control Factor for Crustal Fault Zone (CFZ) Geothermal Reservoir in an Amagmatic System: A 3D Dynamic Numerical Modeling Approach
|
Natural resources research
| 2,022
|
cc-by
| 12,620
|
To cite this version: H. Duwiquet, F. Magri, S. Lopez, T. Guillon, L. Arbaret, et al.. Tectonic Regime as a Control Factor
for Crustal Fault Zone (CFZ) Geothermal Reservoir in an Amagmatic System: A 3D Dynamic Numer-
ical Modeling Approach. Natural Resources Research, 2022, 31 (6), pp.3155-3172. 10.1007/s11053-
022-10116-w. insu-03836300 Tectonic Regime as a Control Factor for Crustal Fault
Zone (CFZ) Geothermal Reservoir in an Amagmatic
System: A 3D Dynamic Numerical Modeling Approach
H. Duwiquet, F. Magri, S. Lopez, T. Guillon, L. Arbaret, M. Bellanger, L.
Guillou-Frottier Tectonic Regime as a Control Factor for Crustal Fault
Zone (CFZ) Geothermal Reservoir in an Amagmatic
System: A 3D Dynamic Numerical Modeling Approach
H. Duwiquet, F. Magri, S. Lopez, T. Guillon, L. Arbaret, M. Bellanger, L. Guillou-Frottier Distributed under a Creative Commons Attribution 4.0 International License HAL Id: insu-03836300
https://insu.hal.science/insu-03836300v1
Submitted on 4 Nov 2022 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 Natural Resources Research ( 2022)
https://doi.org/10.1007/s11053-022-10116-w Original Paper H. Duwiquet
,1,2,3,6,7 F. Magri
,4,5 S. Lopez,1 T. Guillon,1 L. Arbaret
,2 M. Bellanger,3
and L. Guillou-Frottier
1,2 ,1,2,3,6,7 F. Magri
,4,5 S. Lopez,1 T. Guillon,1 L. Arbaret
,2 M. Bellanger,3
u-Frottier
1,2 Received 30 March 2022; accepted 27 August 2022 Crustal fault zones provide interesting geological targets for high-temperature geothermal
energy source in naturally deep-fractured basement areas. Field and laboratory studies have
shown the ability of these systems to let fluid flow down to the brittle–ductile transition. However, several key questions about exploration still exist, in particular the fundamental
effect of tectonic regimes on fluid flow in fractured basement domains. Based on poro-
elasticity assumption, we considered an idealized 3D geometry and realistic physical prop-
erties. We examined a model with no tectonic regime (benchmark experiment) and a model
with different tectonic regimes, namely a compressional, an extensional and a strike-slip
tectonic regime. Compared to the benchmark experiment, the results demonstrate that
different tectonic regimes cause pressure changes in the fault/basement system. The tectonic-
induced pressure changes affect convective patterns, onset of convection as well as the
spatial extent of thermal plumes and the intensity of temperature anomalies. Driven by
poro-elastic forces, temperature anomalies around vertical faults in a strike-slip tectonic
regime have a spatial extent that should be considered in preliminary exploratory phases. KEY WORDS: Crustal Fault Zone, Geothermal energy, Tectonic regime, Poro-elasticity driven force,
3D dynamic THM numerical modeling. 1BRGM, Av. C. Guillemin, BP 36009 45060 Orle´ans Cedex 2,
France.
2Univ. Orle´ans, CNRS, BRGM, ISTO, UMR7327, 45071 Orle´ans,
France.
3TLS-Geothermics, 91 chemin de Gabardie, 31200 Toulouse,
France.
4Division Research/International, BASE, The Federal Office for
the Safety of Nuclear Waste Management, Berlin, Germany.
5Institute of Geological Sciences, Hydrogeology Group, Freie
Universita¨t Berlin, Berlin, Germany.
6IFPEN, 1-4 avenue Bois-Pre´au, 92852 Rueil-Malmaison, France.
7To whom correspondence should be addressed; e-mail: hugo.-
duwiquet@ifpen.fr H. Duwiquet et al. H. Duwiquet et al. such as Soultz-sous-Foreˆts, Upper Rhine Graben,
France (Genter et al., 2010). However, the seismicity
induced by these injection phases have jeopardized
several geothermal projects (Evans et al., 2005;
Deichmann & Giardini, 2009). Between 2020 and
2021 in Alsace (France) a series of induced earth-
quakes
of
magnitude
M = 3,
M = 3.6,
M = 3.9
caused the definitive shutdown of the Vendenheim
geothermal project and raised doubts in the popu-
lation. Since
2020,
the
United
Downs
Deep
Geothermal Project (UDDGP) is attempting to
target a naturally fractured reservoir in the heat-
producing Cornish granite (Ledingham et al., 2019;
Paulillo et al., 2020). Drilling through the sub-ver-
tical, strike-slip Porthtowan fault zone has induced
an earthquake of magnitude M = 1.5 (www.induce
dearthquakes.org). fractures (Chester & Logan, 1986; Schulz & Evans,
1998). Field and laboratory observations show a
succession of damage zones and fault cores corre-
sponding to the multiple fault core conceptual
model initially described by Faulkner et al. (2003). CFZs are present around the globe. In a few
examples, they are found in Chile—Atacama Fault
System (Mitchell and Faulkner, 2009), Germany–
Badenweiler–Lenzkirch Suture (Brockamp et al.,
2015), or Finland–Pasmaja¨rvi Fault Zone (Ojala
et al., 2019). These structures can be located in dif-
ferent geological settings (magmatic or amagmatic)
and different tectonic stress regimes (extensional,
compressional, strike-slip). This paper investigates
the role of tectonic regimes on geothermal reservoirs
within and around CFZs in amagmatic systems. g
y
Although CFZs provide potential renewable
and economic geothermal resources, they remain
largely unexplored and therefore undeveloped. A
thorough understanding of the processes that impact
fluid flow and heat transfer is a prerequisite for
successful geothermal exploration (Rowland & Sib-
son, 2004). Fluid-filled fractures have been observed
down to mid-crustal depths, as shown by the deep
boreholes of the Kola Peninsula (Kozlovsky, 1984),
and the German KTB continental deep drill hole
(Grawinkel & Sto¨ckhert, 1997; Ito & Zoback, 2000). Additionally, Famin et al. (2004) suggested that
massive infiltration of surface-derived fluids oc-
curred in a detachment shear zone of Tinos Island
(Greece), down to a depth of 10–15 km. Similarly,
Siebenaller et al. (2013) demonstrated that meteoric
fluid infiltration occurs down to the BDT (around a
depth of 8 km) in the Naxos detachment fault
(Greece). INTRODUCTION Usually, the formation of a geothermal resource
requires the presence of fluid, a drain and a heat
source. Permeable faults are natural drains for fluids
circulation and attract rising scientific and economic
interest for mineral (e.g., lithium) and geothermal
resources (Saevarsdottir et al., 2014; Liang et al.,
2018; Guillou-Frottier et al., 2020). Crustal fault
zones (CFZs) are hundreds to thousands of meter-
wide geological structures that localize deformation
(Ben-Zion & Rovelli, 2014) and modify the petro-
physical properties of the crust from the surface
down to the brittle–ductile transition (BDT). CFZs
are defined by faults intersection and interconnected 1BRGM, Av. C. Guillemin, BP 36009 45060 Orle´ans Cedex 2,
France. 2Univ. Orle´ans, CNRS, BRGM, ISTO, UMR7327, 45071 Orle´ans,
France. 3 3TLS-Geothermics, 91 chemin de Gabardie, 31200 Toulouse,
France. 6IFPEN, 1-4 avenue Bois-Pre´au, 92852 Rueil-Malmaison, France. 7To whom correspondence should be addressed; e-mail: hugo.-
duwiquet@ifpen.fr 2022 The Author(s) METHODS The Comsol Multiphysics software is based on
the finite element method (FEM) and can model,
among other various physical processes, fluid flow,
heat transfer and elastic deformation of materials in
a 3D geometry. The Comsol Multiphysics is a well-
known tool offering a complete access to the solu-
tion of partial differential equations. Benchmark
tests have already been performed with previous
numerical
codes
(OpenGeoSys,
Comsol
Multi-
physics v3.5a, and Comsol Multiphysics v5.4, see
Guillou-Frottier et al., (2020) and Appendix 1). (
)
pp
)
We
considered
an
idealized
and
synthetic
model that represents a typical vertical fault zone of
400 m thickness, in the middle of a 5.5 km side cubic
volume of homogeneous basement rock. The fault
zone, which corresponded to a multiple fault core,
was treated like continuous porous medium. At very
high fracture density, this assumption is reasonable
(Zareidarmiyan et al., 2020). The mesh was defined
by 15,785 tetrahedra, with mesh sizes of 500 m for
low permeability zones and mesh sizes of 170 m for
high permeability zones. Preliminary convergence
tests showed that a finer mesh size gave the same
results. The transient simulations were run up to
990 kyrs. The vertical faces of the basement were
thermally
insulated
and
fluid
circulation
was
blocked. On the upper horizontal face, a tempera-
ture of 20 C and a pressure of 105 Pa were imposed
(Fig. 1). (
)
Here, we investigate the role of tectonic re-
gimes on the formation of an amagmatic geothermal
reservoir (i.e., the only heat source is the natural
geothermal flux) within a CFZ. We propose a faul-
ted
3D
THM
(thermal–hydraulic–mechanical)
numerical model with a simplified geometry. The
physical properties are realistic and the permeability
is adapted to the fractured environment. In order to
understand the role of tectonic regimes, we first
considered a benchmark experiment that neglects
tectonic stress. This result is compared to several
numerical experiments where tectonic stresses are
implemented. (
g
)
The initial thermal regime corresponded to a
geothermal gradient of 30 C/km. At the base of the
model a heat flux of 100 Mw m2 was imposed. This
heat flux represents the sum of the mantle heat flow
and the heat emitted by the decay of radioactive
elements in the underlying crust. H. Duwiquet et al. Recent work has shown that pressure and
temperature conditions can induce fluid flow close to
the BDT (Violay et al., 2017; Watanabe et al., 2017,
2021). q
g)
The complex nature of the interactions between
thermal (T), hydraulic (H), mechanical (M) and
chemical (C) processes affect the initial state and
dynamic behavior of the geothermal reservoir under
natural conditions. Theoretical and numerical mod-
eling has been used since 1945 to understand the
controlling factors of fluid circulations (Horton &
Rogers, 1945; Katto & Masuoka, 1967; Horne, 1979;
Forster & Smith, 1989; Lo´pez & Smith, 1995;
OSullivan et al., 2001; Magri et al., 2016; Guillou-
Frottier et al., 2020). Tectonic deformation has been
considered as driving forces that influence fluid flow
in different geological contexts (Ord & Oliver, 1997;
Cox, 1999; Rowland & Sibson, 2004). Bethke (1985)
shows that compressive tectonic settings can lead to
increased fluid pressure and favor upward move-
ment. Sibson (1987) links overpressure and upward
movement to fault-valve activity. In extensional
tectonic settings, the generated under-pressure ap-
pears to cause downward fluid migration (McLellan
et al. 2004). Cui et al. (2012) used simplified 2D
models with a fault zone to show that with degrees
of shortening exceeding 1% fluid flow is affected. In
3D models, Eldursi et al. (2020) suggest that during
tectonically active periods, the decrease in pore
pressure can reorient fluid flow in fractured zones. Nevertheless, none of them have looked at the effect
of tectonic regime on fluid flow and on the temper-
ature anomalies in CFZs. The presence of hot fluids, driven by convection
around geothermal wells, is mandatory to reach
production power that leverage the high drilling cost
of deep borehole. When the natural permeability is
insufficient, for reaching economically viable injec-
tion/production flowrates during the plant opera-
tional
phase,
the
strategies
developed
by
the
Enhanced Geothermal
System
(EGS)
technique
consists in increasing the permeability by different
injection phases, among which hydraulic stimula-
tions aim at causing hydro-shearing and/or hydro-
fracturing (e.g., Gischig and Preisig, 2015; Bijay and
Ghazanfari, 2021). These methods have allowed the
development of many geothermal power plants, In CFZs, fluid circulation driven by buoyancy
forces
occurs
through
upward
and
downward
movement localizing positive temperature anoma-
lies at shallow depths (Duwiquet et al., 2019; Guil-
lou-Frottier et al., 2020; Duwiquet, 2022). H. Duwiquet et al. These 2D
and 3D TH (thermal–hydraulic) numerical modeling Tectonic Regime as a Control Factor for Crustal Fault Zone convection, spatial extent of thermal plumes and
intensity of the temperature anomalies. studies have shown that vertical or subvertical deep
deformation zones could
concentrate the most
important temperature anomalies at the lowest
depths. However, these results do not consider
mechanical effects on fluid flow. Among mechanical
processes, the poro-elasticity hypothesis describes
the interaction between fluids and deformation in
the porous medium. Fluids in a reservoir are af-
fected by stresses, whether on their pressure (un-
drained conditions in low-permeable media with,
e.g., increase in pressure under compressive stress
state), or on their circulation (drained conditions in
permeable media with, e.g., convection from more
to less compressed regions). Our previous study,
which included poro-elasticity, suggested that verti-
cal deformation zones oriented at 30 and 70 to a
maximum horizontal stress could correspond to
potential targets for high-temperature geothermal
energy (Duwiquet et al., 2021a; 2022). Thus, the
stress orientation has an effect on fluid flow, as al-
ready suggested by Jiang et al. (2019). In anisotropic
boundary conditions, these effects are expected to
be strongly accentuated. Indeed, they introduce
shearing conditions favorable to dilation. These as-
pects could be treated in mechanical terms, which
would allow the calculation of slip tendency, defined
by the ratio of shear stress to effective normal stress
(Morris et al., 1996). METHODS In
addition, weathering and fracturing processes present
infaultzonestend toincreasethe valueofthePoisson’s
ratio (Heap et al., 2020). The physical properties of the
fluids were identical to the study of Duwiquet et al.,
(2021a), as well as the incorporation of the stress evo-
lution with depth as boundary conditions. However, as
detailed in the following paragraph, here we used
Andersonian assumption to consider different tectonic
regimes in the 3D dynamic numerical models. In order to investigate the influence of tectonic
regimes on fluid flow, we compared a model with no
tectonic stresses applied (which we called the
‘‘benchmark experiment’’) with models where dif-
ferent tectonic regimes were considered. For these
models, we had free boundary condition at the top,
and clamping at the bottom (displacement blocked
in all three directions). For the four vertical sides of
the model, stress boundary conditions were applied. Because we intended to study the impact of the
tectonic regimes, the magnitudes of the boundary
stresses were set so as to be representative of the
three main tectonic regimes namely, compressional,
extensional and strike-slip. We thus built up three
numerical model, one per tectonic regime. For this,
we considered an Andersonian assumption whereby
the principal stresses are expressed with vertical
(Sv), maximum horizontal (SHmax) and minimum
horizontal (Shmin) components, which are used reg-
ularly
in
geomechanical
studies
of
reservoirs
(Anderson, 1905; Zoback et al., 2003): The relative
Table 1. Set of physical parameters used in numerical modeling
Category
Symbols
Fault zone
Basement
Unit
Porosity
U
0.1
0.05
–
Permeability
k
Variable
1016
m2
Thermal conductivity
ks
3
2
W/(m.K)
Heat capacity
Cps
800
800
J/(kg.K)
Bulk density
qs
2700
2700
kg/m3
Young’s modulus
E
5
60
GPa
Poisson’s ratio
m
0.30
0.25
–
Biot–Willis coefficient
aB
0.8
0.8
– Table 1. Set of physical parameters used in numerical modeling Table 1. Set of physical parameters used in numerical modeling
Category
Symbols
Fault zone
Basement
Unit
Porosity
U
0.1
0.05
–
Permeability
k
Variable
1016
m2
Thermal conductivity
ks
3
2
W/(m.K)
Heat capacity
Cps
800
800
J/(kg.K)
Bulk density
qs
2700
2700
kg/m3
Young’s modulus
E
5
60
GPa
Poisson’s ratio
m
0.30
0.25
–
Biot–Willis coefficient
aB
0.8
0.8
– However, in the fault zone we imposed a value of 0.30. Thisvaluecanbeexplainedbytwoaspects,thefirstone
is that the samples on which the Poisson coefficients
were measured were much smaller than the block
considered. METHODS The fluid was as-
sumed to be pure water, and the fluid density de-
pended on pressure and temperature conditions (as
detailed in Duwiquet et al., 2021a). The details on
Darcy law, heat equation and Hooke law can be
found in Duwiquet et al., (2021a). All thermal, hy-
draulic and mechanical parameters are detailed in
Table 1. The effect of the poro-elasticity driven force on
fluid flow will differ from one tectonic regime to an-
other and will impact the formation of a high-temper-
ature geothermal reservoir in fractured environment. The observed differences can be explained mainly by
different lateral fluid pressure variations. This study
highlights the importance of mechanical effects in
geothermal processes and the relevance dealing with
these aspects during the exploratory phase of such
naturally potential reservoir. The tectonic-induced
pressure changes affect fluid flow patterns, onset of The Young’s moduli imposed in numerical mod-
els are suitable for fault zone and basement (Cappa &
Rutqvist, 2011). In the basement, the Poisson’s ratio is
0.25, a value generally accepted in the literature. H. Duwiquet et al. Figure 1. Model setup, boundary conditions and results of the benchmark experiment (+), i.e., without stress application. Pore pressure
(white contour), temperature (color patterns) and flow field (vectors) are displayed. The temperature profile is plotted against depth (right
chart, crosses). When free convection is established, the 150 C isotherm is located at 1.8 km depth. The results with stress application are
shown in Figs. 4 and 5 along the ZX and YX planes. The benchmark experiment reached the steady-state regime at 65 kyrs. Figure 1. Model setup, boundary conditions and results of the benchmark experiment (+), i.e., without stress application. Pore pressure
(white contour), temperature (color patterns) and flow field (vectors) are displayed. The temperature profile is plotted against depth (right
chart, crosses). When free convection is established, the 150 C isotherm is located at 1.8 km depth. The results with stress application are
shown in Figs. 4 and 5 along the ZX and YX planes. The benchmark experiment reached the steady-state regime at 65 kyrs. However, in the fault zone we imposed a value of 0.30. Thisvaluecanbeexplainedbytwoaspects,thefirstone
is that the samples on which the Poisson coefficients
were measured were much smaller than the block
considered. This change of scale tends to increase the
value of the Poisson coefficient (Heap et al., 2020). Tectonic Regime as a Control Factor for Crustal Fault Zone Tectonic Regime as a Control Factor for Crustal Fault Zone magnitudes of these stresses determine the modeled
tectonic regime, thus: <0) brings a more compressive stress (positive
compression convention). We assumed constant
evolution of stress with depth. Notice that, in their
natural state, a strike-slip stress regime holds for
both regions, i.e., r1 ¼ SHmax, r2 ¼ Sv, r3 ¼ SHmin. In
the following, we kept the realistic principal stress
magnitude given in Eqs. 1 and 2 but changed in our
different scenarios the axes along which they oper-
ate. This way, we were able to investigate the effect
of tectonic regimes while keeping realistic stress
ratios. In the end, six numerical models were built
(Table 2) in addition to the benchmark experiment
(with no tectonic stresses applied).
Compressional
(reverse/thrust
faulting),
with
SHmax ‡ Shmin ‡ SV
Extensional
(normal
faulting)
with
SV ‡
SHmax ‡ Shmin
Strike-slip, with SHmax ‡ SV ‡ Shmin Assuming that our model was aligned with the
principal stresses, pure normal stresses were applied on
the lateral boundaries (no shear applied on the bound-
aries). For the compressional regime, the fault was
perpendicular to the SHmax stress applied on the
boundaries. For the extensional regime, the fault was
perpendicular to Shmin. Finally, for the strike-slip one,
thefaultwasat45betweenShminandSHmaxorientation. Notice that in the remainder of this paper, the
color coding used in Eq. 1 and is the same
throughout the study for a better reading of the re-
sults. The red color code corresponds to high stress
intensity and the green color code corresponds to
low stress intensity. In order to allow the conver-
gence of the 3D numerical calculations with this
THM coupling, the application of the stresses was
applied progressively from t = 0 yr until t = 10 yr. The results are shown in steady-state, at 65 kyr for
the benchmark experiment and at 990 kyr for the
model where stresses were applied. In addition, and to understand the possible ef-
fects of stress intensity, stress-depth profiles were
collected on two natural systems, one in the French
Massif Central and one near the San Andreas Fault. Two cases were considered, a low stress intensity
zone (e.g., French Massif Central) and a high stress
intensity zone (e.g., San Andreas Fault). It should be
noted that we assumed that regional stresses prevail
over local stress variations, which is consistent with
our homogeneity assumption. METHODS This change of scale tends to increase the
value of the Poisson coefficient (Heap et al., 2020). In
addition, weathering and fracturing processes present
infaultzonestend toincreasethe valueofthePoisson’s
ratio (Heap et al., 2020). The physical properties of the
fluids were identical to the study of Duwiquet et al.,
(2021a), as well as the incorporation of the stress evo-
lution with depth as boundary conditions. However, as
detailed in the following paragraph, here we used
Andersonian assumption to consider different tectonic
regimes in the 3D dynamic numerical models. ‘‘benchmark experiment’’) with models where dif-
ferent tectonic regimes were considered. For these
models, we had free boundary condition at the top,
and clamping at the bottom (displacement blocked
in all three directions). For the four vertical sides of
the model, stress boundary conditions were applied. Because we intended to study the impact of the
tectonic regimes, the magnitudes of the boundary
stresses were set so as to be representative of the
three main tectonic regimes namely, compressional,
extensional and strike-slip. We thus built up three
numerical model, one per tectonic regime. For this,
we considered an Andersonian assumption whereby
the principal stresses are expressed with vertical
(Sv), maximum horizontal (SHmax) and minimum
horizontal (Shmin) components, which are used reg-
ularly
in
geomechanical
studies
of
reservoirs
(Anderson, 1905; Zoback et al., 2003): The relative In order to investigate the influence of tectonic
regimes on fluid flow, we compared a model with no
tectonic stresses applied (which we called the H. Duwiquet et al. Table 2. Synthesis of the different tectonic regimes and stress intensities considered Stress intensity Tectonic regime Normal stress applied on boundaries
High (Eq 1)
Extensional
Boundaries parallel to fault orientation : Shmin
Boundaries perpendicular to fault orientation : SHmax
Compressional
Boundaries parallel to fault orientation : SHmax
Boundaries perpendicular to fault orientation : Shmin
Strike-slip
Fault orientation oblique towards lateral boundaries
Low (Eq 2)
Extensional
Boundaries parallel to fault orientation : Shmin
Boundaries perpendicular to fault orientation : SHmax
Compressional
Boundaries parallel to fault orientation : SHmax
Boundaries perpendicular to fault orientation : Shmin
Strike-slip
Fault orientation oblique towards lateral boundaries for fractured rock. The ki is permeability (m2) of
intact rock, and kf is permeability (m2) of fractured
rock. The stress state of a fault can be defined
qualitatively in terms of slip tendency, defined by the
ratio of shear stress to effective normal stress
(Morris et al., 1996). In purely elastic domain and
without dissipative phenomena, the slip tendency is
an indicator (high/low general trend) to assess the
stress variation on a fault zone. More specifically,
slip tendency indicates how critically stressed fault
zones are, and how easily they can be reactivated by,
e.g.,
stimulation
methods. Consequently,
some
preferential flow paths may appear under the action
of a given applied tectonic stress (Siler et al., 2019). The slip tendency is defined as: center and two downward circulations at the fault
ends (Fig. 1). As illustrated by colored streamlines
in Figure 1, these fluid circulations transferred heat
by convection. Thermally induced buoyancy forces
drove the flow. The hot and less dense fluid rose to
the surface. As its density increases due to the
cooling effects of the top boundary condition, the
fluid sank within the fault plane leading to a classical
natural convection pattern. Under these conditions,
the free convection generated a thermal disturbance
of the environment. In a purely diffusive setting, the
150 C isotherm was observed at 5 km depth,
whereas here the same isotherm was localized at
1.8 km depth, at the center of the fault. Fluid
velocities were of the order of 1 9 10–9 m.s1 at the
center of the fault and 20 9 10–9 m.s1 at the ends of
the fault. The higher fluid velocities at the bottom
center of the fault were responsible for slight in-
crease in pressure (white lines). H. Duwiquet et al. This trend was al-
ready observed in other numerical TH modeling
(Scott et al., 2017). The temperature profile at the
center of the model was similar to typical tempera-
ture profiles measured in geothermal reservoirs such
as Soultz-sous-Foreˆts (Genter et al., 2010). TS ¼ s
r0n
ð4Þ ð4Þ ð4Þ where s is shear stress and r0n is effective normal
stress defined as: r0
n ¼ rn aB pf
ð
Þ
ð5Þ ð5Þ where rn is total normal stress; aB is the Biot–Willis
coefficient and pf is fluid pressure. Tectonic Regime as a Control Factor for Crustal Fault Zone The stress profiles as a
function of depth corresponded to the following
equations (Cornet & Burlet, 1992; Zoback, 1992): Fluid flow velocities, and thus efficiency of heat
transport by convection through the crust, were
controlled directly by the permeability of the rocks. Permeability is a dynamic and variable parameter
that can change during different geological processes
(Gleeson & Ingebritsen, 2016). The relationship
between permeability and porosity is widely de-
bated. However, although adapted to porous media,
the Kozeny–Carman relationship does not seem to
be appropriate for fractured media (Lamur et al.,
2017; Parisio et al., 2019). Instead, the permeability
k we considered has been empirically demonstrated
to be appropriate for fractured media (Lamur et al.,
2017), thus: High stress intensity:
3 = 1 × 106 + (15000 × (
))
2 = 4 × 106 + (20000 × (
))
1 = 7.5 × 106 + (28667 × (
))
ð1Þ ð1Þ log k ¼ 1 x
ð
Þlogki þ xlogkf
ki ¼ 4:979 1011n3:11
ð3Þ
kf ¼ 1:143 1011n0:64 3 = 0.1 × 106 + (13975 × (
))
2 = 0.1 × 106 + (26225 × (
))
1 = 0.1 × 106 + (28725 × (
))
ð2Þ ð3Þ ð2Þ where x represents the degree of rock fracturing;
x ¼ 1 for fully fractured rock and x ¼ 0 for fully
intact rock. Here, we took x ¼ 0:8 corresponding to
the significant fracturing degree of the CFZ, inter-
spersed with intact zones. The n represents initial
porosity. Here, n = 0.05 for intact rock and n = 0.1 where r1, r2; r3, are the maximum, intermediate and
minimum principal stresses (MPa), respectively; z is
the vertical upwards axis, and an increase in depth ( z H. Duwiquet et al. RESULTS The effects of high and low stress intensity
application (Eqs. 1 and 2 ‘‘method section’’) on the
convective regimes are shown in Figures 2 and 3,
respectively. The results are shown on vertical cross
sections along the fault plane and on horizontal
cross sections, at 2 km depth. For each tectonic re-
gime that was considered, the results showed the
temperature anomalies, the fluid flow pattern and Formation of a High-Temperature Geothermal
Reservoir Within a Vertical Crustal Fault Zone
with No Tectonic Stresses: The Benchmark
Experiment The numerical simulation without stress appli-
cation showed an upward circulation at the fault Tectonic Regime as a Control Factor for Crustal Fault Zone Figure 2. Results of numerical modeling after high stress intensity stresses application (in red, see Eq. 2). The results are shown in
vertical section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. In red, the velocity is the fastest; in blue, the velocity is the slowest. g Figure 2. Results of numerical modeling after high stress intensity stresses application (in red, see Eq. 2). The results are shown in
vertical section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. In red, the velocity is the fastest; in blue, the velocity is the slowest. Figure 2. Results of numerical modeling after high stress intensity stresses application (in red, see Eq. 2). The results are shown in
vertical section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. In red, the velocity is the fastest; in blue, the velocity is the slowest. Formation of a High-Temperature Geothermal
Reservoir Within a Vertical Crustal Fault Zone
with No Tectonic Stresses: The Benchmark
Experiment They differed from the benchmark case by their
number, intensity and lateral extension. the fluid velocities. The results can be compared
directly to the benchmark experiment (Fig. 1). Notable differences existed and are detailed below. In an extensional tectonic regime, two positive
temperature anomalies formed (Figs. 2 and 3). They
were + 55 C in high stress intensity and + 47 C in
low stress intensity. For both stress intensities, the
second temperature anomalies were less intense, +
10 C in high stress intensity and + 7 C in low
stress intensity. For both stress intensities, a negative
temperature anomaly developed at the center of the
fault. This anomaly was 60 C for the high stress Temperature Anomalies Regardless of the considered tectonic regime,
different positive and negative temperature anoma-
lies were observed. In the following, temperature
anomalies correspond to the difference of tempera-
ture resulting from a conductive thermal regime. H. Duwiquet et al. Figure 3. Results of numerical modeling after low stress intensity application (in green, see Eq. 1). The results are shown in vertical
section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. In red, the velocity is the fastest; in blue, the velocity is the slowest. H. Duwiquet et al. Figure 3. Results of numerical modeling after low stress intensity application (in green, see Eq. 1). The results are shown in vertical
section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. In red, the velocity is the fastest; in blue, the velocity is the slowest. Figure 3. Results of numerical modeling after low stress intensity application (in green, see Eq. 1). The results are shown in vertical
section (side view, located in the middle of the fault) and in horizontal section (top view, located at 2 km depth). The scale of
temperature anomalies and fluid flow velocities is different according to the tectonic regimes. For each regime, the maximum and
minimum values of temperatures and fluid flow velocities are indicated. Positive temperature anomalies are colored red, negative
temperature anomalies are colored blue. Fluid circulation is marked by the lines, the direction by the arrows. The color of the lines
corresponds to fluid velocity. Fluid Flow Pattern In extensional tectonic regimes, the fluid flow
pattern was characterized by a downward movement
at the center of the fault and two upward move-
ments at the ends of the fault (Figs. 2 and 3). In high
stress intensity the maximum fluid velocity was
30 9 10–9 m.s1, against
21 9 10–9 m.s1 in low
stress intensity. The minimum fluid velocities were,
for each intensity, 1 9 10–9 m.s1. In horizontal
cross section, at 2 km depth, upward movements
were generated at positive temperature anomalies
while downward movements were generated at
negative temperature anomalies. In high stress
intensity, the maximum fluid velocity was 25 9 10–
9 m.s1, compared to 17 9 10–9 m.s1 in low stress
intensity. The minimum fluid velocity was 5 9 10–
9 m.s1 in high stress intensity, compared to 1 9 10–
9 m.s1 in low stress intensity. In strike-slip regimes and regardless of stress
intensity applied (Figs. 2 and 3), positive tempera-
ture anomaly extended widely along the length of
the fault, from the surface to 4.5 km deep. The
maximum
positive
temperature
anomaly
value
was + 70 C for the high stress intensity (Fig. 2),
and + 62 C for the low stress intensity (Fig. 3). In
horizontal cross section, these temperature anoma-
lies spread largely beyond the fault. Temperature anomalies of + 25 C were found
in the basement, suggesting that in a strike-slip sys-
tem, the positive temperature anomaly, and inde-
pendently from the stress intensity, represents an
important volume. This heat propagation was done
by thermal diffusion from the fault center, where the
temperature anomaly was the most intense. Indeed,
the larger the convection cell inside the fault, the
wider the extent of the diffusive perturbation. At
2 km depth, the maximum value of the temperature
anomaly was + 65 C for the high stress intensity,
and + 61 C for the low stress intensity. Negative
temperature anomalies were present and localized at
the extremities of the fault. They were 45 C for
the high stress intensity and 40 C for the low
stress intensity. In compressional tectonic regimes, for each
stress intensity, there was a slightly different con-
vective pattern. In high stress intensity, there were
two
upward
and
two
downward
movements,
whereas in low stress intensity there were two up-
ward and three downward movements. Temperature Anomalies In red, the velocity is the fastest; in blue, the velocity is the slowest. value
of + 20 C. This + 20 C
anomaly
spread
more
widely
in
the
system
while
the + 41 C
anomaly remained localized on the fault. The neg-
ative anomaly located at the center of the fault was
37 C and extended into the basement in the same
way as the positive + 41 C anomaly. intensity and 57 C for the low stress intensity. For a high stress intensity, at 2 km depth, the hori-
zontal cross section showed a negative temperature
anomaly that reached a maximum of 30 C. This
anomaly covered a large surface area of the fault. The
positive
temperature
anomaly
of + 45 C
occupied the remaining space, but extended further
into the basement. In the basement and up to the
edge of the model, we found a positive temperature
anomaly of + 20 C. For a low stress intensity, at
2 km depth, two positive temperature anomalies and
one negative anomaly were observed. The first
positive anomaly was + 41 C, the second had a In a compressional tectonic regime, indepen-
dently from the stress intensity, two positive tem-
perature anomalies were observed. The value of the
maximum
temperature
anomaly
in
high
stress
intensity was + 60 C and in low intensity it was +
51 C. Depending on the stress intensity, these two
maximum values were spatially slightly shifted Tectonic Regime as a Control Factor for Crustal Fault Zone Tectonic Regime as a Control Factor for Crustal Fault Zone (Figs. 2 and 3). The amplitudes of the second tem-
perature anomaly were + 35 C and + 40 C for low
and high stress intensity, respectively. In the hori-
zontal cross section, the two temperature anomalies
were found for each intensity. In high stress inten-
sity, the values of these two temperature anomalies
were + 56 C and + 47 C. In low stress intensity,
the values were + 47 C and + 35 C. The lateral
extension of these temperature anomalies was lim-
ited. They were surrounded by negative temperature
anomalies that locally reached 47 C in high
intensity and 42 C in low intensity. At a depth of
2 km, the positive temperature anomalies were
much less extended than in extensional tectonic or
strike-slip regimes (see below). Temperature Anomalies temperature distribution, and this was clearly re-
lated to the different convective patterns and fluid
flow velocity, as described below. H. Duwiquet et al. H. Duwiquet et al. of the fault and two downward movements at the
ends of the fault (Figs. 2 and 3). The fluid velocity
varied with stress intensity. In low stress intensity,
the minimum fluid velocity was 1 9 10–9 m.s1 and
the maximum was 12 9 10–9 m.s1 at the downward
movements. In high stress intensity, the minimum
fluid velocity was 1 9 10–9 m.s1 and the maximum
was 17 9 10–9 m.s1. benchmark experiment (Fig. 4). The fluid pressure
varied between the basement and the fault. This
lateral variation differed according to the stress
intensity applied. y
pp
In each of the cases, the tectonic regimes and
the stress intensity generated lateral fluid pressure
variation between the fault and the basement. In
high stress intensity, and for the same horizontal
distance, these lateral pressure differences were 1.5,
0.45, and 0.97 MPa for compressional, strike-slip,
and extensional tectonic regimes, respectively. In
low stress intensity, the lateral pressure differences
were 1.35, 0.3 and 0.91 MPa for compressional,
strike-slip,
and
extensional
tectonic
regimes,
respectively. These lateral fluid pressure differences
drove the fluids from the high-pressure zones (i.e.,
basement) to the low-pressure zone (i.e., fault). With application of tectonic stresses, the fluid
flow pattern was different from the benchmark
experiment,
with
consequences
on
temperature
anomalies and fluid velocities. In the benchmark
experiment, buoyancy was the only driving force for
fluid convection. Here, in the presence of tectonic
stresses, stress induced forces also influenced fluid
flow. Fluid Flow Pattern The maxi-
mum fluid velocity for high stress intensity was
5 9 10–9 m.s1 and for low stress intensity was
10 9 10–9 m.s1. For this tectonic regime, the max-
imum fluid velocity was present when the lowest
stress intensity was applied. In horizontal cross sec-
tion, at 2 km depth and for high stress intensity,
upward movements were localized where tempera-
ture anomalies were positive, whereas downward
movements
were
localized
where
temperature
anomalies were negative. The fluid velocity was
between 1 and 2 9 10–9 m.s1. In low stress inten-
sity, the downward movements were localized at the
extremities and at the center of the fault. The up-
ward
movements
were
localized
between
each
downward movement. The maximum fluid velocity
was 10 9 10–9 m.s1 and the minimum was 1 9 10–
9 m.s1. At a depth of 2 km, fluid velocities varied
from 1 to 2 9 10–9 m.s1. Thus, fluid velocity values
in a compressional regime were the lowest recorded. In the strike slip tectonic regime
as for the To summarize, tectonic regimes influence the
distribution
and
the
amplitude
of
temperature
anomalies. Positive temperature anomalies were
most intense in strike-slip, then in compression and
extension. The spatial extent of positive temperature
anomalies was not identical for each tectonic regime. In strike-slip, these anomalies were largely extended
through the basement. This lateral extension was
less important in the extensive tectonic regime. Fi-
nally, in compressional regime these anomalies were
localized in the near vicinity of the fault. The tec-
tonic regimes have shown to play a key role in In the strike-slip tectonic regime, as for the
benchmark experiment, the fluid flow pattern was
characterized by an upward movement at the center Tectonic Regime as a Control Factor for Crustal Fault Zone Tectonic Regime as a Control Factor for Crustal Fault Zone Figure 5. Time required to reach the steady-state temperature anomalies, depending on the tectonic regimes and the applied
stress intensities. The color code and patterns are the same as those used in the previous figures. Considering the compressional tectonic regime
(solid line), the time needed to reach a steady-state
regime in high stress intensity was 85 kyrs, while it
was 95 kyrs in low stress intensity. This difference
was observed for each regime so that the stress
intensity had a role in the onset of convection. As
can be seen in Figure 5, the compressional regime,
which had the highest lateral fluid pressure differ-
ence, reached the steady-state more quickly than the
other tectonic regimes. The lowest lateral fluid
pressure difference was recorded for the strike-slip
regime. The strike-slip regime reached the steady-
state regime at 203 kyrs for high stress intensity and
223 kyrs for low stress intensity. regime, the temperature anomalies should be more
intense than in a compressive or extensive regime,
but that the onset was the most delayed with respect
to the benchmark case. What effect(s) on the Onset of Positive Temperature? After stress application, the fluid pressure was
different from the benchmark experiment where
pressure was hydrostatic (Fig. 4). Whatever the
stress intensity applied, the experiment with com-
pressional and strike-slip tectonic regimes showed
fluid pressures higher than those in the benchmark
experiment, whereas the extensional tectonic regime
displayed fluid pressures lower than those in the The regional mechanical stresses imposed a
pressure distribution that interacted with buoyancy
forces, which caused free convection patterns. The
lateral fluid pressure differences brought the fluid
from the zones of high pressure to those of low
pressure. However, as shown in Figure 5, the time
needed to set up this temperature anomaly varied. Figure 4. Lateral fluid pressure in high stress intensity application (in red) and low stress intensity application (in green), at 2 km
depth. For comparison, the fluid pressure of the benchmark experiment is indicated (+). With stresses application, the fluid pressure
is different from the benchmark experiment. The lateral fluid pressure variation depends on the tectonic regime and stress intensity. Figure 4. Lateral fluid pressure in high stress intensity application (in red) and low stress intensity application (in green), at 2 km
depth. For comparison, the fluid pressure of the benchmark experiment is indicated (+). With stresses application, the fluid pressure
is different from the benchmark experiment. The lateral fluid pressure variation depends on the tectonic regime and stress intensity. Figure 5. Time required to reach the steady-state temperature anomalies, depending on the tectonic regimes and the applied
stress intensities. The color code and patterns are the same as those used in the previous figures. ectonic Regime as a Control Factor for Crustal Fault Zone Tectonic Regime as a Control Factor for Crustal Fault Zone Permeability and Slip Tendency Variation In the fault, very small permeability variations
were observed as a function of stress intensity and
tectonic regime. For the benchmark experiment, the
fault’s permeability was 1 9 10–12.1 m2. In high
stress intensity, the variation of the permeability
values in the center of the fault was as follows:
0.08%, 0.42% and 0.66% for extensional, strike-slip
and compressional tectonic regimes, respectively. In
low stress intensity, the variation of the permeability
values in the center of the fault was as follows:
0.08%, 0.33%, 0.74% for extensional, strike-slip and
compressional tectonic regimes, respectively. The
effect of stress intensity on permeability remained
negligible. For example, considering the compres-
sional tectonic regime, the difference between high
and low stress intensity was 2.3%. We noted a con-
sistency between the values of fluid velocities and
permeability values. Indeed, the fluid velocities were
the most important for the highest permeability, and The benchmark experiment reached the steady-
state regime as early as 65 kyrs (Fig. 5), and finally
the additional stress input may increase the time to
reach a steady-state in situ. The differences observed
on the different tectonic regimes were consistent
with the differences observed on the lateral fluid
pressure between the fault and the basement. However, pressure differences alone could not ex-
plain the different fluid velocities observed (Figs. 2
and 3), and the mechanical behavior of the base-
ment-fault system after stress application. In a
geothermal exploration context, these numerical
calculations showed that, in a strike-slip tectonic H. Duwiquet et al. Figure 6. Slip tendency in high stress intensity application (in red), and low stress intensity application (in green). Figure 6. Slip tendency in high stress intensity application (in red), and low stress intensity application (in green the least important for the lowest permeability. Once the stresses were applied, the permeability was
slightly different from the benchmark experiment,
and controlled the fluid velocity. between the basement and the fault that the trend
was most important. This result illustrates the
heterogeneity of the mechanical parameters incor-
porated in the numerical model, and it shows further
that the role of the stress intensity on the range of
values
tested
will
not
drastically
change
the
mechanical response of the basement-fault system. The general trend of slip tendency is shown for
each tectonic regime and stress intensity applied
(Fig. 6). Overall, the general trend was higher in
high stress intensity than in low stress intensity ap-
plied. Permeability and Slip Tendency Variation The results showed a distinctive variation in
the fault and in the basement for each tectonic re-
gime. Regardless of the stress intensity applied, the
slip tendency in a strike-slip tectonic regime was the
highest, because it was more critically stressed. In
the strike-slip regime, the slip tendency increased
slightly from the edge of the basement to the level of
the fault zone. At the edge of the fault, the slip
tendency increased significantly. In the middle of the
fault, the slip tendency decreased slightly. This
evolution was the same regardless of the stress
intensity applied. DISCUSSION The presence of fluids and a sufficiently high
permeability are two factors that allow the devel-
opment of a geothermal resource, from a natural
geothermal flux. Considering a simplified geometry,
but realistic physical properties (pressure and tem-
perature-dependent fluid density, temperature-de-
pendent fluid viscosity), our 3D TH and 3D THM
numerical modeling showed that, in a basement
domain, CFZs allow hot fluids to ascend at eco-
nomically viable depth. The comparison of numeri-
cal
models
with
and
without
tectonic
stresses
highlights the non-negligible role of tectonic regime
on the spatial distribution of positive temperature
anomalies. y
pp
In compressional and extensional regimes, the
trend from the edges of the basement to the fault
increased similarly. This increase was slightly greater
in compression than in extension. At the edge of the
fault, the general trend was the same. In compres-
sion, the slip tendency decreased, and in extension it
increased. In extension, the middle of the fault was
less critically stressed than in compression. Regard-
less of the intensity of the stress, this evolution was
the same. Of the three tectonic regimes tested, the
strike-slip regime was the most critically stressed. The general trend showed that it is at the interface Nature and Impact of Poro-elasticity Driven Force of 150 C at 1.8 km depth (Fig. 1). Two downward
movements were located at the ends of the fault. This convective pattern was already observed in 2D
and 3D TH numerical modeling of fault zones in the
crustal domain (Wanner et al., 2019; Guillou-Frot-
tier et al., 2020) and it was called ‘‘bulb-like’’ con-
vective pattern. This particular shape is favored
because hot fluids have less resistance when flowing
upwards (viscosity is smaller at high temperature)
and it can also localize temperature anomalies in the
basement. With a flat topography and without
metamorphic devolatilization and/or magmatic fluid
production that might change fluid pressure (Nur &
Walder, 1990), fluid circulation is driven only by
buoyancy force. With tectonic regimes application,
we saw that other forces are added and modify the
fluid circulation. These numerical models showed that the tested
tectonic regimes had an influence on fluid pressure
variation. The poro-elastic assumption describes the
interaction of fluids and deformation in porous
media. Compressional, extensional, and strike-slip
tectonic regimes induce variable fluid pressures for
each case. The incorporation of heterogeneous
mechanical parameters between the fault and the
basement (see Table 1) led to a different mechanical
response and thus to a heterogeneous variation of
the fluid pressure between the fault and the base-
ment (see Fig. 4). This lateral fluid pressure differ-
ence drove the fluids from the high-pressure zones to
the low-pressure zones. This effect was facilitated by
higher permeability values in the fault than in the
basement. Considering the experiments where tectonic
regimes are accounted for, the obtained results
were different from the benchmark experiment
(Figs. 2 and 3). This highlights the key role of the
relationship between tectonic settings and fluid
flow. In our results, the stress intensity parameter
did not change the general dynamics of convec-
tive
patterns
between
two
identical
regimes. Considering a sensitivity study on a large Pont-
gibaud Crustal Fault Zone (French Massif Cen-
tral), where the stresses boundary magnitudes
were tested while holding their directions, a shift
from one convective pattern to another would
occur for a variation of approximately 40 MPa in
the
maximum
horizontal
stress
magnitude
(Duwiquet et al., 2021a; 2021b). For this last
example and in the light of the observations
made in this study, it could be possible that an-
other force besides buoyancy influences fluid cir-
culation. Tectonic Regime as a Control Factor for Crustal Fault Zone Nature and Impact of Poro-elasticity Driven Force Different Tectonic Regime, Different Fluid Flow Without
stress
application,
the
benchmark
experiment showed an upward movement at the
center of the fault and brought about a temperature Tectonic Regime as a Control Factor for Crustal Fault Zone Tectonic Regime as a Control Factor for Crustal Fault Zone H. Duwiquet et al. H. Duwiquet et al. Duwiquet et al., 2019; 2021a; Gomila et al., 2021). This should be sufficient to allow fluids to flow by
buoyancy forces and transfer heat by free convec-
tion. However, between tectonic regimes, the dif-
ference in permeability cannot explain convective
pattern variability results. Nevertheless, these small
permeability variations influence the velocities of
fluid flow. The fluid velocity decreased with perme-
ability decrease (see Figs. 2, 3, and sub-section
Permeability and Slip Tendency Variation). Actually,
the positive temperature anomalies in the compres-
sion and extensional regime model remained cen-
tered on the fault compared to the strike-slip
tectonic regimes. Therefore, other processes must
limit the development of temperature anomalies in
compressional regime. By concentrating the flow of
fluids on limited spaces, the poro-elasticity driven
force could have the effect of spatially concentrating
temperature anomalies when the difference between
the fluid pressure of the basement and the fault is
large. We used an idealized geometry with single,
vertical fault zone. Hence, the linear stress boundary
conditions give rather homogeneous stress ratios
and states along the fault. In natural systems, the
network of variously oriented and dipping fault
zones brings heterogeneous stress states, even along
each fault zone taken separately. Still, and recalling
that the aim of our study is to better understand the
setup conditions of convection cells, the results al-
ready allow us to highlight the complex impact of
mechanics over convection patterns. Among others,
our conclusions tend to show that within a complex
fault zone network, fault zones undergoing strike-
slip conditions might be the ones to be preferably
explored. Future work, including sensitivity analysis
on parameters, law of behavior, boundary condition
effects, should be conducted to confirm this trend. These fundamental results are generally appli-
cable in nature to any fracture rock that may contain
fluids, gas or oil in its pores, in basement geological
context. However, our results do not take into ac-
count fundamental aspects such as the consideration
of more complex rheology, to start with plastic
phenomena. The effects of fault intersections, pre-
cipitation and dissolution of mineral phases, and
fluid composition were also not taken into account. Moreover, temperature impact on stress distribution
around a fault should be investigated. These inter-
relations induce shear stresses that can reactivate a
fault (Karaog˘lu et al., 2020), and thereafter modify
its permeability. H. Duwiquet et al. The dependence of permeability on
all of these phenomena should probably generate
permeability anisotropy. The development of this
anisotropy can cause a change in the intrinsic
properties of a geothermal reservoir and induce a
change in the heat transfer mode (Sun et al., 2017). In cases where fluid flow is important (i.e., areas of
high permeability), the permeability can take a
particular geometry that minimizes the resistance to
flow and then optimizes fluid flow (Bejan & Lorente,
2011). Fluid salinity could also play an important
role, but effects would be marked above 400 C
(Driesner, 2007), which was beyond our modeled
temperatures. Nature and Impact of Poro-elasticity Driven Force In this study, the stress intensity applied
was not a factor influencing the convection pat-
tern. However, the stress intensity has a role in
the time to reach the steady state. Indeed, the
steady-state was reached faster when the most
intense stresses were applied (Fig. 5), and this
was not explored in the Duwiquet et al. (2021a;
2021b) study. Moreover, the role of tectonics on
the direction of fluid flow has been highlighted. Considering a numerical approach, studies have
shown that, in compression, upward fluid move-
ments are favored (Upton, 1998), whereas, in
extension, downward fluid movements are favored
(Cui et al., 2012). Here, we saw that different
tectonic regimes have a role in the onset of
convection. In compressional tectonic regime, the positive
temperature anomaly was concentrated in the fault,
whereas in strike-slip tectonic regime, this anomaly
extended more widely into the basement (Fig. 4). This is related to the different fluid pressures. The
difference in fluid pressure between the fault and the
basement depended on the tectonic regime. In
compressional tectonic regime, the difference in
fluid pressure between the basement and the fault
was larger than in strike-slip tectonic regime. This
concentrated the fluid flow in a more restricted
volume in compression than in strike-slip. This im-
pacts the spatial extent of the positive temperature
anomaly, and therefore the geothermal potential of
these naturally fractured zones. y
The force that drives fluids from high-pressure
zones to low-pressure zones is similar to the effects
of topography on fluid flow (Forster & Smith, 1989;
Lo´pez & Smith, 1995). This poro-elasticity driven
force will therefore influence the initial fluid motions
(downward and upward movement) and thus the
time required for the steady-state temperature
anomaly to develop. Considering a TH coupling, the
fluid circulation in the benchmark experiment was
driven by free convection. Considering three dif-
ferent tectonic regimes with the same TH coupling,
the fluid circulation was the result of an interplay
between forced and free convection. These changes
were not related to boundary conditions, but to the
tectonic regimes themselves. Forced convection is
here referred to as stress induced convection. After stress applications, the permeability val-
ues obtained were consistent with fractured and al-
tered granitic environment (Sardini et al., 1997; Consequences for High-Temperature Geothermal
Potential of Crustal Fault Zones For
geothermal
exploration,
slip
tendency
analyses can be used to target favorable zones for
natural fluid flow and future enhancement by fault
reactivation (Barton et al., 1995; Morris et al., 1996;
Ito & Zoback, 2000). Our results showed that the
strike-slip regime would be the most favorable to
allow fluid flow. The occurrence of geothermal
reservoirs in such contexts was already known, as for
the Alpine Fault, New Zealand (Boulton et al.,
2012), or in the Geysers geothermal area, California
(Altmann et al., 2013). In the light of our results, an
exploratory phase on the geothermal potential of
fault zones could further consider strike-slip faulted
regime as interesting targets for geothermal power
system. If slip tendency can be used as a potential
qualitative indicator in purely elastic models, a more
accurate interpretation would require incorporating
dissipative
mechanical
behaviors,
such
as
e.g.,
Mohr–Coulomb elasto-plastic law, which would al-
low to quantify the variation in slip tendency. By
considering irreversible mechanical processes, such
as including dilation (opening under shearing) and
fracturing (increase in pore space/fractures), the
highest favorability of strike-slip regime toward
convection is expected to be emphasized. Geothermal energy can become a major asset
for the transition to low-carbon energy sources. Its
development requires, prior to exploration, com-
prehensive understanding of limiting and enabling
factors controlling fluid flow and the location of
temperature anomalies (Jolie et al., 2021). Fault
intersections, triple junctions systems, are, among
others, examples of anomalously permeable zones, Tectonic Regime as a Control Factor for Crustal Fault Zone that localize fluids flow (Person et al. 2012; Karaog˘lu
et al., 2016, 2019). Our numerical results showed
that, within a single permeable fault, and without
other heat sources, the poro-elasticity driven force
provoked by the tectonic regimes causes lateral fluid
pressure variation, allowing for the more or less ra-
pid development of the temperature anomaly, by
mixed, free and forced convection. For example,
strike-slip tectonic regimes would have the largest
temperature anomalies, but would take the longest
time to set up. HD would also like to sincerely thank Patrick Ledru,
Fre´de´ric Donze´, Roland Horne, Christine Souque,
Albert Genter and Fabrice Gaillard who, through
their relevant advice and suggestions within the
framework of the thesis defense, have contributed to
improve the final version of this manuscript. Consequences for High-Temperature Geothermal
Potential of Crustal Fault Zones All
authors would like to thank the anonymous reviewer
(R1) as well as O¨ zgu¨r Karaog˘lu (R2) for their
insightful suggestions as well as their constructive
remarks APPENDIX 1 The calibration of our numerical experiments
was performed based on results from Comsol Mul-
tiphysics version 3.5 (Guillou-Frottier et al., 2020)
as well as the OpenGeoSys numerical code (Magri
et al., 2017). These models consider a 40 m wide
vertical
fault
in
an
impermeable
box
(a
5.5 9 5.5 9 5.5 km side cube). The fluid properties
were identical for all three results with a linear
dependence of temperature with water density, and
an exponential decrease in viscosity with tempera-
ture. This result is shown for a time t0 + 1013 s. The
imposed permeability value was 5 9 10–15 m2. The OPEN ACCESS This article is licensed under a Creative Com-
mons 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://creativecom
mons.org/licenses/by/4.0/. The complex nature of the processes that occur
during the development of a geothermal resource
within a CFZ makes it an environment that requires
a state-of-the-art numerical analysis of the processes
that can control fluid circulation. Such fully coupled
analysis will help exploration phases and ultimately
promote the development of this renewable energy,
not only in anomalously hot areas, but in anomalous
permeable areas, and thus promote the development
of this climate-neutral energy. FUNDINGS The financial support was provided by Bureau
de Recherches Ge´ologiques et Minie`res (BRGM),
Institut des Sciences de la Terre dOrle´ans (ISTO),
TLS-Geothermics and by the ‘‘GERESFAULT’’
project (ANR-19-CE05-0043). DECLARATIONS p
Overall, this work suggests that anomalous
permeable zones, like CFZs, with no external heat
source,
have
significant
energy
potential. The
exploration of vertical CFZs in strike-slip stress re-
gime could accelerate the transition to low-carbon,
renewable and climate-neutral energy. Studies have
shown that major strike-slip fault zones localize
porosity and permeability even beyond the BDT
(Faulkner et al., 2010; Cao & Neubauer, 2016). Our
study confirms that this tectonic regime seems to
favor higher thermal anomalies than compressional
tectonics. Conflict of Interest
The authors declare that they
have no conflict of interest. H. Duwiquet et al. H. Duwiquet et al. Figure 7. Benchmarking of our numerical experiment with the OpenGeoSys Code (Magri et al., 2017) and Comsol Multiphysics.
V3.5a (Guillou-Frottier et al., 2020). Figure 7. Benchmarking of our numerical experiment with the OpenGeoSys Code (Magri et al., 2017) and Comsol Multiphysics.
V3.5a (Guillou-Frottier et al., 2020). Geysers geothermal area. In Proceedings of Thirty-Eighth
Workshop on Geothermal Reservoir Engineering. fluid flow velocity was 2.03 9 10–9 m.s1. The fluid
flow velocity was slightly higher than that described
by Guillou-Frottier et al., (2020), who recorded a
velocity of 1.4 9 10–9 m.s1. Geysers geothermal area. In Proceedings of Thirty-Eighth
Workshop on Geothermal Reservoir Engineering. Anderson, E. M. (1905). The dynamics of faulting. Transactions of
the Edinburgh Geological Society, 8(3), 387–402. Barton, C. A., Zoback, M. D., & Moos, D. (1995). Fluid flow
along potentially active faults in crystalline rock. Geology,
23(8), 683–686. The convective patterns were similar. Fluid
velocity accelerated along the permeable fault and
exhibited upward movement due to the thermal
gradient and buoyancy forces related to lower water
density at depth. The thermal perturbations were
also within the orders of magnitude of previous
studies with 23.04 C and + 31.14 C. Bejan, A., & Lorente, S. (2011). The constructal law and the
evolution of design in nature. Physics of Life Reviews, 8(3),
209–240. Ben-Zion, Y., & Rovelli, A. (2014). Properties and processes of
crustal fault zones: volume I. Pure and Applied Geophysics,
171(11), 2863–2865. Bethke, C. M. (1985). A numerical model of compaction-driven
groundwater flow and heat transfer and its application to the
paleohydrology of intracratonic sedimentary basins. Journal
of Geophysical Research: Solid Earth, 90(B8), 6817–6828. The numerical experiments of Magri et al. (2017) and Guillou-Frottier et al. (2020) used Dar-
cys law in conjunction with the equations of heat. The numerical experiments in this study coupled the
equations of heat and mass transfer with the mod-
ulus of poro-elasticity. The poro-elasticity interface
of the Comsol Multiphysics combines Darcys law
with the linear elastic behavior of porous media
(details can be found in Duwiquet et al. 2021a). Poro-elastic coupling allows boundary stresses to be
imposed, which can be recorded as fluid pressure. In
Figure 7 (right panel), no stress or other mechanical
conditions were applied. Consequently, this experi-
ment corresponded to an identical coupling as in the
previous studies (Fig. 7, left and middle panels). (
)
Bijay, K. C., & Ghazanfari, E. (2021). H. Duwiquet et al. Geothermal reservoir
stimulation through hydro-shearing: An experimental study
under conditions close to enhanced geothermal systems. Geothermics, 96, 102200. Boulton, C., Carpenter, B. M., Toy, V., & Marone, C. (2012). Physical properties of surface outcrop cataclastic fault rocks,
Alpine Fault, New Zealand. Geochemistry, Geophysics,
Geosystems. https://doi.org/10.1029/2011GC003872. Brockamp, O., Schlegel, A., & Wemmer, K. (2015). Complex
hydrothermal alteration and illite K-Ar ages in Upper Visean
molasse sediments and magmatic rocks of the Variscan
Badenweiler-Lenzkirch suture zone, Black Forest Germany. International Journal of Earth Sciences, 104(3), 683–702. Cao, S., & Neubauer, F. (2016). Deep crustal expressions of ex-
humed strike-slip fault systems: Shear zone initiation on
rheological boundaries. Earth-Science Reviews, 162, 155–176. Cappa, F., & Rutqvist, J. (2011). Impact of CO2 geological
sequestration on the nucleation of earthquakes. Geophysical
Research Letters. https://doi.org/10.1029/2011GL048487. Chester, F. M., & Logan, J. M. (1986). Implications for mechan-
ical properties of brittle faults from observations of the
Punchbowl fault zone, California. Pure and applied geo-
physics, 124(1), 79–106. Altmann, J. B., Heidbach, O., & Gritto, R. (2013). Relative
importance of processes leading to stress changes in the ACKNOWLEDGMENTS HG warmly thanks the DGR/GEM team of
BRGM for their hospitality during the preparation
of this manuscript as well as Institut Franc¸ais du
Pe´trole et des Energies Nouvelles (IFPEN) for
permission to submit this manuscript for publication. Tectonic Regime as a Control Factor for Crustal Fault Zone The role of in situ
stress in organizing flow pathways in natural fracture net-
works at the percolation threshold. Geofluids. https://doi.org/
10.1155/2019/3138972. Duwiquet, H., et al. (2019). On the geothermal potential of crustal
fault zones: A case study from the Pontgibaud area (French
Massif Central, France). Geothermal Energy, 7(1), 1–29. Jolie, E., Scott, S., Faulds, J., Chambefort, I., Axelsson, G., Gu-
tie´rrez-Negrı´n, L. C., Regenspurg, S., Ziegler, M., Ayling, B.,
Richter, A., & Zemedkun, M. T. (2021). Geological controls
on geothermal resources for power generation. Nature Re-
views Earth & Environment, 2(5), 324–339. Duwiquet, H., et al. (2021a). Crustal fault zones (CFZ) as
geothermal power systems: A preliminary 3D THM model
constrained by a multidisciplinary approach. Geofluids. http
s://doi.org/10.1155/2021/8855632. g
Eldursi, K., et al. (2020). New insights from 2-and 3-D numerical
modelling on fluid flow mechanisms and geological factors
responsible for the formation of the world-class Cigar Lake
uranium deposit, eastern Athabasca Basin, Canada. Miner-
alium Deposita, 56(7), 1365–1388. ( )
Karaog˘lu, O¨ ., Bayer, O¨ ., Turgay, M. B., & Browning, J. (2020). Thermomechanical
interactions
between
crustal
magma
chambers in complex tectonic environments: Insights from
Eastern Turkey. Tectonophysics, 793, 228607. ¨ Karaog˘lu, O¨ ., Bazargan, M., Baba, A., & Browning, J. (2019). Thermal fluid circulation around the Karliova triple junction:
Geochemical
features
and
volcano-tectonic
implications
(Eastern Turkey). Geothermics, 81, 168–184. ¨ ( )
Evans, K. F., et al. (2005). Microseismicity and permeability
enhancement of hydrogeologic structures during massive
fluid injections into granite at 3 km depth at the Soultz HDR
site. Geophysical Journal International, 160(1), 388–412. Karaog˘lu, O¨ ., Browning, J., Bazargan, M., & Gudmundsson, A. (2016). Numerical modelling of triple-junction tectonics at
Karlıova, Eastern Turkey, with implications for regional
magma transport. Earth and Planetary Science Letters, 452,
157–170. Famin, V., Philippot, P., Jolivet, L., & Agard, P. (2004). Evolution
of hydrothermal regime along a crustal shear zone, Tinos
Island,
Greece. Tectonics. https://doi.org/10.1029/2003T
C001509. Katto, Y., & Masuoka, T. (1967). Criterion for the onset of con-
vective flow in a fluid in a porous medium. International
Journal of Heat and Mass Transfer, 10(3), 297–309. Faulkner, D. R., et al. (2010). A review of recent developments
concerning the structure, mechanics and fluid flow properties
of fault zones. Journal of Structural Geology, 32(11), 1557–
1575. f
f
( )
Kozlovsky, Y. (1984). The Worlds Deepest Well. Scientific
American, 251(6), 98–105. Faulkner, D. R., Lewis, A. C., & Rutter, E. H. (2003). Tectonic Regime as a Control Factor for Crustal Fault Zone Gleeson, T., & Ingebritsen, S. (2016). Crustal permeability. (Eds.)
Wiley. Cox, S. F. (1999). Deformational controls on the dynamics of fluid
flow in mesothermal gold systems. Geological Society, Lon-
don, Special Publications, 155(1), 123–140. Gomila, R., et al. (2021). Quantitative anisotropies of palaeop-
ermeability in a strike-slip fault damage zone: Insights from
micro-CT analysis and numerical simulations. Tectono-
physics, 810, 228873. ( )
Cui, T., Yang, J., & Samson, I. M. (2012). Tectonic deformation
and fluid flow: Implications for the formation of unconfor-
mity-related uranium deposits. Economic Geology, 107(1),
147–163. Grawinkel, A., & Sto¨ckhert, B. (1997). Hydrostatic pore fluid
pressure to 9 km depth-Fluid inclusion evidence from the
KTB deep drill hole. Geophysical Research Letters., 24(24),
3273–3276. Deichmann, N., & Giardini, D. (2009). Earthquakes induced by
the stimulation of an enhanced geothermal system below
Basel (Switzerland). Seismological Research Letters., 80(5),
784–798. Guillou-Frottier, et al. (2020). On the morphology and amplitude
of 2D and 3D thermal anomalies induced by buoyancy-driven
flow within and around fault zones. Solid Earth, 11(4), 1571–
1595. Driesner, T. (2007). The system H2O–NaCl. Part II: Correlations
for molar volume, enthalpy, and isobaric heat capacity from 0
to 1000 C 1 to 5000 bar, and 0 to 1 XNaCl. Geochimica et
Cosmochimica Acta, 71(20), 4902–4919. Heap, M. J., et al. (2020). Towards more realistic values of elastic
moduli for volcano modelling. Journal of Volcanology and
Geothermal Research, 390, 10668. Duwiquet, H. et al. (2021b). Crustal fault zones (CFZ) as
geothermal power systems: 3D variation of permeability and
related processes. In Stanford Geothermal Workshop, (2021b,
February). Horne, R. N. (1979). Three-dimensional natural convection in a
confined porous medium heated from below. Journal of Fluid
Mechanics, 92(4), 751–766. y)
Duwiquet, H. (2022). Crustal fault zones as geothermal power
systems. Contribution of Numerical Modelling and Compar-
ison with Natural Systems (Doctoral dissertation, Universite´
d’Orle´ans). ( )
Horton, C. W., & Rogers, F. T., Jr. (1945). Convection currents in
a porous medium. Journal of Applied Physics, 16(6), 367–370. (
) Ito, T., & Zoback, M. D. (2000). Fracture permeability and in situ
stress to 7 km depth in the KTB scientific drillhole. Geo-
physical Research Letters, 27(7), 1045–1048. Duwiquet, H., et al. (2022). Crustal Fault Zones as underexploited
geothermal resources: Contribution of numerical modelling
and comparison with natural systems. In Stanford Geother-
mal Workshop, (2022, February). Jiang, C., Wang, X., Sun, Z., & Lei, Q. (2019). REFERENCES Cornet, F. H., & Burlet, D. (1992). Stress field determinations in
France by hydraulic tests in boreholes. Journal of Geophys-
ical Research: Solid Earth, 97(B8), 11829–11849. Altmann, J. B., Heidbach, O., & Gritto, R. (2013). Relative
importance of processes leading to stress changes in the H. Duwiquet et al. S. (1997). Mechanical controls on fluid
flow during regional metamorphism: Some numerical models. Journal of Metamorphic Geology, 15(3), 345–359. Violay, M., Heap, M. J., Acosta, M., & Madonna, C. (2017). Porosity evolution at the brittle-ductile transition in the
continental crust: Implications for deep hydro-geothermal
circulation. Scientific reports., 7(1), 1–10. OSullivan, M. J., Pruess, K., & Lippmann, M. J. (2001). State of
the art of geothermal reservoir simulation. Geothermics,
30(4), 395–429. Wanner, C., Diamond, L. W., & Alt-Epping, P. (2019). Quantifi-
cation of 3-D thermal anomalies from surface observations of
an orogenic geothermal system (Grimsel Pass, Swiss Alps). Journal of Geophysical Research: Solid Earth, 124(11),
10839–10854. Parisio, F., Vilarrasa, V., Wang, W., Kolditz, O., & Nagel, T. (2019). The risks of long-term re-injection in supercritical
geothermal systems. Nature communications, 10(1), 1–11. Paulillo, A., Cotton, L., Law, R., Striolo, A., & Lettieri, P. (2020). Geothermal energy in the UK: The life-cycle environmental
impacts of electricity production from the United downs deep
geothermal power project. Journal of Cleaner Production,
249, 119410. Watanabe, N., Abe, H., Okamoto, A., Nakamura, K., & Komai,
T. (2021). Formation of amorphous silica nanoparticles and
its impact on permeability of fractured granite in superhot
geothermal environments. Scientific reports, 11(1), 1–11. Person, M., Hofstra, A., Sweetkind, D., Stone, W., Cohen, D.,
Gable, C. W., & Banerjee, A. (2012). Analytical and
numerical models of hydrothermal fluid flow at fault inter-
sections. Geofluids, 12(4), 312–326. Watanabe, N., Egawa, M., Sakaguchi, K., Ishibashi, T., & Tsu-
chiya, N. (2017). Hydraulic fracturing and permeability
enhancement in granite from subcritical/brittle to supercriti-
cal/ductile conditions. Geophysical Research Letters, 44(11),
5468–5475. Rowland, J. V., & Sibson, R. H. (2004). Structural controls on
hydrothermal flow in a segmented rift system, Taupo Vol-
canic Zone, New Zealand. Geofluids, 4(4), 259–283. Zareidarmiyan, A., Parisio, F., Makhnenko, R. Y., Salarirad, H.,
& Vilarrasa, V. (2020). How equivalent are equivalent por-
ous
media? Geophysical
Research
Letters,
48,
e2020GL0089163. Saevarsdottir, G., Tao, P. C., Stefansson, H., & Harvey, W. (2014). Potential use of geothermal energy sources for the produc-
tion of lithium-ion batteries. Renewable Energy, 61, 17–22. Zoback, et al. (2003). Determination of stress orientation and
magnitude in deep wells. International Journal of Rock
Mechanics and Mining Sciences, 40(7–8), 1049–1076. Sardini, P., Lede´sert, B., & Touchard, G. (1997). Quantification of
microscopic porous networks by image analysis and mea-
surements of permeability in the Soultz-sous-Foreˆts granite
(Alsace, France). H. Duwiquet et al. Magri, F., Mo¨ller, S., Inbar, N., Mo¨ller, P., Raggad, M., Ro¨diger,
T., Rosenthal, E., & Siebert,. (2016). 2D and 3D coexisting
modes of thermal convection in fractured hydrothermal sys-
tems-Implications for transboundary flow in the Lower Yar-
mouk Gorge. Marine and Petroleum Geology., 78, 750–758. Schulz, S. E., & Evans, J. P. (1998). Spatial variability in micro-
scopic deformation and composition of the Punchbowl fault,
southern California: Implications for mechanisms, fluid–rock
interaction, and fault morphology. Tectonophysics, 295(1–2),
223–244. Scott, S., Driesner, T., & Weis, P. (2017). Boiling and condensa-
tion of saline geothermal fluids above magmatic intrusions. Geophysical Research Letters, 44(4), 1696–1705. McLellan, J. G., Oliver, N. H. S., & Schaubs, P. M. (2004). Fluid
flow in extensional environments; numerical modelling with
an application to Hamersley iron ores. Journal of Structural
Geology, 26(6–7), 1157–2117. y
( )
Sibson, R. H. (1987). Earthquake rupturing as a mineralizing
agent in hydrothermal systems. Geology, 15(8), 701–704. Mitchell, T. M., & Faulkner, D. R. (2009). The nature and origin
of off-fault damage surrounding strike-slip fault zones with a
wide range of displacements: A field study from the Atacama
fault system, northern Chile. Journal of Structural Geology,
31(8), 802–816. Siebenaller, L., et al. (2013). Fluid record of rock exhumation
across the brittle–ductile transition during formation of a
metamorphic
core
complex
(Naxos
Island,
Cyclades,
Greece). Journal of Metamorphic Geology, 31(3), 313–338. ( )
Morris, A., Ferrill, D. A., & Henderson, D. B. (1996). Slip-ten-
dency analysis and fault reactivation. Geology, 24(3), 275–
278. Siler, D. L. H., et al. (2019). Three-dimensional geologic mapping
to assess geothermal potential: Examples from Nevada and
Oregon. Geothermal Energy, 7(1), 1–32. Nur, A. M. O. S., & Walder, (1990). Time-dependent hydraulics of
the Earths crust. In The Role of Fluids in Crustal Processes
Washington, DC (Vol. 113). National Academy Press. Sun, Z. X., et al. (2017). Numerical simulation of the heat
extraction in EGS with thermal-hydraulic-mechanical cou-
pling method based on discrete fractures model. Energy, 120,
20–33. Ojala, A. E., Mattila, J., Markovaara-Koivisto, M., Ruskeeniemi,
T., Palmu, J. P., & Sutinen, R. (2019). Distribution and
morphology of landslides in northern Finland: An analysis of
postglacial seismic activity. Geomorphology, 326, 190–201. (
) Upton, P., Baxter, K., & OBrien, G. W. (1998). Coupled
mechanical/fluid flow models of trap integrity and fault
reactivation: Application to the North West Shelf of Aus-
tralia. The APPEA Journal, 38(1), 488–499. Ord, A., & Oliver, N. H. Tectonic Regime as a Control Factor for Crustal Fault Zone On the
internal structure and mechanics of large strike-slip fault
zones: Field observations of the Carboneras fault in south-
eastern Spain. Tectonophysics, 367(3–4), 235–251. Lamur, A., et al. (2017). The permeability of fractured rocks in
pressurised volcanic and geothermal systems. Scientific re-
ports, 7(1), 1–9. Ledingham, P., Cotton, L., & Law, R. (2019). The united downs
deep geothermal power project.In Proc. 44th Work. Geo-
therm. Reserv. Eng, (pp. 1–11). Forster, C., & Smith, L. (1989). The influence of groundwater flow
on thermal regimes in mountainous terrain: A model study. Journal of Geophysical Research: Solid Earth., 94(B7), 9439–
9451. Liang, X., Xu, T., Feng, B., & Jiang, Z. (2018). Optimization of
heat extraction strategies in fault-controlled hydro-geother-
mal reservoirs. Energy, 164, 853–870. Genter, A., Evans, K., Cuenot, N., Fritsch, D., & Sanjuan, B. (2010). Contribution of the exploration of deep crystalline
fractured reservoir of Soultz to the knowledge of enhanced
geothermal systems (EGS). Comptes Rendus Geoscience.,
342(7–8), 502–516. gy
Lo´pez, D. L., & Smith, L. (1995). Fluid flow in fault zones:
Analysis of the interplay of convective circulation and
topographically driven groundwater flow. Water Resources
Research, 31(6), 1489–1503. (
)
Gischig, V. S., & Preisig, G. (2015, May). Hydro-fracturing versus
hydro-shearing: a critical assessment of two distinct reservoir
stimulation mechanisms. In 13th ISRM International Con-
gress of Rock Mechanics. OnePetro. Magri, F., et al. (2017). Thermal convection of viscous fluids in a
faulted system: 3D benchmark for numerical codes. Energy
Procedia, 125, 310–317. H. Duwiquet et al. In Fluid flow and Transport in Rocks, (pp. 171–189). g
(
)
Zoback, M. L. (1992). Stress field constraints on intraplate seis-
micity in eastern North America. Journal of Geophysical
Research: Solid Earth, 97(B8), 11761–11782.
|
https://openalex.org/W4392357838
|
https://sciendo.com/pdf/10.2478/macvetrev-2024-0015
|
English
| null |
External Progesterone Supplementation During the Ovsynch Protocol Reduces the Incomplete Luteolysis in Dairy Cows Under Heat Stress
|
Macedonian Veterinary Review/Macedonian veterinary review
| 2,024
|
cc-by
| 4,345
|
ABSTRACT The present study aimed to determinate the effect of external progesterone (P4) supplementation on luteolysis in cows
under heat stress. Forty-eight (n=48) dairy cows in the period from July–September 2018 were part of and at day 35±3
postpartum scored for BSC, synchronized using PG-3-G + Ovsynch protocol and randomly allocated into two treatments:
PRID group (n=27) treated with external P4 device between G1 and PGF2α and CON group (n=21) left without treatment. Collection of blood samples to assess P4 concentrations was done at Pre-PG, at G1, at PGF2α, at 72 h after PGF2α (at timed
artificial insemination TAI) and at d 21 after TAI. The pregnancy diagnosis was done at d 21 and d 30 after TAI by ultrasound. The average temperature-humidity index (THI) was 79.5±0.6. At G1, the P4 was significantly lower in the PRID group
(1.84±0.99 ng/mL) in comparison to the CON group (2.97±1.82 ng/mL). In contrast, at PGF2α, there was a tendency (p=0.09)
of increased P4 concentration in PRID group compared with the CON group (4.26±1.68 and 3.74±2.39 ng/mL), respectively. At TAI, more PRID cows (p=0.0001) had a lower P4 (0.06±0.03 ng/mL), in comparison to CON (1.28±2.41 ng/mL). At d 21 and d 30 after TAI, more PRID cows were predicted and diagnosed pregnant (16/27 or 59.25% and 13/27 or 48.14%)
compared with the CON group (11/21 or 52.38% and 8/21 or 38.08%) respectively, but without any significant differences. Supplementation of the P4 during the Ovsynch protocol increases the P4 before TAI and reduces the incomplete luteolysis
in heat stressed dairy cows. Key words: cows, heat stress, Ovsynch, luteolysis, PRID Corresponding author: Prof. Branko Atanasov, PhD
E-mail address: batanasov@fvm.ukim.edu.mk
Present address: Faculty of Veterinary Medicine-Skopje,
Ss. Cyril and Methodius University in Skopje,
Lazar-Pop Trajkov 5-7, 1000 Skopje, North Macedonia
Phone: + 38975268148
Copyright: © 2024 Stojanov B. This is an open-access article published
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.
Competing Interests: The authors have declared that no competing
interests exist.
Available Online First: 27 February 2024
Published on: 15 March 2024
https://doi.org/10.2478/macvetrev-2024-0015 Boris Stojanov1, Branko Atanasov2, Juraj Grizelj3, Silvijo Vince3, Ksenija Ilievska2,
Martin Nikolovski2, Toni Dovenski2, Marko Samardžija3 Boris Stojanov1, Branko Atanasov2, Juraj Grizelj3, Silvijo Vince3, Ksenija Ilievska2,
Martin Nikolovski2, Toni Dovenski2, Marko Samardžija3 1Food and Veterinary Agency, Treta Makedonska Brigada 20,
1000 Skopje, North Macedonia
2Faculty of Veterinary Medicine-Skopje, Ss. Cyril and Methodius University in Skopje,
Lazar Pop-Trajkov 5-7, 1000 Skopje, North Macedonia
3Faculty of Veterinary Medicine, University of Zagreb,
Heinzelova 55, 10000 Zagreb, Croatia Received 26 December 2023; Received in revised form 18 February 2024; Accepted 24 February 2024 Mac Vet Rev 2024; 47 (1): 45-50
Available online at
www.macvetrev.mk Mac Vet Rev 2024; 47 (1): 45-50
Available online at
www.macvetrev.mk Mac Vet Rev 2024; 47 (1): 45-50 Mac Vet Rev 2024; 47 (1): 45-50
Available online at
www.macvetrev.mk Macedonian Veterinary Review Ultrasonographic examination, blood collection,
cyclic status, luteolysis, progesterone analysis and
pregnancy diagnosis Ultrasonographic examination, blood collection,
cyclic status, luteolysis, progesterone analysis and
pregnancy diagnosis B-mode scanner (Mindray DP 50, Soma
Technology Inc. USA) equipped with a 7.5 MHz
rectal linear probe was used to map ovarian
structures at G1 and at d 21 after TAI. Collection of
blood samples to evaluate the P4 concentrations was
done at Pre-PG, at G1, at PGF2α, at TAI, and at d 21
after TAI from the coccygeal vein into evacuated
tubes (BD Vacutainer® Plymouth, UK). The samples
were refrigerated at +8 °C and transported to the
lab at the Faculty of Veterinary Medicine–Skopje,
North Macedonia within 3 h after collection. The
sera were collected after centrifuging (1,000 x g for
15 min), and stored at -20 °C until P4 evaluation. The
P4 evaluation was done on an Immuno-scan BDLS
reader using commercially available kit (HUMAN,
Progesterone ELISA Test–GMBH, Germany). The
intra- and inter-assay CV were 4.2% and 7.8%,
respectively. The P4 concentrations collected at
Pre-PG and at G1 were used to determine the cyclic
status of the cows. When both samples contained
P4 level <1 ng/mL, the cows were classified as
acyclic. On contrary, when P4 concentrations were
≥1 ng/mL either at Pre-PG or at G1, the cows were
classified as cyclic. Incomplete luteal regression was
defined to occur when the P4 level was >1 ng/mL
at PGF2α and >0.4 ng/mL at TAI, respectively (14). Although, the pregnancy rate was not the primary
focus of the study due to the small number of cows
included, the ultrasound pregnancy diagnosis was
done at d 21 and d 30 after TAI. Cows that had no
CL and had P4 <0.5 ng/mL on d 21 after TAI, were
predicted to be non-pregnant (17). If there was one
CL >25 mm with a follicle <13 mm, and P4 >2 ng/mL,
the cows were predicted pregnant (17). Positive We hypothesized that supplementation of
exogenous progesterone will increase the P4 level
during the Ovsynch protocol and consequently
might reduce the incomplete luteal regression in
heat stressed dairy cows. Therefore, the objective
was to determine the effect of exogenous P4 on
luteolysis in dairy cows that are under heat stress. INTRODUCTION Several strategies have
been implemented to prevent incomplete luteal
regression: by either increasing the dose of PGF2α
(12) or the frequency of PGF2α treatment (13), or
lengthening the time from G1 to PGF2α for 1 day
(Ovsynch 8) (8, 14) during the Ovsynch protocol. However, the results were inconsistent. body condition (BCS, 1=emaciated and 5=obese)
(15) and pre-synchronized using PG-3-G protocol
as described previously (16). Briefly, the protocol
consisted of injection of PGF2α (Pre-PG; PGF Veyx
Forte, Veyx-Pharma GmbH, Germany) followed
by injection of GnRH (Pre-GnRH; Receptal,
MSD Animal Health, GMBH, Germany) 3 days
later. Seven days after the Pre-GnRH injection, an
Ovsynch 56 protocol was initiated (GnRH1 (G1)–
7 days – PGF2α – 56 h – (GnRH2 (G2) – 16 h – TAI). Cows were allocated randomly into two treatments:
1) PRID group (n=27), treated with external P4
(PRID-progesterone intravaginal releasing device,
CEVA, Sante Animale, France) inserted at G1 and
withdrawal at PGF2α, and 2) CON group (n=21) left
without any additional treatment. the cows with GnRH after AI, due to an increased
number of accessory CLs and thus elevated P4
after AI. Similarly, Mendonca et al. (4) have shown
an improved fertility in heat stressed dairy cows
after treatment with GnRH at day 5 after AI. In
addition, Denicol et al. (5), have demonstrated that
follicles that grew under elevated P4 concentration
(second follicular wave) were more fertile in
comparison with follicles growing under low P4
concentration (first follicular wave). Cumulatively,
all of these studies have shown the importance of
elevated P4 concentration before and after AI that
might positively affect fertility. On the other hand,
increased circulating P4 concentration near AI can
result in a reduced fertility due to an inadequate
luteolysis. The latter has been clearly shown in
numerous studies using timed AI programs (6). Indeed, up to 30% of cows submitted either to an
Ovsynch 56 protocol (G1–7 days – PGF2α – 56 h –
G2 – 16 h – TAI) (7) or to a single PGF2α estrus-
synchronization protocol (8) had an incomplete
luteolysis. Increased concentration of P4 near AI
(>0.4 ng/mL), due to incomplete luteolysis, reduces
fertility (9) because elevated P4 at the time of G2
reduces the magnitude of the GnRH-induced
LH surge (10). Thus, the failure of complete CL
regression limits the achievement of maximal P/AI
(pregnancy per AI) (11). MATERIAL AND METHODS Animals and synchronization protocol INTRODUCTION generally ranges between 16 to 25 °C. The upper
critical point of this range marks the onset of heat
stress. Heat stress adversely affects fertility of
dairy cattle and compromises their reproductive
function through reduced expression of estrus,
disruption of follicular and oocyte function, as
well as increased embryonic mortality (1). In
addition, cows chronically exposed to heat stress
have a reduced progesterone (P4) concentration (2)
leading to decreased fertility due to its importance
on pregnancy maintenance. Therefore, many
hormonal therapy strategies have been implemented
in order to increase the circulating P4 concentration
that might positively affect fertility in heat-stressed
dairy cows. In this regard, López-Gatius et al. (3)
demonstrated an improved fertility after treating In the last few decades, heat stress has become
a global problem that threatens the health and
welfare of animals. The ambient temperature
optimal for good animal health and welfare Stojanov B. et al. the cows with GnRH after AI, due to an increased
number of accessory CLs and thus elevated P4
after AI. Similarly, Mendonca et al. (4) have shown
an improved fertility in heat stressed dairy cows
after treatment with GnRH at day 5 after AI. In
addition, Denicol et al. (5), have demonstrated that
follicles that grew under elevated P4 concentration
(second follicular wave) were more fertile in
comparison with follicles growing under low P4
concentration (first follicular wave). Cumulatively,
all of these studies have shown the importance of
elevated P4 concentration before and after AI that
might positively affect fertility. On the other hand,
increased circulating P4 concentration near AI can
result in a reduced fertility due to an inadequate
luteolysis. The latter has been clearly shown in
numerous studies using timed AI programs (6). Indeed, up to 30% of cows submitted either to an
Ovsynch 56 protocol (G1–7 days – PGF2α – 56 h –
G2 – 16 h – TAI) (7) or to a single PGF2α estrus-
synchronization protocol (8) had an incomplete
luteolysis. Increased concentration of P4 near AI
(>0.4 ng/mL), due to incomplete luteolysis, reduces
fertility (9) because elevated P4 at the time of G2
reduces the magnitude of the GnRH-induced
LH surge (10). Thus, the failure of complete CL
regression limits the achievement of maximal P/AI
(pregnancy per AI) (11). INTRODUCTION Several strategies have
been implemented to prevent incomplete luteal
regression: by either increasing the dose of PGF2α
(12) or the frequency of PGF2α treatment (13), or
lengthening the time from G1 to PGF2α for 1 day
(Ovsynch 8) (8, 14) during the Ovsynch protocol. However, the results were inconsistent. Animals and synchronization protocol Forty-eight (n=48) Holstein dairy cows from
one herd during July-September 2018 were enrolled
in the study. Cows were housed in free-stall barn,
fed a total mixed ration (TMR) twice daily to meet
or exceed production of on average 30 L of milk per
day. Cows were milked twice daily on a 12 hours
interval and had free access to water. All cows
at day 35±3 after parturition, were evaluated for 46 Progesterone supplementation reduces the luteal regression in heat-stressed dairy cows pregnancy diagnosis at d 30 was declared when an
embryo with a visible heartbeat was observed. In
addition, cows were further clustered according to
their BCS (<2.75 and ≥2.75). Statistical analysis T-test and Duncan multiple range test were
performed to compare the data between PRID and
CON. Correlations analysis was performed for BCS
and P4 for PRID and CON groups. Values were
presented as means ±standard deviation. At G1, the P4 concentrations was lower in the
PRID group (1.84±0.99 ng/mL) in comparison to
the CON groups (2.97±1.82 ng/mL, p=0.004). In
contrast, at PGF2a, the PRID group of cows tended
to have (p=0.09) an increased P4 concentration
compared with the CON group (4.26±1.68 and
3.74±2.39 ng/mL, respectively). In addition, at TAI,
the P4 concentration differed between the treatments
(p=0.0001), with more PRID cows having a lower
P4 concentration (0.06±0.03 ng/mL) compared with
the CON group (1.28±2.41 ng/mL), implying that
more PRID cows underwent a complete luteolysis
in comparison with the CON group (Table 1). At
d 21 no differences (p=0.54) were detected in the P4
concentration between the treatments. Temperature
and
relative
humidity
measurements: Temperature
and
relative
humidity
measurements: The data for Relative humidity (RH) and
average daily temperature (T) values were collected
from the nearest meteorological station and the
temperature-humidity index (THI) was calculated
as THI = (1.8 x T + 32) – ((0.55 - 0.0055 x RH) x
(1.8 x T - 26)) (18). Table 1. Progesterone (P4) concentration between the PRID and CON group
Treatment†
P4 concentration
PRID (n=27)
CON (n=21)
p-value
At G1
1.84±0.99*
2.97±1.82
0.004
At PGF2α
4.26±1.68
3.74±2.39
0.09
At TAI
0.06±0.03*
1.28±2.41
0.0001
†In all cow the estrous cycles were presynchronized using PG-3-G protocol (Pre-PGF2α and 3 days later a Pre-GnRH). Ten days
after the Pre-PGF2α, injection, an Ovsynch protocol (G1 –7 days – PGF2α – 56 h – G2 – 16 h – TAI) was initiated with either PRID
insertion between G1 and PGF2α (PRID group) or without treatment (CON group)
Table 2. Percentage of cows predicted or diagnosed pregnant at day 21 and 30 after timed artificial insemination
(TAI), respectively in PRID and CON group
Treatment†
Conception rate
PRID (n=27)
CON (n=21)
p-value
At d 21
59.25% (16/27)
52.38% (11/21)
0.23
At d 30
48.14% (13/27)
38.08% (8/21)
0.12
†In all cow the estrous cycles were presynchronized using PG-3-G protocol (Pre-PGF2α and 3 days later a Pre-GnRH). RESULTS The average THI during the experiment was
79.5±0.6, showing that cows were under heat stress
condition. Overall, all cows were classified as cyclic
according to the P4 concentration (P4 >1 ng/mL) at
PRE-PG and G1. Statistical analysis Ten days
after the Pre-PGF2α, injection, an Ovsynch protocol (G1 –7 days – PGF2α – 56 h – G2 – 16 h – TAI) was initiated with either PRID
insertion between G1 and PGF2α (PRID group) or without treatment (CON group)
Table 3. Correlation between body condition score (BCS) and progesterone (P4) concentration at 21 day after
timed artificial insemination (TAI)
Group
BCS (n=27)
(mean ±std)
P4 (n=21)
(mean ±std)
r
PRID
2.90±0.27
2.76±2.08
-0.20
CON
2.79±0.26
2.71±2.36
0.29
†In all cow the estrous cycles were presynchronized using PG-3-G protocol (Pre-PGF2α and 3 days later a Pre-GnRH). Ten days
after the Pre-PGF2α, injection, an Ovsynch protocol (G1 –7 days – PGF2α – 56 h – G2 – 16 h – TAI) was initiated with either PRID
insertion between G1 and PGF2α (PRID group) or without treatment (CON group) Table 1. Progesterone (P4) concentration between the PRID and CON group Table 2. Percentage of cows predicted or diagnosed pregnant at day 21 and 30 after timed artificial inseminatio
TAI), respectively in PRID and CON group Table 3. Correlation between body condition score (BCS) and progesterone (P4) concentration at 21 day after
timed artificial insemination (TAI)
Group
BCS (n=27)
(mean ±std)
P4 (n=21)
(mean ±std)
r
PRID
2.90±0.27
2.76±2.08
-0.20
CON
2.79±0.26
2.71±2.36
0.29
†In all cow the estrous cycles were presynchronized using PG-3-G protocol (Pre-PGF2α and 3 days later a Pre-GnRH). Ten days
after the Pre-PGF2α, injection, an Ovsynch protocol (G1 –7 days – PGF2α – 56 h – G2 – 16 h – TAI) was initiated with either PRID
insertion between G1 and PGF2α (PRID group) or without treatment (CON group) 47 Stojanov B. et al. At d 21 after TAI, more PRID cows were
predicted pregnant (16/27 or 59.25%) compared
with the CON group (11/21 or 52.38%) but the
differences were not significant. Similarly, at d
30 after TAI, more PRID cows were diagnosed
pregnant (13/27 or 48.14%) compared with CON
group of cows (8/21 or 38.08%, Table 2). It was
assumed that cows that were diagnosed pregnant at
d 21 but non-pregnant at d 30, had experienced late
embryonic mortality. rates were not the primary focus of the present
study as a small number of cows were included in
the study. Nevertheless, our results showed a 10%
increase in pregnancy rate in PRID treated cows
compared with CON group of cows. Xu et al. Statistical analysis (22)
reported similar results where cows observed in
estrus and inseminated after treatment with PGF2α
during early diestrus (d 5 to 9, low P4) had lower
pregnancy rates compared with cows inseminated
in estrus after PGF2α treatment given on or after
d 10 of the estrous cycle (high P4) (22). When
cows
were
additionally
clustered
according to their BCS and correlated with the P4
concentration at G1, at PGF2α, and at d 21 after TAI,
no differences were detected (Table 3). Similarly,
no differences were observed in the pregnancy
rates at d 30 among the cows with the same BCS
between the PRID and the CON group of cows. Based on the ultrasound examination and
P4 level, a higher percentage of cows (more than
50%) were predicted for pregnancy at d 21 in both
groups. Nevertheless, more cows in CON group at
d 30 experienced an embryonic mortality compared
with the PRID group of cows. Similarly, Cunha
et al. (23) observed an increase in pregnancy loss
between d 29 and d 57 when cows had low P4 (14.3%
loss) vs high P4 (6.8% loss) during growth of the
follicular wave that produces the ovulatory follicle
before AI. Nevertheless, the exact underlying
physiological mechanisms of increased pregnancy
loss in heat-stressed cows remain unclear. We are
speculating that a partial luteal regression after
single PGF2α injection, as shown in the present
study, elevate the P4 concentration (near the AI)
that might increase the total α-inhibin production
by the cumulus–oocyte complex, which may
reduce the embryo development after cleavage (24)
and thus, increase the pregnancy loss. In addition,
elevated P4 concentration might affect the uterine
blood flow and thus functionality of the uterus that
could results in reduced embryo development and
increased pregnancy loss (25). DISCUSSION The present study aimed to observe whether
supplementation of exogenous P4 concentration
during the Ovsynch protocol will reduce the
percentage of cows with incomplete luteal regression
in heat-stressed dairy cows. The results have
shown that higher number of PRID-treated cows
underwent a complete luteal regression compared
to the not-treated CON group of cows with a single
PGF2α application in the Ovsynch protocol. In
addition, our results showed that PRID treatment
in heat-stressed cows is sufficient to increase the
P4 level at the time preceding PGF2α application. The latter is in agreement with the results observed
by Bisinotto and Santos (19), in which single insert
delivers sufficient P4 by incrementing plasma
concentrations during the development of the
ovulatory follicle (near the time preceding PGF2α
application). Similarly, greater P4 concentrations
at the time of PGF2α were associated with greater
probability of luteolysis after PGF2α treatment
(20) as shown in the current study. Cumulatively,
increased P4 concentration during the follicular
growth accompanied with completed luteolysis
after PGF2α treatment and ovulatory success after
G2 were associated with greater pregnancy rate (21). Cows that were induced to ovulate the dominant
follicle of the second follicular wave (high P4) have
an increased pregnancy rate compared with the
cows that were induced to ovulate the dominant
follicle of the first follicular wave (low P4) (5). Indeed, we have shown that PRID treated cows
have an increased pregnancy rate at d 30 compared
with CON group of cows. Although, the pregnancy The BCS was not related either with the
luteolysis or with the pregnancy rate during the heat
stress. These results do not corroborate with the
results from our recent study (26), where we have
found that thinner cows (BCS<2.75) have greater
failure in luteolysis in comparison to the cows
with BCS≥2.75. It is unclear how body condition
might influence luteolysis thus future studies need
to be conducted to evaluate the aforementioned
relationship. AUTHORS CONTRIBUTION TD, MS, BS planed the study design. BA and TD
performed the ultrasonographic examination of the cows. SV, KI and JG performed the proofreading. MN have run
the statistical analysis. BS, TD and BA have synchronized
the cows. BA and BS collected the blood samples. 8. Atanasov, B., Hostens, M., Celeska, I., Ilieska, K.,
Opsomer, G., Dovenski, T. (2015). Follicular dynamics
following induced luteolysis and transvaginal
ultrasound-guided aspiration of the largest follicle in
dairy cows. Vet Arhiv. 85(3): 247- 260. 9. Wiltbank, M.C., Souza, A.H., Carvalho, P.D.,
Cunha, A.P., Giordano, J.O., Fricke, P.M., Baez, G.M.,
Diskin, M.G. (2014). Physiological and practical
effects of progesterone on reproduction in dairy
cattle. Animal 8 (Suppl. 1): 70-81. https://doi.org/10.1017/S1751731114000585
PMid:24703103 CONCLUSION Supplementation of the exogenous P4 during
the standard Ovsynch protocol increases the
P4 concentration before TAI and reduces the
incomplete luteal regression in heat stressed dairy
cows. In addition, elevated P4 concentrations before 48 Progesterone supplementation reduces the luteal regression in heat-stressed dairy cows Progesterone supplementation reduces the luteal regression in heat-stressed dairy cows 5. Denicol, A.C., Lopes Jr., G., Mendonça, L.G.D.,
Rivera, F.A., Guagnini, F., Perez, R.V., Lima, J.R.,
et al. (2012). Low progesterone concentration during
the development of the first follicular wave reduces
pregnancy per insemination of lactating dairy cows. J Dairy Sci. 95(4): 1794-1806. https://doi.org/10.3168/jds.2011-4650
PMid:22459828 TAI increases the pregnancy rates and reduces the
pregnancy loss in heat-stressed cows. Nevertheless,
in order to confirm our findings, further studies are
warranted, including a larger number of cows. CONFLICT OF INTERESTS The authors declare that they have no known conflict
of interest in the conduction of the current study. 6. Bisinotto, R.S., Chebel, R.C., Santos, J.E.P. (2010). Follicular wave of the ovulatory follicle and
notcyclic status influences fertility of dairy cows. J Dairy Sci. 93(8): 3578-3587. https://doi.org/10.3168/jds.2010-3047
PMid:20655426 ACKNOWLEDGMENTS We would like to acknowledge ZK Pelagonija Dairy
Farm (Bitola, R.M) for allowing us the use of their
facilities. 7. Wiltbank, M.C., Pursley, J.R. (2014). The cow as an
induced ovulator: timed AI after synchronization of
ovulation. Theriogenology 81(1): 170-185. https://doi.org/10.1016/j.theriogenology.2013.09.017
PMid:24274420 REFERENCES 1. Hansen, P.J. (2011). Managing reproduction during
heat stress in dairy cows.In C.A. Risco, P. Melendez
Retamal (Eds.) Dairy production medicine, First
Edition (pp. 153-164). John Wiley & Sons, Inc. https://doi.org/10.1002/9780470960554.ch13 10. Pulley, S.L., Keisler, D.H., Stevenson, J.S. (2015). Concentrations of luteinizing hormone and ovulatory
responses in dairy cows before timed artificial
insemination. J Dairy Sci. 98(9): 6188-6201. https://doi.org/10.3168/jds.2015-9473
PMid:26142861 2. Wolfenson, D., Roth, Z., Meidan, R. (2000). Impaired reproduction in heat - stressed cattle: basic
and applied aspects. Anim Reprod Sci. (60 - 61): 535-547. https://doi.org/10.1016/S0378-4320(00)00102-0
PMid:10844222 3. López-Gatius, F., Santolaria, P., Martino, A.,
Delétang, F., De Rensis, F. (2006). The effects of
GnRH treatment at the time of AI and 12 days later
on reproductive performance of high producing
dairy cows during the warm season in northeastern
Spain. Theriogenology 65(4): 820-830. https://doi.org/10.1016/j.theriogenology.2005.07.002
PMid:16112722 11. Stevenson, J.S. (2016). Physiological predictors
of ovulation and pregnancy risk in a fixed-time
artificial insemination program. J Dairy Sci. 99(12):
10077-10092. https://doi.org/10.3168/jds.2016-11247
PMid:27720155 12. Giordano, J.O., Wiltbank, M.C., Fricke, P.M., Bas, S.,
Pawlisch, R., Guenther, J.N., Nascimento, A.B. (2013). Effect of increasing GnRH and PGF2α dose
during Double-Ovsynch on ovulatory response,
luteal regression, and fertility of lactating dairy
cows. Theriogenology 80(7): 773-783. https://doi.org/10.1016/j.theriogenology.2013.07.003
PMid:23932174 4. Mendonça, L.G.D., Mantelo, F.M., Stevenson, J.S. (2017). Fertility of lactating dairy cows treated
with gonadotropin-releasing hormone at AI, 5 days
after AI, or both, during summer heat stress. Theriogenology. 91, 9-16. https://doi.org/10.1016/j.theriogenology.2016.11.032
PMid:28215691 49 Stojanov B. et al. 13. Wiltbank, M.C., Baez, G.M., Cochrane, F., Barletta, R.V.,
Trayford, C.R., Joseph, R.T. (2015). Effect of a
second treatment with prostaglandin F2α during the
Ovsynch protocol on luteolysis and pregnancy in
dairy cows. J Dairy Sci. 98(12): 8644-8654. https://doi.org/10.3168/jds.2015-9353
PMid:26433418 20. Martins, J.P.N., Policelli, R.K., Neuder, L.M.,
Raphael, W., Pursley, J.R. (2011). Effects of
cloprostenol sodium at final prostaglandin F2α of
Ovsynch on complete luteolysis and pregnancy per
artificial insemination in lactating dairy cows. J Dairy Sci. 94(6): 2815-2824. https://doi.org/10.3168/jds.2010-3652
PMid:21605751 14. Atanasov, B., Dovenski, T., Celeska, I., Stevenson, J. S. (2021). Luteolysis, progesterone, and pregnancy per
insemination after modifying the standard 7-day
Ovsynch program in Holstein-Friesian and Holstein
cows. J Dairy Sci. 104(6): 7272-7282. https://doi.org/10.3168/jds.2020-19922
PMid:33773782 21. Bisinotto, R.S., Castro, L.O., Pansani, M.B., Narciso,
C.D., Martinez, N., Sinedino, L.D.P., Pinto, T.L.C.,
et al. (2015). Progesterone supplementation to
lactating dairy cows without a corpus luteum at
initiation of the Ovsynch protocol. J. Dairy Sci. 98(4): 2515-2528. https://doi.org/10.3168/jds.2014-9058
PMid:25682137 15. Please cite this article as: Stojanov B., Atanasov B., Grizelj J., Vince S., Ilievska K., Nikolovski M., Dovenski T., Samardžija M.
External progesterone supplementation during the ovsynch protocol reduces the incomplete luteolysis ın dairy cows under heat
stress. Mac Vet Rev 2024; 47 (1): 45-50. https://doi.org/10.2478/macvetrev-2024-0015 REFERENCES Ferguson, J.D., Galligan, D.T., Thomsen, N. (1994). Principal descriptors of body condition score in
Holstein cows. J Dairy Sci. 77(9): 2695-2703. https://doi.org/10.3168/jds.S0022-0302(94)77212-X
PMid:7814740 22. Xu, Z.Z., Burton, L.J., MacMillan, K.L. (1997). Reproductive performance of lactating dairy cows
following estrus synchronization regimens with
PGF2α and P4. Theriogenology 47(3): 687-701. https://doi.org/10.1016/S0093-691X(97)00027-7
PMid:16728021 16. Stevenson, J.S., Sauls, J.A., Mendonça, L.G.D.,
Voelz, B.E. (2018). Dose and frequency of PGF2α
administration to lactating dairy cows exposed
to pre-synchronization and either five- or seven
day Ovsynch protocols: ovulation, luteolysis, and
pregnancy rates. J Dairy Sci. 101(10): 9575-9590. https://doi.org/10.3168/jds.2018-14653
PMid:30100501 23. Cunha, A.P., Guenther, J.N., Maroney, M.J.,
Giordano, J.O., Nascimento, A.B., Bas, S., Ayres, H.,
Wiltbank, M.C. (2008). Effects of high vs. low
progesterone concentrations during Ovsynch on
double ovulation rate and pregnancies per AI in high
producing dairy cows. J Dairy Sci. 91 (Suppl. 1): 246. 24. Silvia, W.J., Lewis, G.S., McCracken, J.A., Thatcher, W.W.,
Wilson, Jr. L. (1991). Hormonal regulation of uterine
secretion of prostaglandin F2α during luteolysis in
ruminants. Biol Reprod. 45(5): 655-663. https://doi.org/10.1095/biolreprod45.5.655
PMid:1756203 17. Dovenski, T., Atanasov, B., Ilievska, K., Stojanov, B.,
Jasari, B. (2016). Implementation of “on-farm”
method for early detection of non- pregnant cows by
a single ultrasonographic examination of the ovaries
on day 21 after artificial insemination. Reprod
Domest Anim. 51(S2): 73. 25. Souza, A.H., Silva, E.P.B., Cunha, A.P., Gumen, A.,
Ayres,
H.,
Brusveen,
D.J.,
Guenther,
J.N.,
Wiltbank, M.C. (2011). Ultrasonographic evaluation
of endometrial thickness near timed AI as a
predictor of fertility in high-producing dairy cows. Theriogenology 75(4): 722-733. https://doi.org/10.1016/j.theriogenology.2010.10.013
PMid:21196031 18. Vitali, A., Segnalini, M., Bertocchi, L., Bernabucci, U.,
Nardone, A., Lacetera, N. (2009). Seasonal pattern
of mortality and relationships between mortality
and temperature-humidity index in dairy cows. J Dairy Sci. 92(8): 3781-3790. https://doi.org/10.3168/jds.2009-2127
PMid:19620660 26. Atanasov, B., Adamov, N., Celeska, I., Ilievska, K.,
Angjelovski, B., Trbogazov, Z., Davkov, F., et al. (2020). Modification of the standard 7-day
Ovsynch protocol to increase the luteolytic and
synchronization risks in dairy cows. Mac Vet Rev. 43(2): 161-167. https://doi.org/10.2478/macvetrev-2020-0028 19. Bisinotto, R.S., Santos, E.P. (2011). The use of
endocrine treatments to improve pregnancy rates in
cattle. Reprod Fertil Dev. 24(1): 258-266. https://doi.org/10.1071/RD11916
PMid:22394967 50
|
https://openalex.org/W332540842
|
https://europepmc.org/articles/pmc3998529?pdf=render
|
English
| null |
2,3-Bis[(3-methylbiphenyl-4-yl)imino]butane
|
Acta crystallographica. Section E
| 2,014
|
cc-by
| 2,830
|
organic compounds Experimental
Crystal data
C30H28N2
Mr = 416.54
Orthorhombic, Pbca
a = 8.347 (3) A˚
b = 7.063 (3) A˚
c = 39.946 (16) A˚
V = 2355.0 (16) A˚ 3
Z = 4
Mo K radiation
= 0.07 mm1
T = 293 K
0.19 0.18 0.15 mm
Data collection
Bruker APEXII CCD
diffractometer
Absorption correction: multi-scan
(SADABS; Sheldrick, 2004)
Tmin = 0.987, Tmax = 0.990
15668 measured reflections
2200 independent reflections
1251 reflections with I > 2(I)
Rint = 0.088
Refinement
R[F 2 > 2(F 2)] = 0.069
wR(F 2) = 0.135
S = 1.01
2200 reflections
148 parameters
H-atom parameters constrained
max = 0.15 e A˚ 3
min = 0.16 e A˚ 3 Experimental
Crystal data
C30H28N2
Mr = 416.54
Orthorhombic, Pbca
a = 8.347 (3) A˚
b = 7.063 (3) A˚
c = 39.946 (16) A˚
Data collection
Bruker APEXII CCD
diffractometer
Absorption correction: multi-scan
(SADABS; Sheldrick, 2004)
Tmin = 0.987, Tmax = 0.990
Refinement
R[F 2 > 2(F 2)] = 0.069
wR(F 2) = 0.135
S = 1.01
2200 reflections Acta Crystallographica Section E
Structure Reports
Online
ISSN 1600-5368 Acta Crystallographica Section E V = 2355.0 (16) A˚ 3
Z = 4
Mo K radiation
= 0.07 mm1
T = 293 K
0.19 0.18 0.15 mm Jingjing Chen, Jianchao Yuan,* Jie Zhao, Weibing Xu and
Yanqiong Mu 15668 measured reflections
2200 independent reflections
1251 reflections with I > 2(I)
Rint = 0.088 15668 measured reflections
2200 independent reflections
1251 reflections with I > 2(I)
Rint = 0.088 Bruker APEXII CCD
diffractometer
Absorption correction: multi-scan
(SADABS; Sheldrick, 2004)
Tmin = 0.987, Tmax = 0.990 Key Laboratory of Eco-Environment-Related Polymer Materials of the Ministry of
Education, Key Laboratory of Polymer Materials of Gansu Province, College of
Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070,
People’s Republic of China
Correspondence e-mail: jianchaoyuan@nwnu.edu.cn Refinement
R[F 2 > 2(F 2)] = 0.069
wR(F 2) = 0.135
S = 1.01
2200 reflections 148 parameters
H-atom parameters constrained
max = 0.15 e A˚ 3
min = 0.16 e A˚ 3 Received 3 March 2014; accepted 13 March 2014 Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.004 A˚;
R factor = 0.069; wR factor = 0.135; data-to-parameter ratio = 14.9. Data collection: APEX2 (Bruker, 2008); cell refinement: SAINT
(Bruker, 2008); data reduction: SAINT; program(s) used to solve
structure: SHELXTL (Sheldrick, 2008); program(s) used to refine
structure: SHELXTL; molecular graphics: SHELXTL; software used
to prepare material for publication: SHELXTL. The title compound, C30H28N2, is a product of the condensa-
tion reaction of 2-methyl-4-phenylaniline and butane-2,3-
dione. The molecule lies on a crystallographic inversion
centre. The C
N bond has an E conformation. The dihedral
angle between the two benzene rings of the 4-phenyl-2-
methylphenyl group is 29.19 (76). The 1,4-diazabutadiene
plane makes an angle of 70.1 (10) with the N-bonded
methylphenyl ring and an angle of 81.08 (97) with the
terminal phenyl group. We thank the National Natural Science Foundation of
China (20964003) for funding. We also thank the Key
Laboratory of Eco Environment-Related Polymer Materials
of the Ministry of Education, Key Laboratory of Polymer
Materials of Gansu Province (Northwest Normal University),
for financial support. Supporting information for this paper is available from the IUCr
electronic archives (Reference: FK2080). Supporting information for this paper is available from the IUCr
electronic archives (Reference: FK2080). Related literature The title compound was synthesized as an -diimine ligand for
applications in olefin polymerization Ni(II)--diimine cata-
lysts, see: Johnson et al. (1995); Killian et al. (1996); Wang et al. (2013); Ionkin & Marshall (2004); Meinhard et al. (2007). For
the effect of the ligand structure on the activity of the catalyst
and the properties of the products, see: Popeney & Guan
(2005); Yuan et al. (2005); Helldo¨rfer et al. (2003). For related
structures, see: Yuan et al. (2013). 1. Comment In recent years, a variety of α-diimine ligands containing various ortho and para position substituted N-aryl rings
(Johnson et al. 1995; Killian et al.. 1996; Popeney et al.. 2005; Yuan et al., 2005; Wang et al. 2013) and backbone effects
(Helldörfer et al.. 2003) and teraryl substituted-α-diimine ligands (Ionkin et al.. 2004; Meinhard et al.. 2007) were
employed to study their influence on the catalytic activity of α-diimine-Ni(II) complexes. In this study, we designed and
synthesized the title compound as a bidentate ligand. The molecule lies on a crystallographic inversion centre. The single
bond of 1, 4-diazabutadiene fragment is (E)-configured. The dihedral angles between the 1,4-diazabutadiene plane and
the benzene ring bonded to the N atom are 70.12 (96)° and 81.08 (97)° for the terminal phenyl group, resp. The dihedral
angle between both aromatic ring planes is 29.19 (76)° (Figure 1). The crystal packing shows stacking of molecules along
a-axis (Figure 2), however, no significant intermolecular H-bonding is observed. A very similar molecular structure is
known from Yuan et al. (2013). 2. Experimental Formic acid (0.5 ml) was added to a stirred solution of 2-methyl-4-phenylaniline (0.916 g, 2.2 mmol) and 2,3-Butane-
dione (0.086 g, 1 mmol) in 20 ml anhydrous ethanol (20 ml). The mixture was stirred at 50 oC for 24 h, then cooled, and
the precipitate was separated by filtration. The solid was recrystallized from ethanol/dichloromethane (v/v= 8:1), washed
with cold ethanol and dried under vacuum to give the title compound. Yield is 86%. Crystals suitable for X-ray structure
determination were grown from a cyclohexane/dichloromethane (v:v= 1:2) solution. Anal. Calc. for C30H28N2: C, 86.50;
H, 6.78; N, 6.72. Found: C, 86.62; H, 6.57; N, 6.58. References Bruker (2008). SAINT, APEX2 and SADABS. Bruker AXS Inc., Madison,
Wisconsin, USA. Helldo¨rfer, M., Backhaus, J. & Alt, H. G. (2003). Inorg. Chim. Acta, 351, 34–42. Ionkin, A. S. & Marshall, W. J. (2004). Organometallics, 23, 3276–3283. Johnson, L. K., Killian, C. M. & Brookhart, M. (1995). J. Am. Chem. Soc. 117,
6414–6415. Killian, C. M., Tempel, D. J., Johnson, L. K. & Brookhart, M. (1996). J. Am. Chem. Soc. 118, 11664–11665. Meinhard, D., Wegner, M., Kipiani, G., Hearley, A., Reuter, P., Fischer, S
Marti, O. & Rieger, B. J. (2007). J. Am. Chem. Soc, 129, 9182–9191. Marti, O. & Rieger, B. J. (2007). J. Am. Chem. Soc, 129, 9182–9191. Popeney, C. S. & Guan, Z. B. (2005). Organometallics, 24, 1145–1155. Sheldrick, G. M. (2004). SADABS. University of Go¨ttingen, Germany. (
)
Popeney, C. S. & Guan, Z. B. (2005). Organometallics, 24, 1145–1155. Sheldrick, G. M. (2004). SADABS. University of Go¨ttingen, Germany. (
)
Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Wang, F. Z., Yuan, J. C., Song, F. Y., Li, J., Jia, Z. & Yuan, B. N. (2013). Appl. Organomet. Chem. 27, 319–327. g
Yuan, J. C., Jia, Z., Li, J., Song, F. Y., Wang, F. Z. & Yuan, B. N. (2013). Transition Met. Chem. 38, 341–350. Yuan, J. C., Silva, L. C., Gomes, P. T., Valerga, P., Campos, J. M., Ribeiro, M. R.,
Chien, J. C. W. & Marques, M. M. (2005). Polymer, 46, 2122–2132. Chen et al. o455 o455 Acta Cryst. (2014). E70, o455 Chen et al. doi:10.1107/S1600536814005686 supplementary materials 2,3-Bis[(3-methylbiphenyl-4-yl)imino]butane Jingjing Chen, Jianchao Yuan, Jie Zhao, Weibing Xu and Yanqiong Mu Acta Cryst. (2014). E70, o455 [doi:10.1107/S1600536814005686] Acta Cryst. (2014). E70, o455 [doi:10.1107/S1600536814005686] Acta Cryst. (2014). E70, o455 [doi:10.1107/S1600536814005686] 3. Refinement Positions of the methyl H atoms were derived from Fourier maps (HFIX 137), with C–H 0.96 Å and Uiso(H) = 1.5Ueq(C). All other H atoms were placed in geometrically idealized positions and constrained to ride on their parent atoms with C–
H distances distances of 0.93 Å and Uiso(H) = 1.2Ueq(C). sup-1 Acta Cryst. (2014). E70, o455 supplementary materials Figure 1
The title molecule with displacement ellipsoids plotted at 50% probability level. Atoms with label "a" are related by the
symmetry code (-x+1, -y+1, -z). Figure 2
Crystal packing viewed along the a-axis. 2,3-Bis[(3-methylbiphenyl-4-yl)imino]butane
Crystal data
C30H28N2
Mr = 416.54
Orthorhombic, Pbca
a = 8.347 (3) Å
b = 7.063 (3) Å
c = 39.946 (16) Å
V = 2355.0 (16) Å3
Z = 4
F(000) = 888
Dx = 1.175 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 1674 reflections
θ = 2.6–21.7°
µ = 0.07 mm−1
T = 293 K
Block, yellow
0.19 × 0.18 × 0.15 mm
Data collection
Bruker APEXII CCD
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
φ and ω scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2004)
Tmin = 0.987, Tmax = 0.990
15668 measured reflections
2200 independent reflections
1251 reflections with I > 2σ(I)
Rint = 0.088
θmax = 25.5°, θmin = 2.6°
h = −10→10
k = −8→8
l = −48→47 Figure 1 Figure 1
The title molecule with displacement ellipsoids plotted at 50% probability level. Atoms with label "a" are related by the
symmetry code (-x+1, -y+1, -z). The title molecule with displacement ellipsoids plotted at 50% probability level. Atoms with label "a" are related by the
symmetry code (-x+1, -y+1, -z). Figure 2 Crystal packing viewed along the a-axis. supplementary materials Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.069
wR(F2) = 0.135
S = 1.01
2200 reflections
148 parameters
0 restraints
Primary atom site location: structure-invariant
direct methods
Secondary atom site location: difference Fourier
map
Hydrogen site location: difference Fourier map
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0366P)2 + 1.4273P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max < 0.001
Δρmax = 0.15 e Å−3
Δρmin = −0.16 e Å−3
Extinction correction: SHELXTL (Sheldrick,
2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Extinction coefficient: 0.0025 (6) Refinement
Refinement on F2
Least-squares matrix: full
R[F2 > 2σ(F2)] = 0.069
wR(F2) = 0.135
S = 1.01
2200 reflections
148 parameters
0 restraints
Primary atom site location: structure-invarian
direct methods Secondary atom site location: difference Fourier
map
Hydrogen site location: difference Fourier map
H-atom parameters constrained
w = 1/[σ2(Fo2) + (0.0366P)2 + 1.4273P]
where P = (Fo2 + 2Fc2)/3
(Δ/σ)max < 0.001
Δρmax = 0.15 e Å−3
Δρmin = −0.16 e Å−3
Extinction correction: SHELXTL (Sheldrick,
2008), Fc*=kFc[1+0.001xFc2λ3/sin(2θ)]-1/4
Extinction coefficient: 0.0025 (6) 2,3-Bis[(3-methylbiphenyl-4-yl)imino]butane Crystal data
C30H28N2
Mr = 416.54
Orthorhombic, Pbca
a = 8.347 (3) Å
b = 7.063 (3) Å
c = 39.946 (16) Å
V = 2355.0 (16) Å3
Z = 4
F(000) = 888
Dx = 1.175 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 1674 reflections
θ = 2.6–21.7°
µ = 0.07 mm−1
T = 293 K
Block, yellow
0.19 × 0.18 × 0.15 mm
Data collection
Bruker APEXII CCD
diffractometer
Radiation source: fine-focus sealed tube
Graphite monochromator
φ and ω scans
Absorption correction: multi-scan
(SADABS; Sheldrick, 2004)
Tmin = 0.987, Tmax = 0.990
15668 measured reflections
2200 independent reflections
1251 reflections with I > 2σ(I)
Rint = 0.088
θmax = 25.5°, θmin = 2.6°
h = −10→10
k = −8→8
l = −48→47 Dx = 1.175 Mg m−3
Mo Kα radiation, λ = 0.71073 Å
Cell parameters from 1674 reflections
θ = 2.6–21.7°
µ = 0.07 mm−1
T = 293 K
Block, yellow
0.19 × 0.18 × 0.15 mm sup-2 Acta Cryst. (2014). E70, o455 supplementary materials Acta Cryst. (2014). E70, o455 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full
covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and
torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2,
conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used
only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2
are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq
C1
0.6218 (4)
−0.0240 (5)
0.17712 (7)
0.0644 (9)
H1
0.7117
−0.0468
0.1640
0.077*
C2
0.6029 (4)
−0.1219 (5)
0.20680 (8)
0.0716 (10)
H2
0.6792
−0.2106
0.2133
0.086*
C3
0.4725 (4)
−0.0893 (5)
0.22677 (7)
0.0663 (9)
H3
0.4597
−0.1548
0.2468
0.080*
C4
0.3612 (4)
0.0413 (4)
0.21680 (7)
0.0594 (9)
H4
0.2726
0.0652
0.2303
0.071*
C5
0.3788 (4)
0.1375 (4)
0.18713 (6)
0.0504 (8)
H5
0.3012
0.2248
0.1807
0.061*
C6
0.5099 (3)
0.1070 (4)
0.16654 (6)
0.0442 (7)
C7
0.5276 (3)
0.2107 (4)
0.13451 (6)
0.0425 (7)
C8
0.6072 (3)
0.1298 (4)
0.10744 (7)
0.0509 (8)
H8
0.6563
0.0124
0.1099
0.061*
C9
0.6142 (3)
0.2216 (4)
0.07704 (7)
0.0532 (8)
H9
0.6678
0.1652
0.0592
0.064*
C10
0.5427 (3)
0.3961 (4)
0.07266 (6)
0.0423 (7)
C11
0.4688 (3)
0.4856 (4)
0.09956 (6)
0.0432 (7)
C12
0.4629 (3)
0.3887 (4)
0.12980 (6)
0.0443 (7)
H12
0.4126
0.4468
0.1479
0.053*
C13
0.3949 (4)
0.6774 (4)
0.09594 (8)
0.0674 (10)
H13A
0.2882
0.6648
0.0873
0.101*
H13B
0.4580
0.7522
0.0808
0.101*
H13C
0.3912
0.7384
0.1174
0.101* ic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2)
x
y
z
Uiso*/Ueq Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) sup-3 Acta Cryst. (2014). Acta Cryst. (2014). E70, o455 Special details E70, o455 supplementary materials pp
y
4
C14
0.4850 (3)
0.4425 (4)
0.01552 (6)
0.0440 (7)
C15
0.3729 (4)
0.2792 (5)
0.01278 (8)
0.0773 (11)
H15A
0.3722
0.2102
0.0335
0.116*
H15B
0.4073
0.1972
−0.0050
0.116*
H15C
0.2670
0.3248
0.0081
0.116*
N1
0.5572 (3)
0.4954 (3)
0.04174 (5)
0.0486 (6)
Atomic displacement parameters (Å2)
U11
U22
U33
U12
U13
U23
C1
0.068 (2)
0.078 (3)
0.0472 (19)
0.013 (2)
0.0012 (16)
0.0083 (18)
C2
0.086 (3)
0.075 (2)
0.054 (2)
0.016 (2)
−0.0096 (19)
0.012 (2)
C3
0.092 (3)
0.064 (2)
0.0429 (18)
−0.005 (2)
−0.0080 (19)
0.0083 (17)
C4
0.075 (2)
0.063 (2)
0.0400 (17)
−0.0046 (19)
0.0066 (16)
−0.0005 (16)
C5
0.0651 (19)
0.0476 (19)
0.0386 (17)
−0.0013 (16)
0.0034 (14)
0.0006 (15)
C6
0.0502 (17)
0.0475 (18)
0.0349 (15)
0.0018 (15)
−0.0037 (13)
−0.0025 (14)
C7
0.0388 (15)
0.0504 (19)
0.0381 (16)
−0.0011 (15)
−0.0014 (13)
−0.0022 (14)
C8
0.0587 (18)
0.0529 (19)
0.0410 (17)
0.0119 (16)
0.0057 (14)
0.0047 (16)
C9
0.0593 (18)
0.058 (2)
0.0424 (18)
0.0049 (17)
0.0088 (15)
−0.0019 (16)
C10
0.0417 (15)
0.0495 (18)
0.0356 (16)
−0.0052 (15)
−0.0010 (13)
0.0013 (14)
C11
0.0430 (15)
0.0456 (17)
0.0410 (17)
−0.0007 (14)
−0.0023 (13)
−0.0011 (14)
C12
0.0474 (16)
0.0479 (18)
0.0377 (16)
0.0014 (15)
0.0034 (13)
−0.0058 (14)
C13
0.088 (2)
0.055 (2)
0.059 (2)
0.0099 (19)
0.0034 (18)
0.0063 (17)
C14
0.0404 (15)
0.0522 (19)
0.0395 (16)
−0.0047 (14)
0.0022 (13)
0.0024 (13)
C15
0.090 (2)
0.086 (3)
0.055 (2)
−0.041 (2)
−0.0109 (19)
0.0185 (19)
N1
0.0521 (14)
0.0561 (16)
0.0375 (13)
−0.0075 (12)
0.0012 (12)
0.0028 (12)
Geometric parameters (Å, º)
C1—C6
1.381 (4)
C9—C10
1.380 (4)
C1—C2
1.382 (4)
C9—H9
0.9300
C1—H1
0.9300
C10—C11
1.391 (3)
C2—C3
1.369 (4)
C10—N1
1.426 (3)
C2—H2
0.9300
C11—C12
1.389 (3)
C3—C4
1.368 (4)
C11—C13
1.496 (4)
C3—H3
0.9300
C12—H12
0.9300
C4—C5
1.374 (4)
C13—H13A
0.9600
C4—H4
0.9300
C13—H13B
0.9600
C5—C6
1.386 (4)
C13—H13C
0.9600
C5—H5
0.9300
C14—N1
1.265 (3)
C6—C7
1.481 (4)
C14—C15
1.489 (4)
C7—C12
1.381 (4)
C14—C14i
1.504 (5)
C7—C8
1.392 (3)
C15—H15A
0.9600
C8—C9
1.378 (4)
C15—H15B
0.9600
C8—H8
0.9300
C15—H15C
0.9600
C6—C1—C2
121.4 (3)
C8—C9—H9
119.6
C6—C1—H1
119.3
C9—C10—C11
120.0 (3)
C2—C1—H1
119.3
C9—C10—N1
120.8 (2)
C3—C2—C1
120.4 (3)
C11—C10—N1
118.9 (3) Atomic displacement parameters (Å2) sup-4 Acta Cryst. (2014). Special details E70, o455 supplementary materials C3—C2—H2
119.8
C10—C11—C12
117.6 (3)
C1—C2—H2
119.8
C10—C11—C13
121.3 (3)
C4—C3—C2
118.9 (3)
C12—C11—C13
121.1 (3)
C4—C3—H3
120.5
C7—C12—C11
123.6 (3)
C2—C3—H3
120.5
C7—C12—H12
118.2
C3—C4—C5
120.8 (3)
C11—C12—H12
118.2
C3—C4—H4
119.6
C11—C13—H13A
109.5
C5—C4—H4
119.6
C11—C13—H13B
109.5
C4—C5—C6
121.3 (3)
H13A—C13—H13B
109.5
C4—C5—H5
119.4
C11—C13—H13C
109.5
C6—C5—H5
119.4
H13A—C13—H13C
109.5
C1—C6—C5
117.2 (3)
H13B—C13—H13C
109.5
C1—C6—C7
121.9 (3)
N1—C14—C15
126.1 (3)
C5—C6—C7
120.9 (3)
N1—C14—C14i
116.4 (3)
C12—C7—C8
117.0 (3)
C15—C14—C14i
117.5 (3)
C12—C7—C6
121.9 (3)
C14—C15—H15A
109.5
C8—C7—C6
121.1 (3)
C14—C15—H15B
109.5
C9—C8—C7
120.8 (3)
H15A—C15—H15B
109.5
C9—C8—H8
119.6
C14—C15—H15C
109.5
C7—C8—H8
119.6
H15A—C15—H15C
109.5
C10—C9—C8
120.9 (3)
H15B—C15—H15C
109.5
C10—C9—H9
119.6
C14—N1—C10
122.1 (2)
C6—C1—C2—C3
−0.8 (5)
C8—C9—C10—C11
3.0 (4)
C1—C2—C3—C4
0.2 (5)
C8—C9—C10—N1
176.7 (3)
C2—C3—C4—C5
0.5 (5)
C9—C10—C11—C12
−3.4 (4)
C3—C4—C5—C6
−0.6 (5)
N1—C10—C11—C12
−177.2 (2)
C2—C1—C6—C5
0.7 (5)
C9—C10—C11—C13
178.0 (3)
C2—C1—C6—C7
−179.0 (3)
N1—C10—C11—C13
4.2 (4)
C4—C5—C6—C1
0.0 (4)
C8—C7—C12—C11
2.3 (4)
C4—C5—C6—C7
179.7 (3)
C6—C7—C12—C11
−176.1 (3)
C1—C6—C7—C12
−152.1 (3)
C10—C11—C12—C7
0.7 (4)
C5—C6—C7—C12
28.3 (4)
C13—C11—C12—C7
179.4 (3)
C1—C6—C7—C8
29.6 (4)
C15—C14—N1—C10
2.3 (5)
C5—C6—C7—C8
−150.0 (3)
C14i—C14—N1—C10
−178.4 (3)
C12—C7—C8—C9
−2.7 (4)
C9—C10—N1—C14
71.9 (4)
C6—C7—C8—C9
175.7 (3)
C11—C10—N1—C14
−114.4 (3)
C7—C8—C9—C10
0.1 (4)
Symmetry code: (i) −x+1, −y+1, −z. Symmetry code: (i) −x+1, −y+1, −z. sup-5 Acta Cryst. (2014). E70, o455
|
https://openalex.org/W3131178200
|
https://www.nature.com/articles/s41598-021-83374-y.pdf
|
English
| null |
Effect of palmitoylation on the dimer formation of the human dopamine transporter
|
Scientific reports
| 2,021
|
cc-by
| 14,655
|
www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ also includes the serotonin and norepinephrine transporters (SERT and NET), which are all located in the presyn-
aptic plasma membrane of neurons2–4. The MATs have a conserved architecture consisting of 12 transmembrane
helices (TM) (Fig. 1a) with a central substrate binding site5,6. For neurotransmitter translocation the MATs use the
transmembrane sodium gradient as a driving force7. Dysregulation of the MATs has been linked to several major
diseases such as depression8, Parkinson disease9 and attention deficit hyperactive disorder10. The transporters
have also been implicated in drug addiction11,12. The means of regulation and supramolecular organization of
the MATs in the plasma membrane is therefore of great interest for application in drug development13. p
g
pp
g
p
Membrane protein oligomerization is increasingly observed to play an important role in protein func-
tion and regulation, and the MATs are no exception14,15. The MATs have been experimentally shown to form
homooligomers16,17, and specifically in the synaptic plasma membrane several oligomeric states have been
detected18. MAT oligomerization is presumed necessary for endoplasmic reticulum export16 and for DAT it
has been suggested that the oligomeric state can have a cooperative effect on inhibitor binding and substrate
transport19. From cysteine cross-linking experiments there is evidence suggesting that SERT and DAT form a
homotetramer presumably consisting of a dimer of dimers20,21, but from more recent single-molecule experiments
other oligomeric states have also been observed for hSERT18, and for hDAT only the monomeric and dimeric
states have been detected in the plasma membrane20. p
Currently, no consensus exists with regard to the oligomeric interfaces present within and across MATs in the
plasma membrane. Symmetric interfaces involving either TM4, TM6, or TM2 have been suggested for DAT from
cysteine cross-linking and mutagenesis experiments17,20,22,23. Computational protein–protein docking studies
indicate that TM2, TM6, and TM11 in union constitute a symmetrical dimeric interface in hDAT23. For SERT,
FRET experiments of the TM fragments TM1-2 and TM11-12 point to these pairs forming a symmetrical dimer
interaction site24. Crystal structures of a prokaryotic NSS member, the leucine transporter (LeuT), presents a
symmetrical dimer consisting of an interface involving TM9, TM12, and extracellular loop 2 (EL2)25. In sum-
mary, a wide range of different interfaces have been detected and proposed to be important for hDAT and hSERT
oligomerization. Collectively, the studies do not point to a unique single dimer model for all MATs, rather several
interfaces for the same transporter are likely to exist. www.nature.com/scientificreports/ p
y
Recently, we successfully presented data indicating that a likely dimer candidate for hSERT in a 1-palmitoyl-
2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane consists of a symmetrical TM12/TM12 interface (the
naming convention applied throughout the manuscript for indicating which helices are found at the dimer inter-
face. TM helices contacting from the same protomer are separated by commas, while a slash is used to indicate
the second protomer’s interacting helices)14. The TM12/TM12 interface is in partial agreement with previous
FRET studies24 and the interface observed in crystal structure of the prokaryotic NSS member, the leucine
transporter (LeuT)25. We employed a combination of multiple self-assembly simulations followed by umbrella
sampling replica exchange molecular dynamics (US-REMD) simulations on a subset of highly populated hSERT
dimer conformations to determine potential of mean force (PMF) free energy profiles of the interfaces14. From
the same work, we further show that the lipid composition of the embedding membrane largely affected the
strength of the TM12/TM12 interface. Lipids bound to the surface of membrane proteins in general, could
therefore either stabilize or destabilize certain dimeric or higher order oligomeric states26,27. In the case of DAT,
a palmitoyl (palm) chain is reversibly attached at TM12 on Cys581 (Fig. 1a) and has been shown to regulate DAT
surface expression and turnover28,29. Palm is a post-translational modification involving the thioesterification
of palmitate, a 16-carbon fatty acid, to a cysteine residue. For integral membrane proteins, this modification is
reversible and regulates a variety of properties including functional activity, trafficking, turnover, membrane raft
targeting, and cholesterol binding30–34. Due to the location of the palm group on TM12, which has been shown
from both LeuT crystal structures and hSERT self-assembly simulations to be involved in the dimer interface,
we speculated that the palm group may have a regulatory mechanism on hDAT dimer formation, similar to what
has been observed for some GPCRs35. In this contribution, we employ our previous approach used for hSERT to
investigate the impact of S-palmitoylation on hDAT oligomeric behavior14,36. We identify several interfaces and
show that the stability of dimer interfaces involving TM12 changes when TM12 is palmitoylated. We also compare
the LeuT dimer from crystal structures to those obtained from self-assembly simulations and find that similar
helices are involved at the interface for LeuT, hDAT and hSERT. www.nature.com/scientificreports/ Collectively, we propose that the MATs share a
common dimer pattern, which mainly involves the helices TM9, TM11, and TM12, and that S-palmitoylation
may act as a functional switch. transporter Talia Zeppelin1, Kasper B. Pedersen1,2, Nils A. Berglund1, Xavier Periole1,3* &
Birgit Schiøtt1,2* The human dopamine transporter (hDAT) is one in three members of the monoamine transporter
family (MAT). hDAT is essential for regulating the dopamine concentration in the synaptic cleft
through dopamine reuptake into the presynaptic neuron; thereby controlling hDAT dopamine
signaling. Dysfunction of the transporter is linked to several psychiatric disorders. hDAT and the other
MATs have been shown to form oligomers in the plasma membrane, but only limited data exists on
which dimeric and higher order oligomeric states are accessible and energetically favorable. In this
work, we present several probable dimer conformations using computational coarse-grained self-
assembly simulations and assess the relative stability of the different dimer conformations using
umbrella sampling replica exchange molecular dynamics. Overall, the dimer conformations primarily
involve TM9 and/or TM11 and/or TM12 at the interface. Furthermore, we show that a palmitoyl group
(palm) attached to hDAT on TM12 modifies the free energy of separation for interfaces involving
TM12, suggesting that S-palmitoylation may change the relative abundance of dimers involving TM12
in a biological context. Finally, a comparison of the identified interfaces of hDAT and palmitoylated
hDAT to the human serotonin transporter interfaces and the leucine transporter interface, suggests
similar dimer conformations across these protein family. Abbreviations
AA
All atom
CG
Coarse grain
DAT
Dopamine transporter
EL2
Extracellular loop 2
hDAT, hNET, hSERT
Human DAT, NET or SERT is indicated with a ‘h’
LASA
Lipid accessible surface area
LeuT
Leucine transporter
MAT
Monoamine transporter
NSS
Neurotransmitter Sodium Symporter
NET
Norepinephrine transporter
palm
Palmitoylation
PMF
Potential of mean force
SERT
Serotonin transporter
TM
Transmembrane
US-REMD
Umbrella sampling replica exchange molecular dynamics
POPC
1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine The dopamine transporter (DAT) mediates reuptake of the neurotransmitter dopamine from the synaptic cleft
back into the presynaptic neuron and thereby terminates dopaminergic signaling. DAT is part of the monoamine
transporter family (MAT) and the larger Neurotransmitter Sodium Symporter (NSS) family1. The MAT family 1Department of Chemistry, Aarhus University, Aarhus C, Denmark. 2Interdisciplinary Nanoscience Center,
Aarhus University, Aarhus C, Denmark. 3Present address: School of Biological Sciences, University of Auckland &
Canterbury, Auckland & Christchurch, New Zealand. *email: x.periole.science@gmail.com; birgit@chem.au.dk Scientific Reports | (2021) 11:4164 | https://doi.org/10.1038/s41598-021-83374-y www.nature.com/scientificreports/ Results
P l
i esu ts
Palmitoylation of hDAT barely affects the interhelical contacts formed in self‑assembly simu-
lations. In order to study which helices are most prevalent at the oligomeric interfaces of hDAT and how
these are affected by hDAT being S-palmitoylated (hDAT-palm) on TM12, we performed 10 self-assembly simu-
lations each lasting 30 µs and containing 16 hDAT or 16 hDAT-palm proteins embedded in a pure POPC mem-
brane bilayer (Fig. 1b). Visual inspection of the last frames of the different repeat simulations indicates that both
hDAT and hDAT-palm form dimers and higher order oligomers on a µs timescale (Fig. 1a and Supplementary
Fig. S1 for a full overview)14.i g
To investigate which helices are most prevalent at the interface, interhelical contacts were quantified between
protein pairs. Interhelical contacts were calculated by counting the number of times a helix from one protomer
is in contact with another helix of another protomer, but not considering the palmitoyl group. The interhelical
contacts were summed across all frames and repeat simulations to consider all possible dimer pairs. Illustrated
in Fig. 1c (central panel) is the proportion of times each helix (TM1 to TM12) is found to form a contact
with any other helix in another protein. It is observed that in the simulations of hDAT with and without the
S-palmitoylation, the helices that frequently form contacts are similar in the two systems (Fig. 1c, middle panel). Scientific Reports | (2021) 11:4164 | https://doi.org/10.1038/s41598-021-83374-y www.nature.com/scientificreports/ Figure 1. System setup and interhelical contact maps of hDAT and hDAT-palm excluding the palmitoyl group. Figure 1. System setup and interhelical contact maps of hDAT and hDAT-palm excluding the palmitoyl group. (a) The hDAT CG structures with and without a palm group (orange spheres) attached to TM12. Shown in (b) is
a top view of one of a system a(top view with respect to the membrane) at 0 µs (left) and after a 30 µs simulation
(right). In the two systems 16 hDAT and hDAT-palm molecules were initially equally spaced and randomly
orientated with respect to each other in a pure POPC membrane. hDAT and hDAT-palm were simulated for
30 µs and repeated 10 times. Palm, TM9, TM11, and TM12 are highlighted in orange, blue, purple, and magenta,
respectively. The green dots represent the GL1 bead of POPC in the nearest periodic images for the system. Results
P l
i Illustrated in (c), hDAT (left) and hDAT-palm (right), are the per helix contact maps calculated for all possible
dimer pairs. An interhelical contact was considered when any residue in one protomer’s helix was within 7 Å of
any residue in the other protomer’s helix (not considering the palm group). The contacts have been normalized
by the total number of contacts calculated for the hDAT system. The 1D plot immediately above the contact map
illustrates the sum of all TM contacts calculated for each helix given in %. The top 1D plot illustrates the average
lipid accessible surface area in nm2 of the different helices in the two systems using a probe of size 0.26 nm. The
standard deviations are not visible in the plots due to their small sizes (they vary between 0.23 and 0.71). Figure 1. System setup and interhelical contact maps of hDAT and hDAT-palm excluding the palmitoyl group. (a) The hDAT CG structures with and without a palm group (orange spheres) attached to TM12. Shown in (b) is
a top view of one of a system a(top view with respect to the membrane) at 0 µs (left) and after a 30 µs simulation
(right). In the two systems 16 hDAT and hDAT-palm molecules were initially equally spaced and randomly
orientated with respect to each other in a pure POPC membrane. hDAT and hDAT-palm were simulated for
30 µs and repeated 10 times. Palm, TM9, TM11, and TM12 are highlighted in orange, blue, purple, and magenta,
respectively. The green dots represent the GL1 bead of POPC in the nearest periodic images for the system. Illustrated in (c), hDAT (left) and hDAT-palm (right), are the per helix contact maps calculated for all possible
dimer pairs. An interhelical contact was considered when any residue in one protomer’s helix was within 7 Å of
any residue in the other protomer’s helix (not considering the palm group). The contacts have been normalized
by the total number of contacts calculated for the hDAT system. The 1D plot immediately above the contact map
illustrates the sum of all TM contacts calculated for each helix given in %. The top 1D plot illustrates the average
lipid accessible surface area in nm2 of the different helices in the two systems using a probe of size 0.26 nm. Results
P l
i In contrast, TM9, which is the most
contact-forming helix, is only marginally exposed. It is also notable that TM7, which is more exposed than TM9,
forms few contacts. These observations suggest that the exposure of a TM is not the main factor determining
the formation of contacts. Specifically, helix contacts involving TM9, TM11, and TM12 and to some extent TM4-6 are predominant in both
systems. We then assessed whether the helices frequently in contact are correlated with being more accessible
to the surrounding lipid environment by computing the lipid accessible surface area (LASA) of the 12 differ-
ent helices using a spherical probe with a radius of 0.26 nm (Fig. 1c, top panels). From the LASA, it is detected
that the helices, TM4, TM11, and TM12, which are frequently present at the dimer interface, are also the most
exposed to the lipid environment of all the TM helices (Fig. 1c, top panels). In contrast, TM9, which is the most
contact-forming helix, is only marginally exposed. It is also notable that TM7, which is more exposed than TM9,
forms few contacts. These observations suggest that the exposure of a TM is not the main factor determining
the formation of contacts. Illustrated in the bottom panels of Fig. 1c are 2D contact maps revealing interhelical pairs in the simulations
that most frequently form contacts. Interhelical contact matrices share a comparable pattern in the two systems
(Fig. 1c, bottom panels) indicating that similar helices are found at the dimer interface in the two systems. How-
ever in the simulations where hDAT is palmitoylated, TM12 of one protomer is in less contact with TM12 of
another protomer compared to simulations without palmitoylation. Otherwise the two systems agree on TM12
of one protomer being frequently in contact with TM4-5 and TM9 of the other protomer. TM9 being frequently
in contact with TM4, TM9, TM11, and TM12, and TM11 being frequently in contact with TM4 and TM11. The
relative distributions of the helix pairs are, however, slightly different in the two systems. p
g
yf
y
Two motifs have been suggested to promote integral protein oligomerization: a leucine zipper motif and
a GXXXG motif 37,38. The MATs contain a conserved GXXXG motif in both TM6 and EL3, and hDAT has an
additional leucine zipper in TM9 (the locations of the motifs are highlighted in Supplementary Fig. 2). Results
P l
i The
standard deviations are not visible in the plots due to their small sizes (they vary between 0.23 and 0.71). https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ Figure 2. Dimer cluster comparison between the two systems, hDAT and hDAT-palm. Depicted in (a)
is a transmembrane (TM) overview of hDAT shown from the side with respect to the membrane normal. The helices are individually color-coded using the same coloring scheme as applied throughout the article. Highlighted in bold spheres is the palmitoyl group (palm) covalently bound to TM12. The two angles, ϕ1 and
ϕ3, are used for describing the individual protomer’s relative orientation. Depicted in (b) are the common top
clusters observed in both the hDAT and hDAT-palm simulations, with the exception of the TM12/TM12 dimer,
which is only detected 0.1% of the time in the hDAT-palm simulations. For a full view of the representative
dimers of all top clusters of the hDAT and hDAT-palm system see Supplementary Fig. S5-6. Figure 2. Dimer cluster comparison between the two systems, hDAT and hDAT-palm. Depicted in (a)
is a transmembrane (TM) overview of hDAT shown from the side with respect to the membrane normal. The helices are individually color-coded using the same coloring scheme as applied throughout the article. Highlighted in bold spheres is the palmitoyl group (palm) covalently bound to TM12. The two angles, ϕ1 and
ϕ3, are used for describing the individual protomer’s relative orientation. Depicted in (b) are the common top
clusters observed in both the hDAT and hDAT-palm simulations, with the exception of the TM12/TM12 dimer,
which is only detected 0.1% of the time in the hDAT-palm simulations. For a full view of the representative
dimers of all top clusters of the hDAT and hDAT-palm system see Supplementary Fig. S5-6. Specifically, helix contacts involving TM9, TM11, and TM12 and to some extent TM4-6 are predominant in both
systems. We then assessed whether the helices frequently in contact are correlated with being more accessible
to the surrounding lipid environment by computing the lipid accessible surface area (LASA) of the 12 differ-
ent helices using a spherical probe with a radius of 0.26 nm (Fig. 1c, top panels). From the LASA, it is detected
that the helices, TM4, TM11, and TM12, which are frequently present at the dimer interface, are also the most
exposed to the lipid environment of all the TM helices (Fig. 1c, top panels). www.nature.com/scientificreports/ The TM9/TM9 interface is the highest excluded lipid exposed area for both
the hDAT and hDAT-palm interfaces. However, in agreement with the contact analysis, there is no clear pat-
tern between the dimers observed to be among the top clusters and the degree in which their interface is lipid
exposed (Table 1).i A limitation of the MARTINI force field is the inherent stickiness of the model40, despite this, however, we
do see protein unbinding events. Moreover, the protein surface probed is similar when comparing the two sets
of five repeat simulations (see Supplementary Fig. S8), suggesting a reasonably converged surface exploration. To evaluate in a more quantitative manner the strength of the highly populated dimer interfaces detected in the
cluster analysis, we computed the PMF along protomer separation using US-REMD simulations. Palmitoylation of TM12 changes the energetics of dimer interfaces involving TM12. In the
following section, we compare the interface’s strength of a subset of different dimer conformations by generating
their PMF energy profiles from US-REMD simulations. The hDAT dimer conformations selected to be evalu-
ated through PMF energy profiles consisted of TM9,TM12/TM9,TM12 (cluster 1), TM4,TM9/TM11 (cluster
3,4), TM9/TM9 (cluster 6), and TM12/TM12 (cluster 9). These interfaces are found among the most popu-
lated clusters of the hDAT self-assembly simulations (Fig. 2b). Similarly, the hDAT-palm interfaces TM9,TM12/
TM9,TM12 (cluster 10), TM12/TM12 (cluster 53), and TM9/TM9 (cluster 1) were also evaluated, to further
understand the effect of palm on the dimer interface strength. The hDAT-palm TM9/TM9 interface was pri-
marily used as a control, since palm is not involved at this interface. The energetics is therefore expected to be
unchanged between the hDAT and the palmitoylated hDAT system. The protocol used for generating the PMF’s
has previously been applied by Periole et al. on hSERT and on GPCRs36 and is briefly described in the Methods
section 14. It should be highlighted that the aim was to rank the conformations and not to establish absolute free
energies.hfi g
The PMFs for the different hDAT interfaces are presented in the first panel in Fig. 3a (see Supplementary
Table S1-2 for further details concerning the US-REMD settings). The three hDAT dimer interfaces, TM12/
TM12, TM4,TM9/TM11, and TM9/TM9, have a similar value of free energy of dissociation of 55 ± 10 kJ/mol. The TM9,TM12/TM9,TM12 interface is weaker with a dissociation free energy of ~ 40 kJ/mol. www.nature.com/scientificreports/ populated clusters for both hDAT and hDAT-palm dimers primarily involve TM9 and/or TM11 helices at the
interface (see Supplementary Fig. S5-6). The TM12/TM12 interface of the palmitoylated hDAT is also presented
in Fig. 2b, although it is only detected 0.1% of the time during simulations according to the cluster analysis. The
TM12/TM12 interface is included and studied further in a later section because I) it resembles the hSERT TM12/
TM12 interface14 and II) it involves TM12 on which palm is attached. Based on the cluster analysis, TM12 is
involved more frequently at dimer interfaces found in the hDAT system compared to the hDAT-palm system. The reason for this, is due to the presence of the long flexible palm group that prevents the formation of interpro-
tein contacts. Thus, only short-lived contacts can form at much longer distances than the center-of-mass (COM)
cut-off distance applied for defining a dimer pair used in the cluster analysis (see Methods and “TM12/TM12
dynamics” sections for further details).f y
For describing the relative orientation of the individual protomers with respect to each other in the differ-
ent dimer conformations two dihedral angles were used36, ϕ1 and ϕ3 (Fig. 2a) as defined in the Virtual Bond
Algorithm39. The values of ϕ1 and ϕ3 for the different dimer conformations are given in Table 1. In principle, a
total of three dihedral angles (see Fig. 2a) are necessary for thoroughly understanding two rigid bodies relative
orientation, however, it is observed in our system that the ϕ2 angle does not vary more than ± 25°, due to the
proteins being embedded in a membrane. The ϕ2 angle is therefore not considered further. When the ϕ1 and
ϕ3 values are similar, the dimers can be regarded as symmetrical with respect to a C2 axis perpendicular to the
membrane plane e.g. the dimers TM9/TM9, TM12/TM12, and TM9,TM12/TM9,TM12. Whereas when the ϕ1
and ϕ3 values are dissimilar the dimers are anti-symmetric e.g. the dimers TM4,TM9/TM11 and TM4,TM9/
TM2. In addition, it can also be observed which clusters are closely related e.g. cluster 1,2 and 9 for hDAT (see
Table 1 and Supplementary Fig. S7).hf pp
y
g
The average degree of surface area occlusion of the dimer interfaces found in the different clusters varies
between 12 and 20.7 nm2 and was computed by subtracting the LASA of the full dimer complex from the LASA
of the two individual monomers. Results
P l
i However,
the two conserved GXXXG motifs are buried inside the protein and cannot partake in a dimer interface. The
leucine zipper motif in TM9 of hDAT is exposed to the membrane milieu and the majority of leucine residues
can readily form contacts to another protomer (see Supplementary Fig. 3). It is therefore noteworthy that TM9/
TM9 contacts are frequently observed for both the hDAT and hDAT-palm system. hDAT with and without a palm group results in similar dimers. To characterize further the dimer
conformations formed in the self-assembly simulations of hDAT and palmitoylated hDAT, we performed a clus-
ter analysis of the proteins in contact (see Methods for further details and Supplementary Fig. S4). In agree-
ment with the contact analysis, the cluster analysis shows that similar protein dimers exist for the hDAT and
hDAT-palm systems with slightly differing relative populations (Fig. 2 and Table 1). The predominant dimer
conformations found in both systems consist of the following interfaces evaluated by visual inspection: TM9/
TM9, TM4,TM9/TM11, TM4,TM9/TM2, TM9,TM12/TM9,TM12 and finally TM12/TM4 (Fig. 2b). The most https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Interestingly,
the TM12/TM12 interface seems to have a second minimum (double well) at a separation distance of ~ 0.4 nm
(clear in panel 3 in Fig. 3a).i p
g
When comparing the PMF profiles of the TM9/TM9 interface between hDAT and hDAT-palm (Fig. 3a,
second panel), it is observed that the profiles are very similar, emphasizing that the sampling of the dimer con-
formations along the reaction coordinate is sufficient. This similarly demonstrates that Cys581 S-palmitoylation
of TM12 has no global effect on dimer binding. In contrast, the PMF profiles of the interfaces involving TM12
change with hDAT palmitoylation. The TM9,TM12/TM9,TM12 interface is relatively stabilized upon TM12
S-palmitoylation, whereas the TM12/TM12 interface is relatively destabilized. Furthermore, the global minimum
for the hDAT-palm TM12/TM12 interface is solely found in the second long distance minimum observed for
the hDAT TM12/TM12 interface (Fig. 3a, third panel).f g
p
To further understand the mechanism by which S-palmitoylation affects the energetics of the TM12/TM12
and TM9,TM12/TM9,TM12 interfaces, we determined the average number of (residue-based) contacts formed
in the US-REMD simulations for both the hDAT and hDAT-palm systems (Fig. 3b). We assessed palm’s involve-
ment at the two interfaces by both including and excluding the group from the contact analysis. As expected,
the number of contacts increases as the distance between the proteins decreases, and palm adds a significant
contribution to the total number of contacts formed at both interfaces (Fig. 3b). It is observed at the free energy
minimum of the two interfaces (d = 0 nm) that palm increases the number of non-palm interprotein contacts
for the TM9,TM12/TM9,TM12 interface, but decreases the number of non-palm interprotein contacts for the https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 www.nature.com/scientificreports/ Figure 3. Potential of mean force energy profiles and contact analysis of hDAT and hDAT-palm interfaces. Illustrated in the first panel in (a) are all the PMFs of the different hDAT interfaces: TM9/TM9 (brown), Figure 3. Potential of mean force energy profiles and contact analysis of hDAT and hDAT-palm interfaces. Illustrated in the first panel in (a) are all the PMFs of the different hDAT interfaces: TM9/TM9 (brown),
TM9,TM12/TM9,TM12 (light green), TM12,TM12 (pink), and TM4, TM9/TM11 (light brown). Shown in the
subsequent plots are PMF comparisons of the interfaces TM9/TM9, TM9,TM12/TM9,TM12, and TM12/TM12
between hDAT and hDAT-palm (green). www.nature.com/scientificreports/ Shown in (b) are the average number of residue contacts calculated
within 1 Å distance bins along hDAT dimer separation relative to the distance at energy minimum as detected
from the PMF energy profiles. The number of residue contacts for the two systems, hDAT and hDAT-palm, at
the two interfaces, TM12/TM12 and TM9, TM12/TM9, TM12, were monitored. For both interfaces the Cys581
residue where palm is attached is either included (incl.) or excluded (excl.) from the contacts analysis. A contact
was counted when the distance between two residue pairs was below 7 Å. Each contact was unbiased. Error
bars have been included by performing bootstrapping on the data using 10 iterations, although they are barely
visible. Shown in (c) are representative dimer conformations of hDAT-palm at different separation distances
for the TM9, TM12/TM9, TM12 interface (top) and the TM12/TM12 interface (bottom). Highlighted are
helices TM3, TM9, TM11 and TM12. The green mesh corresponds to the palm occupancies calculated using
the Volmap plugin in VMD for the dimer conformations found around the given separation distance. For the
Volmap calculations the size of the system beads were set to 2.6 Å and the “occupancy” setting was selected. The
arrows indicate the movement of the proteins with respect to each other. Figure 3. Potential of mean force energy profiles and contact analysis of hDAT and hDAT-palm interfaces. Illustrated in the first panel in (a) are all the PMFs of the different hDAT interfaces: TM9/TM9 (brown),
TM9,TM12/TM9,TM12 (light green), TM12,TM12 (pink), and TM4, TM9/TM11 (light brown). Shown in the
subsequent plots are PMF comparisons of the interfaces TM9/TM9, TM9,TM12/TM9,TM12, and TM12/TM12
between hDAT and hDAT-palm (green). Shown in (b) are the average number of residue contacts calculated
within 1 Å distance bins along hDAT dimer separation relative to the distance at energy minimum as detected
from the PMF energy profiles. The number of residue contacts for the two systems, hDAT and hDAT-palm, at
the two interfaces, TM12/TM12 and TM9, TM12/TM9, TM12, were monitored. For both interfaces the Cys581
residue where palm is attached is either included (incl.) or excluded (excl.) from the contacts analysis. A contact
was counted when the distance between two residue pairs was below 7 Å. Each contact was unbiased. Error
bars have been included by performing bootstrapping on the data using 10 iterations, although they are barely
visible. www.nature.com/scientificreports/ Thus, the strength of the TM12/TM12 and the TM9,TM12/TM9,TM12 interfaces
is correlated with the total number of non-palm protein–protein contacts.h The number of non-palm interprotein contacts in the hDAT-palm TM9,TM12/TM9,TM12 interface is higher
than for the corresponding hDAT interface, indicating that the two proteins can form additional contacts when
palm is present, in contrast to what one might expect (Fig. 3b, top panel). We computed the average ϕ1 and ϕ3
angle values for all dimers found in the TM9,TM12/TM9,TM12 clusters (cluster 1 vs. cluster 10, see Table 1) for
the hDAT and hDAT-palm systems, respectively, and detected a 5.4° average difference. This slight difference
in relative orientation between the hDAT and the hDAT-palm TM9,TM12/TM9,TM12 dimer interface could
explain why additional contacts can form in the hDAT-palm system. In fact, when visualizing the palm 3D occu-
pancy maps around the energy minimum, we observe that palm is located in the nook formed between TM9 and
TM12 (Fig. 3c, top panel). Therefore, palm does not seem to disrupt the formation of non-palm inter-protein con-
tacts, rather it could result in better protein–protein packing possibly due to this slight dimer reorientation. For
both the hDAT and the hDAT-palm systems a small shoulder is observed in the contact analysis at d = ~ 0.6 nm
(Fig. 3a, fourth panel), which corresponds to a reorientation of the TM9,TM12/TM9,TM12 interface resulting
in loss of contacts to a single TM9/TM12 pair (Fig. 3c, top panel). g
p
g
p p
Looking instead at the contact analysis for the hDAT-palm TM12/TM12 interface, a decrease in non-palm
interprotein contacts at the free energy minimum (d = 0 nm) relative to the hDAT system is observed (Fig. 3b,
bottom panel). This decrease may easily be rationalized by palm getting squeezed between the TM12 helix of one
protomer and the protein of the other, thereby preventing certain non-palm interprotein contacts from forming
(Fig. 3c, bottom panel). At the distance of the second free energy minimum of the hDAT TM12/TM12 interface
(d = ~ 0.4 nm), the number of protein contacts in the hDAT and hDAT-palm (excluding palm) system are similar. However, when palm is included in the analysis, a higher number of contacts is detected for the hDAT-palm sys-
tem (Fig. 3b). www.nature.com/scientificreports/ PALM interface
Cluster number
Cluster
size/%
Φ1, Φ3/°
ΔLASA/nm2
−
+
−
+
−
+
−
+
TM9,TM12/TM9,TM12
1, 2
10
13.4
3.0
− 31, − 24
− 34, − 28
16.0
17.6
TM4,TM9/TM11
3, 4
2, 3
10.0
13.0
30, − 127
26, − 123
15.5
16.3
TM9/TM9
6
1
4.3
11.1
3, 0
0, 0
20.8
20.7
TM12/TM12
9
53
3.0
0.1
− 46, − 47
− 41, − 48
12.5
15.2
TM4,TM9/TM2
7, 8
8, 9
7.0
6.1
177, 15
165, 4
18.7
18.7
TM4/TM12
10, 11
11, 12
5.5
4.5
34, − 47
18, − 29
12.5
12.0 PALM interface
Cluster number
Cluster
size/%
Φ1, Φ3/°
ΔLASA/nm2
−
+
−
+
−
+
−
+
TM9,TM12/TM9,TM12
1, 2
10
13.4
3.0
− 31, − 24
− 34, − 28
16.0
17.6
TM4,TM9/TM11
3, 4
2, 3
10.0
13.0
30, − 127
26, − 123
15.5
16.3
TM9/TM9
6
1
4.3
11.1
3, 0
0, 0
20.8
20.7
TM12/TM12
9
53
3.0
0.1
− 46, − 47
− 41, − 48
12.5
15.2
TM4,TM9/TM2
7, 8
8, 9
7.0
6.1
177, 15
165, 4
18.7
18.7
TM4/TM12
10, 11
11, 12
5.5
4.5
34, − 47
18, − 29
12.5
12.0 PALM interface
Cluster number
Cluster
size/%
Φ1, Φ3/°
ΔLASA/nm2
−
+
−
+
−
+
−
+
TM9,TM12/TM9,TM12
1, 2
10
13.4
3.0
− 31, − 24
− 34, − 28
16.0
17.6
TM4,TM9/TM11
3, 4
2, 3
10.0
13.0
30, − 127
26, − 123
15.5
16.3
TM9/TM9
6
1
4.3
11.1
3, 0
0, 0
20.8
20.7
TM12/TM12
9
53
3.0
0.1
− 46, − 47
− 41, − 48
12.5
15.2
TM4,TM9/TM2
7, 8
8, 9
7.0
6.1
177, 15
165, 4
18.7
18.7
TM4/TM12
10, 11
11, 12
5.5
4.5
34, − 47
18, − 29
12.5
12.0 Table 1. hDAT and hDAT-palm cluster properties. The cluster number and cluster size for the different
dimers is given for the system with ( +) and the system without (−) palm. The dimers relative orientation is
given by the ϕ1 and ϕ3 angle (see Fig. 2a) and the average ΔLASA for the different dimer interfaces is listed. Notice that some clusters come in pairs e.g. cluster 7,8 due to symmetry considerations, which is elaborated
upon in the Methods section under clustering. TM12/TM12 interface (Fig. 3b). www.nature.com/scientificreports/ At the d = ~ 0.4 nm distance, palm seems to stabilize the TM12/TM12 interface and it is observed
that palm-palm contacts can form, by bridging across the TM12/TM12 interface (Fig. 3c, bottom panel). y
g
g
g
To summarize, the PMFs show that palm changes the energetics of the interfaces involving TM12 and the
contact analysis along the interface separation distance shows that this is caused by palm being present at the
interface and thereby i) providing, in all cases, additional contacts to form ii) increasing existing protein–protein
contacts (in the TM9,TM12/TM9,TM12 interface) or iii) preventing protein–protein contacts to form (in the
TM12/TM12 interface). The dynamics of the TM9/TM12 interface and the effect of palmitoylation. Visual inspection
of the last frames of the 10 different hDAT and hDAT-palm repeat simulations shows that both single TM9/
TM12 interfaces and double TM9,TM12/TM9,TM12 interfaces are formed, as highlighted in Fig. 4a by pink and
purple boxes, respectively (see Supplementary Fig. S1 for a full overview). To evaluate the dynamics of the TM9/
TM12 interfaces we monitored the formation of single TM9/TM12 dimer interfaces as a function of simulation
time for all TM9/TM12 pairs that were in contact continuously for more than 500 ns during the course of the
simulation. Figure 4b illustrates that TM9/TM12 dimers are formed 33 times in the hDAT system and 35 times
in hDAT-palm system. If Cys581, on which palm is bound is excluded from the contact analysis (Fig. 4b, middle
column) then the number of dimers forming a TM9/TM12 contact in the hDAT-palm system decrease from 35
to 23. In the hDAT system, this change is less prominent, 33 vs. 30, respectively. Collectively, the total number
of frames forming TM9/TM12 contacts drops by 0.1% in the hDAT system and 47% in the hDAT-palm system
when Cys581 is excluded from the contact analysis. These results indicate that the flexible palm group helps
TM9/TM12 contacts to form at longer distances, but that palm at these longer distances is the primary group
forming interprotein contacts.i g
p
While monitoring the formation of the TM9,TM12/TM9,TM12 interface over time, it was revealed that first
single TM9/TM12 contacts form followed by the remaining TM9/TM12 helices coming together (Fig. 4b,c). This dynamical behavior is similar to that observed in the US-REMD simulations (Fig. 3c, top panel). www.nature.com/scientificreports/ Shown in (c) are representative dimer conformations of hDAT-palm at different separation distances
for the TM9, TM12/TM9, TM12 interface (top) and the TM12/TM12 interface (bottom). Highlighted are
helices TM3, TM9, TM11 and TM12. The green mesh corresponds to the palm occupancies calculated using
the Volmap plugin in VMD for the dimer conformations found around the given separation distance. For the
Volmap calculations the size of the system beads were set to 2.6 Å and the “occupancy” setting was selected. The
arrows indicate the movement of the proteins with respect to each other. https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ Table 1. hDAT and hDAT-palm cluster properties. The cluster number and cluster size for the different
dimers is given for the system with ( +) and the system without (−) palm. The dimers relative orientation is
given by the ϕ1 and ϕ3 angle (see Fig. 2a) and the average ΔLASA for the different dimer interfaces is listed. Notice that some clusters come in pairs e.g. cluster 7,8 due to symmetry considerations, which is elaborated
upon in the Methods section under clustering. www.nature.com/scientificreports/ In both
systems the formation of the double TM9,TM12/TM9,TM12 interface takes multiple µs to form after the single https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ Figure 4. Dynamics of the TM9/TM12 interface. In (a) the last frame of a single representative repeat
simulation is shown for hDAT (MD1, top) and hDAT-palm (MD4, bottom). The last frames for all repeat
simulations are supplied in Supplementary Fig S1. Within the pink boxes are TM9/TM12 interfaces and
illustrated in the purple box is a symmetrical interface involving both TM9 and TM12 helices, dubbed TM9,
TM12/TM9, TM12. Highlighted in the black dotted box is a symmetrical tetramer. The green dots correspond
to the GL1 bead in POPC and are depicted for the nearest periodic images to the system. For the single
simulation box the dots have been omitted. The helices TM9, TM11, and TM12 are highlighted in blue, purple,
and mauve, respectively. Illustrated in (b) is the formation of single TM9/TM12 contacts both considering and
not considering Cys581 (wo. Cys581) on which palm is attached in the analysis (the first and second column,
respectively). Double TM9,TM12/TM9,TM12 contacts are also monitored for the same representative repeat
simulations as depicted in (a) for hDAT (top) and hDAT-palm (bottom)(third column). Each color in the same
plot represents a different dimer pair and the total number of dimers across all repeat simulations that form
the given interface are noted in the plots. The TM9, TM12/TM9, TM12 interface is further subdivided into two
similar color shades representing the minimum distance for each TM9/TM12 helix pair located in the same
dimer. The contact plots for the different interfaces across all repeat simulations are supplied in Supplementary
Fig. S10-14. In (c) the formation of the symmetrical TM9, TM12/TM9, TM12 interface is shown. It is observed
that POPC lipids are associated with the interface. Illustrated in (d) is a close-up of the symmetrical tetramer. Figure 4. Dynamics of the TM9/TM12 interface. In (a) the last frame of a single representative repeat
simulation is shown for hDAT (MD1, top) and hDAT-palm (MD4, bottom). The last frames for all repeat
simulations are supplied in Supplementary Fig S1. Within the pink boxes are TM9/TM12 interfaces and
illustrated in the purple box is a symmetrical interface involving both TM9 and TM12 helices, dubbed TM9,
TM12/TM9, TM12. Highlighted in the black dotted box is a symmetrical tetramer. www.nature.com/scientificreports/ The green dots correspond
to the GL1 bead in POPC and are depicted for the nearest periodic images to the system. For the single
simulation box the dots have been omitted. The helices TM9, TM11, and TM12 are highlighted in blue, purple,
and mauve, respectively. Illustrated in (b) is the formation of single TM9/TM12 contacts both considering and
not considering Cys581 (wo. Cys581) on which palm is attached in the analysis (the first and second column,
respectively). Double TM9,TM12/TM9,TM12 contacts are also monitored for the same representative repeat
simulations as depicted in (a) for hDAT (top) and hDAT-palm (bottom)(third column). Each color in the same
plot represents a different dimer pair and the total number of dimers across all repeat simulations that form
the given interface are noted in the plots. The TM9, TM12/TM9, TM12 interface is further subdivided into two
similar color shades representing the minimum distance for each TM9/TM12 helix pair located in the same
dimer. The contact plots for the different interfaces across all repeat simulations are supplied in Supplementary
Fig. S10-14. In (c) the formation of the symmetrical TM9, TM12/TM9, TM12 interface is shown. It is observed
that POPC lipids are associated with the interface. Illustrated in (d) is a close-up of the symmetrical tetramer. Figure 4. Dynamics of the TM9/TM12 interface. In (a) the last frame of a single representative repeat
simulation is shown for hDAT (MD1, top) and hDAT-palm (MD4, bottom). The last frames for all repeat
simulations are supplied in Supplementary Fig S1. Within the pink boxes are TM9/TM12 interfaces and
illustrated in the purple box is a symmetrical interface involving both TM9 and TM12 helices, dubbed TM9,
TM12/TM9, TM12. Highlighted in the black dotted box is a symmetrical tetramer. The green dots correspond
to the GL1 bead in POPC and are depicted for the nearest periodic images to the system. For the single
simulation box the dots have been omitted. The helices TM9, TM11, and TM12 are highlighted in blue, purple,
and mauve, respectively. Illustrated in (b) is the formation of single TM9/TM12 contacts both considering and
not considering Cys581 (wo. Cys581) on which palm is attached in the analysis (the first and second column,
respectively). Double TM9,TM12/TM9,TM12 contacts are also monitored for the same representative repeat
simulations as depicted in (a) for hDAT (top) and hDAT-palm (bottom)(third column). www.nature.com/scientificreports/ p
In the self-assembly simulations a total of eight dimers form double TM9,TM12/TM9,TM12 interfaces in
the hDAT system and two in the hDAT-palm system (Fig. 4b, see Supplementary Fig. S10 and Fig S12 for a full
overview). Excluding Cys581 from the contact calculations in Fig. 4b does not change the number of dimers
forming symmetrical TM9,TM12/TM9,TM12 dimers in the two systems. Moreover, according to US-REMD
simulations, once non-palm interprotein contacts form, the interface becomes more stable in the presence of
palm (Fig. 3b). Nonetheless, the TM9,TM12/TM9,TM12 interface is formed more rarely in the hDAT-palm self-
assembly simulations, possibly due to the exclusion of POPC molecules from the interface space having a higher
energy barrier in the presence of palm and/or palm’s increased flexibility requires a higher amount of sampling
to thoroughly probe the TM9,TM12/TM9,TM12 interface. g
y p
Of potential interest, is the observation that a symmetrical tetramer in two hDAT repeat simulations forms
consisting of TM9/TM12 contacts in addition to TM4/TM12 helix contacts (Fig. 4d). A similar dimer of dimers
construction has previously been suggested for hDAT20. Dynamics of the TM12/TM12 interface and the effect of palm. The TM12/TM12 interface was
previously found to be the most energetically favorable for hSERT and was also the most frequently formed in
hSERT self-assembly simulations (18%)14 and has been partly observed in X-ray structures5. The TM12/TM12
interface is similarly formed in the hDAT and hDAT-palm self-assembly simulations according to the per helix
contact analysis, cluster analysis and visually based on the last frame of the different repeat simulations (Fig. 1b,
Fig. 2b and Fig. 5a, respectively). It is, however, not as populated as other interfaces (3.0% and 0.1% for hDAT
and hDAT-palm, respectively), nor markedly more stable based on the PMF energy profiles (Fig. 3a). The num-
ber of TM12/TM12 pairs that form contacts lasting more than 500 ns in the hDAT simulations both considering
and not considering a contact formed by Cys581 is unchanged (11 in Fig. 5b). In the hDAT-palm simulations,
a higher number of TM12/TM12 dimers form compared to in the hDAT simulations, 34 vs. 11, respectively. However, these dimers are mostly engaged by palm itself and are therefore short-lived and unstable. Only 11 out
of 34 dimers remain if palm (Cys581) is excluded from the contact analysis. Corresponding to a 74% drop in the
relative number of frames forming TM12/TM12 contacts (Fig. 5b). www.nature.com/scientificreports/ In (b) the formation of single TM12/TM12 contacts both
considering and not considering Cys581 (wo. Cys581) on which palm is attached in the analysis. The TM12/
TM12 pairs that were monitored were evaluated as being in contact continuously for 500 ns during the course of
the simulation. A contact was defined when the minimum distance between TM12 helices was below 7 Å. The
contact plots for the different interfaces across all repeat simulations are supplied in Supplementary Fig. S15-17. Shown in (c) is the palm 3D occupancy computed using the Volmap plugin in VMD for all dimers captured in
the TM12/TM12 hDAT-palm cluster (cluster 53 in Table 1). Figure 5. Dynamics of the TM12/TM12 interface. In (a) the last frame of a single representative repeat
simulation is shown for hDAT (MD2, top) and hDAT-palm (MD3, bottom). The last frames for all repeat
simulations are supplied in Supplementary Fig. S1. Similar interfaces are observed across the two systems
involving TM12 on which palm (orange) is attached. Within the blue box a TM12/TM12 interface is observed. The green dots correspond to the GL1 bead in POPC and are shown for the nearest periodic images to the
system. For the single simulation box the dots have been omitted. The helices TM9, TM11, and TM12 are
highlighted in blue, purple and mauve, respectively. In (b) the formation of single TM12/TM12 contacts both
considering and not considering Cys581 (wo. Cys581) on which palm is attached in the analysis. The TM12/
TM12 pairs that were monitored were evaluated as being in contact continuously for 500 ns during the course of
the simulation. A contact was defined when the minimum distance between TM12 helices was below 7 Å. The
contact plots for the different interfaces across all repeat simulations are supplied in Supplementary Fig. S15-17. Shown in (c) is the palm 3D occupancy computed using the Volmap plugin in VMD for all dimers captured in
the TM12/TM12 hDAT-palm cluster (cluster 53 in Table 1). to enter the otherwise enclosed space. Once again illustrating that the TM9,TM12/TM9,TM12 interface is very
dynamic. On the other hand, it could also be indicative of the interface not being stable and thus not necessarily
biologically relevant. However, more likely, the dimer rearrangement is caused by the difficulty in equilibrating
the interfacial lipids. www.nature.com/scientificreports/ Each color in the same
plot represents a different dimer pair and the total number of dimers across all repeat simulations that form
the given interface are noted in the plots. The TM9, TM12/TM9, TM12 interface is further subdivided into two
similar color shades representing the minimum distance for each TM9/TM12 helix pair located in the same
dimer. The contact plots for the different interfaces across all repeat simulations are supplied in Supplementary
Fig. S10-14. In (c) the formation of the symmetrical TM9, TM12/TM9, TM12 interface is shown. It is observed
that POPC lipids are associated with the interface. Illustrated in (d) is a close-up of the symmetrical tetramer. TM9/TM12 helices have come together, and the closure of the interface results in the encapsulation of POPC
lipids in the space between the proteins (highlighted by a black ellipsoid in Fig. 4c). The number of POPC lipids
that get encapsulated by the TM9,TM12/TM9,TM12 dimer are approximately five and eight for the hDAT-palm
and hDAT system, respectively.h TM9/TM12 helices have come together, and the closure of the interface results in the encapsulation of POPC
lipids in the space between the proteins (highlighted by a black ellipsoid in Fig. 4c). The number of POPC lipids
that get encapsulated by the TM9,TM12/TM9,TM12 dimer are approximately five and eight for the hDAT-palm
and hDAT system, respectively.h y
p
y
The TM9,TM12/TM9,TM12 hDAT dimer was converted to atomistic resolution and simulated for 530 ns. During the simulation we observe a slight reorientation of the proteins in which contacts to a single TM9/TM12
pair is lost (see Supplementary Fig. S9). This loss in contacts allows for POPC lipids from the surrounding milieu https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Dynamics of the TM12/TM12 interface. In (a) the last frame of a single representative repeat
simulation is shown for hDAT (MD2, top) and hDAT-palm (MD3, bottom). The last frames for all repeat
simulations are supplied in Supplementary Fig. S1. Similar interfaces are observed across the two systems
involving TM12 on which palm (orange) is attached. Within the blue box a TM12/TM12 interface is observed. The green dots correspond to the GL1 bead in POPC and are shown for the nearest periodic images to the
system. For the single simulation box the dots have been omitted. The helices TM9, TM11, and TM12 are
highlighted in blue, purple and mauve, respectively. www.nature.com/scientificreports/ Each system contains 16 LeuT (PDB ID: 2A65) proteins
equally spaced but randomly orientated in a pure POPE membrane and has been simulated for 30 µs. The
helices TM9, TM11, and TM12 are highlighted in blue, purple, and mauve, respectively. Emphasized by pink
boxes are dimers containing TM12/TM12 and to some extent TM9 contacts. The green dots correspond to the
GL1 bead in POPE and are shown for the nearest periodic image to the system. For the central simulation box
the dots have been omitted. To the right in (c) is a top view with respect to the membrane normal of the central
dimer conformation observed in cluster 1. Notice that the dimer achieved from our simulations has an interface
consisting of TM12/TM12, TM9 contacts, due to a slight change in the two proteins relative orientation. This
change in orientation relative to the LeuT crystal structure dimer results in a loss of contacts to one of the TM9
helices. Represented in (d) is the stable hSERT TM12/TM12 dimer previously detected14. Finally, in (e) is the
PMF of the hSERT TM9,TM12/TM9,TM12 interface. tacts at longer distances which are not stabilized by protein–protein contacts and that in the presence of palm,
the TM12/TM12 interface becomes less stable. From the cluster analysis only 0.1% TM12/TM12 hDAT-palm
dimers are observed, which further illustrates that the TM12/TM12 interface is difficult to capture using the
clustering method, due to palm increasing the interprotein distance and the relative protein orientations that
lead to TM12/TM12 contacts. Comparison of the LeuT interface to the hDAT TM9,TM12/TM9,TM12 and hSERT TM12/TM12
interface. The TM9,TM12/TM9,TM12 dimer interface observed to be among the top clusters for both
hDAT and hDAT-palm (Fig. 2b) is similar to that present in crystal structures of a MAT bacterial homologue,
LeuT (Fig. 6a). However, the hDAT TM9,TM12/TM9,TM12 dimer differs in the relative orientation of the TM12
and TM9 helices in addition to TM12 being kinked in hDAT and being straight in LeuT (Fig. 6b). When align-
ing two hDAT monomers on the LeuT crystal structure (PDB ID: 2A65)25 dimer using Pymol’s align command
the symmetrical dimer achieved (hDAT-on-LeuT) is surprisingly similar to the central dimer conformation of
cluster 1 from the hDAT self-assembly simulations (Fig. 2b). The RMSD between the two dimers Cα atoms is
only 3 Å. www.nature.com/scientificreports/ By computing the 3D occupancy of palm
using the TM12/TM12 dimer conformations captured in the cluster analysis, it is clearly observed that palm is
involved at the interface (Fig. 5c). Taken together, this data illustrates that the palm group promotes dimer con- https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 6. LeuT and MATs have a similar TM12/TM12 and TM12,TM9/TM12,TM9 interface. Shown in (a) is
a top view of the LeuT crystal structure dimer (PDB ID: 2A65) with helices TM9 and TM12 highlighted. In (b)
the central hDAT dimer structure from cluster 1 (see Fig. 2b) is superimposed on hDAT monomers aligned to
the LeuT dimer. Depicted in (c) is a representative last frame of the 10 LeuT self-assembly repeat simulations
(MD1). See Supplementary Fig. S18 for a full overview. Each system contains 16 LeuT (PDB ID: 2A65) proteins
equally spaced but randomly orientated in a pure POPE membrane and has been simulated for 30 µs. The
helices TM9, TM11, and TM12 are highlighted in blue, purple, and mauve, respectively. Emphasized by pink
boxes are dimers containing TM12/TM12 and to some extent TM9 contacts. The green dots correspond to the
GL1 bead in POPE and are shown for the nearest periodic image to the system. For the central simulation box
the dots have been omitted. To the right in (c) is a top view with respect to the membrane normal of the central
dimer conformation observed in cluster 1. Notice that the dimer achieved from our simulations has an interface
consisting of TM12/TM12, TM9 contacts, due to a slight change in the two proteins relative orientation. This
change in orientation relative to the LeuT crystal structure dimer results in a loss of contacts to one of the TM9
helices. Represented in (d) is the stable hSERT TM12/TM12 dimer previously detected14. Finally, in (e) is the
PMF of the hSERT TM9,TM12/TM9,TM12 interface. Figure 6. LeuT and MATs have a similar TM12/TM12 and TM12,TM9/TM12,TM9 interface. Shown in (a) is
a top view of the LeuT crystal structure dimer (PDB ID: 2A65) with helices TM9 and TM12 highlighted. In (b)
the central hDAT dimer structure from cluster 1 (see Fig. 2b) is superimposed on hDAT monomers aligned to
the LeuT dimer. Depicted in (c) is a representative last frame of the 10 LeuT self-assembly repeat simulations
(MD1). See Supplementary Fig. S18 for a full overview. Discussion As with all scientific studies, this one is also not without its limitations. The main concerns are linked to the
reliance on homology modelling as well as the inherent limitations of computational techniques such as CG
MD and free energy calculations40. The protein model used in this study has been thoroughly validated in a
previous publication and whilst the stickiness of the MARTINI model could cause artifacts40, we fortunately see
both binding and unbinding events, increasing the reliability of our results. Free energy calculations using the
MARTINI model should be guiding, providing trends across systems to emphasize that the values represented
here are not absolute but relative energies of dimer dissociation. Sampling issues may arise in all MD studies and
to avoid this we made use of both the MARTINI and atomistic models. Fully equilibrating complex systems with
membranes can be challenging due to the slow lipid dynamics under our time scale and membrane heterogeneity. In this work we do not see fully equilibrated protein–protein interfaces possibly due to the presence of lipids, and
thus it is possible that we miss relevant interfaces as observed for the hSERT self-assembly simulations (Fig. 6e). Nonetheless, it is beyond current computing capabilities to achieve extreme convergence within such complex
systems and we therefore rely on this method of ranking interfaces based on the available timescales, which has
been shown to reproduce experimentally relevant interfaces5. One can also notice the obvious lack of asymptote
at long distances in the binding free energies. It has been taken into account but is not as a major limitation. In summary, we have presented several probable hDAT and hDAT-palm interfaces primarily involving TM9
and/or TM11, and/or TM12, in good agreement with the LeuT crystal structure dimer and FRET data24,25. Spe-
cifically, the interfaces conserved among the two systems are TM9/TM9, TM4,TM9/TM11, TM4,TM9/TM2,
TM9,TM12/TM9,TM12, TM12/TM4, and to a lesser extent TM12/TM12. The hDAT dimers with the interfaces
TM12/TM12, TM9/TM9, TM4,TM9/TM11 and TM9,TM12/TM9,TM12, were further evaluated using US-
REMD for separating the two protomers and generating PMF free energy profiles14. The four interfaces separation
energy resulted in similar PMF values (Fig. 3a) to what has previously been published for other dimer proteins,
indicating that these interfaces could potentially be biologically relevant14,36. Discussion Furthermore, we have shown that
the stability of the interfaces involving TM12, TM12/TM12 and TM9,TM12/TM9,TM12, change when hDAT
is palmitoylated, suggesting that palm may change the relative abundance of dimers involving TM12 in a bio-
logical setup. We anticipate that changes of the membrane composition, e.g. inserting cholesterol, might have
similar effects on protein association. The particularity of lipids properties, especially cholesterol, will directly
or indirectly affect protein–protein interactions by altering membrane mechanical properties. f
According to cysteine cross-linking experiments, TM6/TM6 and TM4/TM4 supposedly form a dimer inter-
face in DAT20,22. The authors show by reintroducing the residues Cys243 (in TM4) or Cys306 (in TM6) in cysteine
depleted DAT proteins that cross-links are formed across DAT molecules, suggesting that these endogenous
cysteine residues located in TM4 and TM6, respectively, are situated at a symmetrical dimer interface22. However,
in our simulations TM4/TM4 and TM6/TM6 interhelical contacts only rarely form (Fig. 1c). Specifically, the
cross-linking residues Cys243 and Cys306 are only found to be symmetrically in contact with the same cysteine
in another protomer 0.0% and 0.4% of the time in the hDAT simulations, respectively, and 0.0% and 1.5% in
the hDAT-palm simulations, respectively. Nonetheless, this does not necessarily disregard the relevance of our
findings. Like all methods, cysteine cross-linking is not without its caveats. A cross-link between proteins will
form irrespective of whether the dimer interface is strong or not, as long as the reactive cysteine residues come
in to contact with each other during random diffusion, the covalent bond will form and keep the two monomers
together. g
Similarly, a hDAT dimer was recently constructed using the ClusPro protein–protein docking webserver
that contained a C2 symmetrical TM2,TM6,TM11/TM2,TM6,TM11 interface where the two Cys306 residues
are in close contact with each other23. However, it should be noted that the ClusPro program has primarily been
optimized for proteins in solution and the authors state that when docking homology models even moderate
errors in key residue side chains or loops may substantially reduce the accuracy of the docking results41. It is
therefore important to thoroughly review the results from several studies when determining biologically relevant
protein dimer interfaces. p
Based on our results we propose that a hDAT dimer consisting of TM9/TM12 contacts could be biologically
relevant. From both self-assembly simulations and subsequent PMF calculations it is observed that the sym-
metrical TM9,TM12/TM9,TM12 dimer is energetically favorable. www.nature.com/scientificreports/ and − 12.2° for the central dimer conformation of the most populated cluster and − 7.3° and − 7.3° for the LeuT
crystal structure dimer (PDB ID: 2A65), respectively (see Supplementary Fig. S21). From visual inspection of
the central LeuT self-assembly dimer of cluster 1 aligned to the Cα atoms of the crystal structure of LeuT (RMSD
value of 4.4 Å), the slight change in the two dihedral angles reflects the LeuT dimer having a TM9,TM12/TM12
interface rather than a symmetrical TM9,TM12/TM9,TM12 interface in self-assembly simulations. Interestingly,
this denotes to a mix of the hDAT TM9,TM12/TM9,TM12 interface and the hDAT and hSERT TM12/TM12
interface (Fig. 6d). In fact, the MAT TM12/TM12 and TM9,TM12/TM9,TM12 interfaces are closely related when
comparing their ϕ1 and ϕ3 angle values, which do not differ more than ~ 10° (Table 1). p
g
ϕ
ϕ
gf
Due to the strength and prevalence of the TM9,TM12/TM9,TM12 interface we revisited our hSERT self-
assembly data and did in fact observe a single TM9,TM12/TM9,TM12 interface being formed. Using this dimer,
we calculated its PMF and found that the TM9,TM12/TM9,TM12 interface is in fact also stable for hSERT and
even stronger than the TM12/TM12 interface, ~ 75 kJ/mol vs. ~ 55 kJ/mol, respectively14 (Fig. 6e). Collectively,
the high similarity between the populated LeuT self-assembly dimer and the crystal structure dimer further
emphasizes the importance of both the TM12/TM12 interface and the TM9,TM12/TM9,TM12 interface in a
biological context for LeuT itself, but also for hDAT and hSERT and potentially also for hNET. www.nature.com/scientificreports/ The main difference is that TM12 is shifted slightly closer to TM9 in the dimer conformation observed
in our simulations as compared to the hDAT-on-LeuT dimer (Fig. 6b). p
( g
)
LeuT has repeatedly been observed to crystallize as a TM9,TM12/TM9,TM12 dimer25. However, this does not
necessarily make it biologically relevant, and the dimer may simply be influenced by the crystallization environ-
ment. Using MS/MS techniques it has been confirmed that LeuT forms a dimer in a lipid environment26, but it is
yet unclear whether this dimer consists of the TM9,TM12/TM9,TM12 interface observed in crystal structures. To
evaluate whether LeuT forms a similar interface during self-assembly simulations, we ran 10 repeat simulations
of LeuT (PDB ID: 2A65) randomly orientated in a POPE membrane and subsequently clustered the generated
dimers. Strikingly, the dimer conformation observed in the top cluster consists of TM12/TM12 contacts with
some involvement of TM9 and occurs 12% of the time (see Fig. 6c and Supplementary Fig. S18-20 for additional
information on the clustering of the LeuT dimers). Using the ϕ1 and ϕ3 dihedral angles for describing the rela-
tive orientation of the LeuT protomers in the different dimers observed, we found that these angles were − 13.2° https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 www.nature.com/scientificreports/ www.nature.com/scientificreports/ In
addition, they observe interfaces involving EL2 loop contacts in three out of eight cases. These interfaces could
potentially be biologically relevant, although when using the MARTINI model an elastic network is usually
applied, which constraints natural loop flexibility. Thus, different dimer conformations involving loop contacts
will inevitably arise depending on the conformation in which the loop has been constrained in. We therefore
propose that such contacts should be regarded with care, especially as hDAT presented here and in Jayaramen’s
study are homology models. The overall conclusion drawn by the authors was that dimer contacts primarily
involve the scaffold region of hDAT (TM 3-5, 8-12) rather than the bundle region (TM1-2,6-7). Although we see
different dimer orientations in our simulations, the overall conclusion drawn by Jayaramen et al. also agrees with
our results. Finally, we typically first detected full closure of the TM9, TM12/TM9, TM12 interface after ~ 10 µs. Therefore, the same interface cannot be expected to be observed by Jayaramen et al., who only simulated for 2 µs. The different dimers of high population identified for hDAT and hDAT-palm are similar to those observed for
hSERT by Periole et al.14. From hSERT self-assembly simulations the top clusters consisted of dimer conforma-
tions with TM12/TM12, TM7/TM12, TM4,TM9/TM11,TM2, and finally EL2/EL2 interfaces. Similar interfaces to
these have also been observed among the conformations in the top clusters for either hDAT (see Supplementary
Fig. S5) or hDAT-palm (see Supplementary Fig. S6) dimers. Interestingly, the separation energy for the hSERT
TM4,TM9/TM11,TM2 interface resulted in ~ 20 kJ/mol, whereas the similar hDAT TM4,TM9/TM11 interface
resulted in a much higher value of ~ 60 kJ/mol. The TM12/TM12 separation energy for hDAT and hSERT are
approximately the same ~ 60 kJ/mol. The TM9/TM9 interface observed frequently in both hDAT and hDAT-
palm self-assembly simulations was not among the top dimer clusters of the hSERT self-assembly simulations. A
possible explanation for the TM9/TM9 interface being frequently observed for hDAT, but not for hSERT, could
be that TM9 in hDAT contains a leucine zipper motif, which is not present in hSERT. Jayaramen et al. have published results on hDAT oligomerization using the MARTINI 2.2 FF, but with a dif-
ferent approach to ours, coined the DAFT protocol15. www.nature.com/scientificreports/ Rather than studying systems containing multiple proteins
as presented here, they used the DAFT protocol in which multiple repeat simulations are performed of two
hDAT proteins randomly orientated in a POPC membrane15. We ran 10 repeats of the hDAT and hDAT-palm
system each lasting 30 µs, whereas Jayaramen et al. simulated 512 repeats each lasting 2 µs. The DAFT and 16
proteins in a membrane approach have previously converged on similar results for rhodopsin when studying
dimers of high occurrence42. For hDAT, there are some similarities in the top dimer candidates predicted using
the two methods, but overall they differ. The conserved interfaces are TM9/TM9 and EL2/EL2 (detected in our
self-assembly, but only presented in Supplementary Fig. S5-6). Jayaramen et al. frequently observe symmetrical
dimers containing C-terminal helix contacts, which is somewhat similar to our TM12/TM12 interface with the
exception of the two protomers being further separated and thereby not exhibiting direct TM12 contacts. In
addition, they observe interfaces involving EL2 loop contacts in three out of eight cases. These interfaces could
potentially be biologically relevant, although when using the MARTINI model an elastic network is usually
applied, which constraints natural loop flexibility. Thus, different dimer conformations involving loop contacts
will inevitably arise depending on the conformation in which the loop has been constrained in. We therefore
propose that such contacts should be regarded with care, especially as hDAT presented here and in Jayaramen’s
study are homology models. The overall conclusion drawn by the authors was that dimer contacts primarily
involve the scaffold region of hDAT (TM 3-5, 8-12) rather than the bundle region (TM1-2,6-7). Although we see
different dimer orientations in our simulations, the overall conclusion drawn by Jayaramen et al. also agrees with
our results. Finally, we typically first detected full closure of the TM9, TM12/TM9, TM12 interface after ~ 10 µs. Therefore, the same interface cannot be expected to be observed by Jayaramen et al., who only simulated for 2 µs.hfi h
p
y
y
y
µ
The different dimers of high population identified for hDAT and hDAT-palm are similar to those observed for
hSERT by Periole et al.14. From hSERT self-assembly simulations the top clusters consisted of dimer conforma-
tions with TM12/TM12, TM7/TM12, TM4,TM9/TM11,TM2, and finally EL2/EL2 interfaces. Similar interfaces to
these have also been observed among the conformations in the top clusters for either hDAT (see Supplementary
Fig. S5) or hDAT-palm (see Supplementary Fig. www.nature.com/scientificreports/ S6) dimers. Interestingly, the separation energy for the hSERT
TM4,TM9/TM11,TM2 interface resulted in ~ 20 kJ/mol, whereas the similar hDAT TM4,TM9/TM11 interface
resulted in a much higher value of ~ 60 kJ/mol. The TM12/TM12 separation energy for hDAT and hSERT are
approximately the same ~ 60 kJ/mol. The TM9/TM9 interface observed frequently in both hDAT and hDAT-
palm self-assembly simulations was not among the top dimer clusters of the hSERT self-assembly simulations. A
possible explanation for the TM9/TM9 interface being frequently observed for hDAT, but not for hSERT, could
be that TM9 in hDAT contains a leucine zipper motif, which is not present in hSERT. www.nature.com/scientificreports/ self-assembly simulations25. Furthermore, it involves interfacial lipids getting encapsulated between the two
protomers and it is regulated by a TM12-palm group as shown by the estimation made from PMFs calcula-
tion. Lipids involvement in oligomer formation is becoming increasingly studied14,26,27. In fact, it has recently
been shown by Gupta et al. that the LeuT dimer contains interfacial lipids consisting of one cardiolipin and six
phospholipids26. When LeuT is in the presence of a dilipidating detergent only the monomeric state is formed,
suggesting that the lipids are necessary for dimer formation. It is possible that this could be extended to DAT. In agreement with both hSERT experimental24 and computational14 data, we observe that hDAT forms an
energetically stable interface consisting of TM12/TM12 interactions. From experimental FRET studies of SERT
double TM fragments, it has been shown that the TM11-12 fragment forms a symmetrical interface24. This has
also been reproduced by computational hSERT self-assembly simulations in which the TM12/TM12 interface
was the most energetically favorable14. In our study, we observe hDAT TM12/TM12 dimers, although when
TM12 becomes palmitoylated the frequency of TM12/TM12 interprotein contacts drops drastically and the
free energy of association is decreased. Illustrating how the S-palmitoyl group can have a regulatory effect on
hDAT dimer formation. Jayaramen et al. have published results on hDAT oligomerization using the MARTINI 2.2 FF, but with a dif-
ferent approach to ours, coined the DAFT protocol15. Rather than studying systems containing multiple proteins
as presented here, they used the DAFT protocol in which multiple repeat simulations are performed of two
hDAT proteins randomly orientated in a POPC membrane15. We ran 10 repeats of the hDAT and hDAT-palm
system each lasting 30 µs, whereas Jayaramen et al. simulated 512 repeats each lasting 2 µs. The DAFT and 16
proteins in a membrane approach have previously converged on similar results for rhodopsin when studying
dimers of high occurrence42. For hDAT, there are some similarities in the top dimer candidates predicted using
the two methods, but overall they differ. The conserved interfaces are TM9/TM9 and EL2/EL2 (detected in our
self-assembly, but only presented in Supplementary Fig. S5-6). Jayaramen et al. frequently observe symmetrical
dimers containing C-terminal helix contacts, which is somewhat similar to our TM12/TM12 interface with the
exception of the two protomers being further separated and thereby not exhibiting direct TM12 contacts. Conclusion In conclusion, several energetically favorable hDAT dimer interfaces are accessible and form in a model mem-
brane on a µs time scale. The majority of these dimers contain TM9 and/or TM11 and/or TM12 at the interface,
all of which have been experimentally shown to be located at the MAT dimer interfaces24,25. Furthermore, we have
shown that the energetic changes for interfaces involving TM12 upon hDAT being S-palmitoylated on Cys581,
due to palm interfering with the formation of non-palm interprotein contacts. Palm may therefore change the
propensity for different dimers that are generated in a biological setting. Overall, it is detected that hDAT and
hSERT form similar interfaces and that both the TM12/TM12 and the TM9,TM12/TM9,TM12 interface share
a high similarity with the LeuT crystal structure dimer.h g
y
y
The indication that palmitoylation has a modulatory role on dimer formation in hDAT could have numerous
biological implications both in terms of protein function and protein surface expression. The regulation of dimer
interfaces through S-palmitoylation may alter the reuptake of dopamine from the synaptic cleft or the increased
affinity for rafts as a result of palmitoylation may aid in the clustering of hDATs. The exact impact on function
however is difficult to assess, mutation studies and an interdisciplinary approach would be highly beneficial to
understand this fundamental process. Discussion What makes this interface further interesting
is that the contacts resemble that of the LeuT crystal structure dimer and can further be reproduced by LeuT Scientific Reports | (2021) 11:4164 | https://doi.org/10.1038/s41598-021-83374-y www.nature.com/scientificreports/ Methods Homology Modelling and System setup – 16 proteins in a membrane. The data presented here is
produced using a hDAT homology model built using a dDAT template (PDB ID: 4XP1). A detailed description
of the model building and validation process can be found in an earlier publication43. Substrate and coordinating
ions were all removed from the atomistic hDAT protein prior to converting it into a MARTINI-v2.2 coarse grain https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 www.nature.com/scientificreports/ model using the martinize script44. The CG protein was inserted into a pure POPC membrane bilayer using the
insane script45. The MARTINI force field for lipids46 and its extension to proteins44,47 was used in combination
with the ElNeDyn48 approach using a 0.9 nm cut-off and a 500 kJ/mol/nm2 force constant for maintaining the
secondary and tertiary structure of the protein. A single hDAT molecule embedded in a POPC membrane in a
1:99 ratio was copied on a four-by-four grid resulting in a system with 16 hDAT molecules. The orientation of
hDAT was randomized by applying a 1000 kJ/mol position restraint on a central residue (BB bead of Leu322)
and running a 3.5 µs simulation. Frames at 3.1, 3.2, 3.3, 3.4 and 3.5 µs were selected as starting points for a 30 µs
simulation, in which the position restraint was removed, and new velocities were generated with a random seed. Two repeat simulations of each starting frame were conducted resulting in ten repeats totaling in 300 µs simula-
tion time. The same procedure was applied using hDAT S-palmitoylated at Cys581 and LeuT (PDB ID: 2A65). The incorporated parameters for S-palmitoyl have recently been published49. In the LeuT self-assembly simula-
tions, a POPE membrane was used instead of a POPC. System setup – PMF calculations. The CG dimers used for calculating PMF profiles of hDAT, hSERT,
and hDAT-palm interfaces were extracted from the self-assembly simulations and first equilibrated in a POPC
membrane of dimensions ~ 9 × 19 × 11 nm3 and solvated with water and 0.15 M NaCl. Prior to running the US-
REMD simulations the systems were equilibrated by applying a position restraint on each hDAT molecule such
that the proteins remained in the center of the short side of the simulation box (10 kJ/mol on the x-axis of the
BB bead of Leu322). Methods The dimer interfaces were probed using the distance between the two protomers as the
reaction coordinate and simultaneously restraining the dimers relative orientation using the ϕ1 and ϕ3 angle
(see Fig. 2a). The distance, ϕ1 and ϕ3 angles where defined using the BB beads of Leu322, Ile393 and Thr456
in hDAT, corresponding to a/A, b/B and c/C, respectively, in Fig. 2a. All dimers at each umbrella started from
a bound conformation and became gradually more unbound with increasing distance of separation, d. The
umbrella windows were generally separated by 0.1 nm, although in some cases this resulted in a low number of
exchanges (> 10%) and a smaller spacing was therefore introduced between these windows (see Supplementary
Table S1-2). Ideally, convergence in this type of setup should be evaluated by starting the proteins from both
an unbound and a bound conformation and observing the PMF profiles approaching each other. However, as
Periole et al. showed for hSERT14, the unbound conformation never seemed to approach the bound, due to lipids
getting stuck at the interface. Instead, we evaluated system convergence by looking at the PMFs in µs simula-
tion windows (see Supplementary Fig. S22-23). When these approached each other, we concluded the systems
to be converged. Typically, this occurred after 3 µs and therefore only the last 7 µs were used for generating the
PMFs. Using the distance distributions in the different umbrella windows, the PMF curves were generated using
a bootstrap procedure in combination with an in-house implementation of WHAM that takes into account the
use of multiple restraints (see Supplementary Fig. S24-25 for the distance histograms). For future unbiasing work
the authors recommend using pyemma50. Molecular dynamics simulations. All MD simulations were performed using the GROMACS simulation
package version 5.1.2 compiled with plumed version 2.151,52. The systems containing 16 hDAT molecules were
simulated using a 20 fs time step during the production run. The electrostatic and van der Waals non-bonding
interactions were treated using a 1.1 nm cut-off and were shifted to 0 using the potential-shift-Verlet scheme as
implemented in GROMACS. A dielectric screening constant of 15 was applied. The pressure was regulated at
1 bar semi-isotropically using the Berendsen barostat with a 2.0 ps coupling time constant and the compress-
ibility set to 3 × 10–4 bar-1. Methods For regulating the temperature, the solvent, protein and membrane were coupled inde-
pendently to an external heat bath set to 310 K using the velocity rescaling scheme and a 1 ps coupling constant.hfh The PMF simulations were performed using a slightly different approach. The time step was 20 and 10 fs
during equilibration and production runs, respectively. The long range electrostatics were treated using the
reaction-field method53. Furthermore, the Berendsen barostat was only applied during a 1 µs equilibration step
and instead the Parrinello-Rahmen pressure coupling was applied during the 10 µs production run54. The frames
were saved every 100 ps during production runs. Because the dimers started from a bound state, the first 1–3
μs simulation time was necessary for the individual umbrella windows to equilibrate at the different interface
distances. The umbrella windows were exchanged every 20 ps. h
It is important to keep in mind that the energies (free energies) obtained within the PMF analysis presented
here should be evaluated relative to each other and that such free energy calculations may dependent extremely
on the method and the environment, here the membrane bilayer. The force field may also affect the results. Thus
all our interpretations are relative and should be interpreted with care. Clustering. For clustering the different dimers observed in the self-assembly simulation, all possible protein
pairs were first extracted and combined into a single trajectory. From this trajectory all frames in which the
distance between the center-of-mass of the BB beads of each protomer was above 5.3 nm were removed. All
remaining frames were symmetrized whereby coordinates in protein A were swapped with the coordinates in
protein B, which allows for symmetrically related dimer pairs to also be considered. Finally, the trajectory con-
taining the non-swapped and swapped coordinates were combined and clustered using the GROMOS clustering
algorithm and a 0.4 nm cutoff. Contact analysis. For evaluating all contact data the gmx mindist tool was applied. A total of 12 × 16 index
groups were generated containing the indices for the residues that constitute the 12 different helices in hDAT for
the 16 different proteins, respectively. In all cases a contact was defined when the distance between any two index
groups between protomers was below 7 Å. Scientific Reports | (2021) 11:4164 | https://doi.org/10.1038/s41598-021-83374-y www.nature.com/scientificreports/ Data availability
Th d
d ata a a ab ty
The datasets generated and/or analyzed during the current study are available from the corresponding author
on reasonable request. Received: 22 May 2020; Accepted: 1 February 2021 Received: 22 May 2020; Accepted: 1 February 2021 References erences
Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011). Norregaard, L. & Gether, U. The monoamine neurotransmitter transporters: structure, conformational changes and molecular References
1. Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011). 2. Norregaard, L. & Gether, U. The monoamine neurotransmitter transporters: structure, conformational changes and molecular
gating. Curr. Opin. Drug. Disc. Dev. 4, 591–601 (2001). References
1. Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol. Rev. 63, 585–640 (2011) 1. Kristensen, A. S. et al. SLC6 neurotransmitter transporters: structure, function, and regulation. ,
S
S C
p
,
,
g
,
(
)
2. Norregaard, L. & Gether, U. The monoamine neurotransmitter transporters: structure, conformational changes and molecular
gating. Curr. Opin. Drug. Disc. Dev. 4, 591–601 (2001). g
g
p
g
3. Pramod, A. B., Foster, J., Carvelli, L. & Henry, L. K. SLC6 transporters: Structure, function, regulation, disease association and
therapeutics. ABCs membr. Transp. Health Dis. (SLC Ser.) 34, 197–219 (2013). 3. Pramod, A. B., Foster, J., Carvelli, L. & Henry, L. K. SLC6 transporters: Structure, function,
therapeutics. ABCs membr. Transp. Health Dis. (SLC Ser.) 34, 197–219 (2013). p
p
4. Torres, G. E., Gainetdinov, R. R. & Caron, M. G. Plasma membrane monoamine transporters: structure, regulation and function
Nat. Rev. Neurosci. 4, 13–25 (2003). 5. Coleman, J. A., Green, E. M. & Gouaux, E. X-ray structures and mechanism of the human serotonin transporter. Nature 532,
334–339 (2016). 6. Penmatsa, A., Wang, K. H. & Gouaux, E. X-ray structure of dopamine transporter elucidates antidepressant mechanism. Nature
503, 85–90 (2013). 7. Chen, N.-H., Reith, M. E. A. & Quick, M. W. Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter
transporter family SLC6. Pflügers Arch. Eur. J. Physiol. 447, 519–531 (2004). p
y
fl g
y
8. Andersen, J., Kristensen, A. S., Bang-Andersen, B. & Strømgaard, K. Recent advances in the understanding of the interaction of
antidepressant drugs with serotonin and norepinephrine transporters. Chem. Commun. 25, 3677–3692 (2009). 9. Seeman, P. & Niznik, H. B. Dopamine receptors and transporters in Parkinson’s disease and schizophrenia. FASEB J. 4, 2737–2744
(1990).i 0. Sharp, S. I., McQuillin, A. & Gurling, H. M. D. Genetics of attention-deficit hyperactivity disorder (ADHD). Neuropharmacology
57, 590–600 (2009). 1. Ritz, M. C., Lamb, R. J., Goldberg, S. R. & Kuhar, M. J. References Symmetrical dimer of the human dopamine transporter revealed by cross-linking Cys-306
at the extracellular end of the sixth transmembrane segment. Proc. Natl. Acad. Sci. U.S.A. 98, 10055–10060 (2001). 23. Cheng, M. H., Garcia-Olivares, J., Wasserman, S., DiPietro, J. & Bahar, I. Allosteric modulation of human dopamine transp
activity under conditions promoting its dimerization. J. Biol. Chem. 292, 12471–12482 (2017).i 24. Just, H., Sitte, H. H., Schmid, J. A., Freissmuth, M. & Kudlacek, O. Identification of an additional interaction domain on transmem-
brane domains 11 and 12 that supports oligomer formation in the human serotonin transporter. J. Biol. Chem. 279, 6650–6657
(2004). 25. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y. & Gouaux, E. Crystal structure of a bacterial homologue of Na+/Cl–depen
neurotransmitter transporters. Nature 437, 215–223 (2005).h p
26. Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421 (2017). . Gupta, K. et al. The role of interfacial lipids in stabilizing memb 27. Anderluh, A. et al. Direct PIP 2 binding mediates stable oligomer formation of the serotonin transporter. Nat. Commun. 8,
(2017). 28. Rastedt, D. E., Vaughan, R. A. & Foster, J. D. Palmitoylation mechanisms in dopamine transporter regulation. J. Chem. Neuroanat. 83, 3–9 (2017). 29. Foster, J. D. & Vaughan, R. A. Palmitoylation controls dopamine transporter kinetics, degradation, and protein kinase C-dependent
regulation. J. Biol. Chem. 286, 5175–5186 (2011). g
(
)
0. El-Husseini, A.E.-D. & Bredt, D. S. Protein palmitoylation: a regulator of neuronal development and function. Nat. Rev. Neurosci
3, 791 (2002). 31. Fang, C. et al. GODZ-mediated palmitoylation of GABAA receptors is required for normal assembly and function of GABAergic
inhibitory synapses. J. Neurosci. 26, 12758–12768 (2006). y y
32. Greaves, J. & Chamberlain, L. H. DHHC palmitoyl transferases: substrate interactions and (patho) physiology. Trends Biochem. Sci. 36, 245–253 (2011).fi ,
(
)
3. Huang, K. & El-Husseini, A. Modulation of neuronal protein trafficking and function by palmitoylation. Curr. Opin. Neurobiol
15, 527–535 (2005). 34. Lai, J. & Linder, M. E. Oligomerization of DHHC protein S-acyltransferases. J. Biol. Chem. 288, 22862–22870 (2013). 34. Lai, J. & Linder, M. E. Oligomerization of DHHC protein S-ac 35. Zheng, H. et al. Palmitoylation and membrane cholesterol stabilize μ-opioid receptor homodimerization and G protein co
BMC Cell Biol. 13, 6 (2012). (
)
36. Periole, X., Huber, T., Marrink, S.-J. & Sakmar, T. P. References Cocaine receptors on dopamine transporters are related to self-adminis-
tration of cocaine. Science 237, 1219–1223 (1987). 12. Elliott, J. M. & Beveridge, R. J. R. Psychostimulants and monoamine transporters: upsetting the balance. Curr. Opin. Pharm. 5,
94–100 (2005). 13. Zeppelin, T., Ladefoged, L. K., Sinning, S. & Schiøtt, B. Substrate and inhibitor binding to the serotonin transporter: Insights
computational, crystallographic, and functional studies. Neuropharmacology 161, 107548 (2019).f y
g
gy
4. Periole, X., Zeppelin, T. & Schiøtt, B. Dimer interface of the human serotonin transporter and effect of the membrane composition
Sci. Rep. 8, 5080 (2018).f p
5. Jayaraman, K. et al. Dopamine transporter oligomerization involves the scaffold domain, but spares the bundle domain. PLoS
Comput. Biol. 14, e1006229 (2018). p
6. Sorkina, T., Doolen, S., Galperin, E., Zahniser, N. R. & Sorkin, A. Oligomerization of dopamine transporters visualized in living
cells by fluorescence resonance energy transfer microscopy. J. Biol. Chem. 278, 28274–28283 (2003).fii yl
gy
py
17. Torres, G. E. et al. Oligomerization and trafficking of the human dopamine transporter. Mutational analysis identifies critical
domains important for the functional expression of the transporter. J. Biol. Chem. 278, 2731–2739 (2003). p
p
p
8. Fjorback, A. W. et al. Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor
emission FRET and fluorescence lifetime imaging microscopy. Biochem. Biophys. Res. Commun. 380, 724–728 (2009).t l
9. Zhen, J. & Reith, M. E. A. Functional properties of dopamine transporter oligomers after copper linking. J. Neurochem. 144
162–171 (2018).h (
)
20. Hastrup, H., Sen, N. & Javitch, J. A. The human dopamine transporter forms a tetramer in the plasma membrane: cross-linking
of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs. J. Biol. Chem. 278, 45045–45048 (2003). 20. Hastrup, H., Sen, N. & Javitch, J. A. The human dopamine transporter forms a tetramer in the plasma membrane: cross-linking
of a cysteine in the fourth transmembrane segment is sensitive to cocaine analogs. J. Biol. Chem. 278, 45045–45048 (2003). 21. Kilic, F. & Rudnick, G. Oligomerization of serotonin transporter and its functional consequences. Proc. Natl. Acad. Sci. U.S.A. 97, 1. Kilic, F. & Rudnick, G. Oligomerization of serotonin transporter and its functional consequences. Proc. Natl. Acad. Sci. U.S.A. 97
3106–3111 (2000). 22. Hastrup, H., Karlin, A. & Javitch, J. A. www.nature.com/scientificreports/ GROMACS: High performance molecular simulations through multi-level parallelism from laptops to
supercomputers. SoftwareX 1–2, 19–25 (2015). p
p
ft
2. Bonomi, M. et al. PLUMED: A portable plugin for free-energy calculations with molecular dynamics. Comput. Phys. Commun
180, 1961–1972 (2009).i 53. Tironi, I. G., Sperb, R., Smith, P. E. & van Gunsteren, W. F. A generalized reaction field method for molecular dynamics simula-
tions. J. Chem. Phys. 102, 5451–5459 (1995). 54. Parrinello, M. & Rahman, A. Polymorphic transitions in single crystals: A new molecular dynamics method. J. Appl. Phys. 52,
7182–7190 (1981). Acknowledgementsh The authors would like to acknowledge Jose C. Flores-Canales for having proofread the manuscript. Moreover, the
research was made possible by the funding from the Danish Council for Independent Research Natural Sciences
(DFF-4002-00502 and DFF-7014-00192B) and Medical Sciences (DFF – 4004-00309), the Lundbeck Foundation,
and the Carlsberg Foundation. Computational resources were provided from the Novo Nordisk Foundation
(NF18OC0032608) through allocations of time at the Centre for Scientific Computing Aarhus and Abacus 2.0. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 37. Landschulz, W. H., Johnson, P. F. & McKnight, S. L. The leucine zipper: a hypothetical structure common to a new class of DNA
binding proteins. Science 240, 1759–1764 (1988).h g p
8. Russ, W. P. & Engelman, D. M. The GxxxG motif: a framework for transmembrane helix-helix association1. J. Mol. Biol. 296
911–919 (2000). 9. Boresch, S., Tettinger, F., Leitgeb, M. & Karplus, M. Absolute binding free energies: a quantitative approach for their calculation
J. Phys. Chem. B 107, 9535–9551 (2003).h R. et al. Pitfalls of the martini model. J. Chem. Theory Comput. 10 h
y
p
41. Kozakov, D. et al. The ClusPro web server for protein–protein docking. Nat. Protoc. 12, 255 (2017). 42. Wassenaar, T. A. et al. High-throughput simulations of dimer and trimer assembly of membrane proteins. The DAFT appro
J. Chem. Theory Comput. 11, 2278–2291 (2015). h
43. Zeppelin, T., Ladefoged, L. K., Sinning, S., Periole, X. & Schiøtt, B. A direct interaction of cholesterol with the dopamine transporter
prevents its out-to-inward transition. PLoS Comput. Biol. 14, e1005907 (2018).ih p
p
4. de Jong, D. H. et al. Improved parameters for the martini coarse-grained protein force field. J. Chem. Theory Comput. 9, 687–697
(2012). 45. Wassenaar, T. A., Ingólfsson, H. I., Böckmann, R. A., Tieleman, D. P. & Marrink, S. J. Computational lipidomics with insane: a
versatile tool for generating custom membranes for molecular simulations. J. Chem. Theory Comput. 11, 2144–2155 (2015). g
gh
y
p
46. Marrink, S. J., de Vries, A. H. & Mark, A. E. Coarse grained model for semiquantitative lipid simulations. J. Phys. Chem. B 108,
750–760 (2004).hih (
)
7. Monticelli, L. et al. The MARTINI coarse-grained force field: extension to proteins. J. Chem. Theory Comput. 4, 819–834 (2008). hih
8. Periole, X., Cavalli, M., Marrink, S.-J. & Ceruso, M. A. Combining an elastic network with a coarse-grained molecular force field
Structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009). y
gh
y
p
49. Atsmon-Raz, Y. & Tieleman, D. P. Parameterization of palmitoylated cysteine, farnesylated cysteine, geranylgeranylated cyst
and myristoylated glycine for the martini force field. J. Phys. Chem. B 121, 11132–11143 (2017).th i
0. Scherer, M. K. et al. PyEMMA 2: A software package for estimation, validation, and analysis of Markov models. J. Chem. Theory
Comput. 11, 5525–5542 (2015). 1. Abraham, M. J. et al. Author contributions T.Z., X.P. and B.S. designed the research. T.Z. prepared the models, performed the simulations and analyzed the
trajectories. K.B.P. performed and analyzed the LeuT self-assembly simulations. T.Z, K.B.P., N.A.B., X.P. and B.S. contributed to manuscript writing, and all authors read and approved the submitted version. References G protein-coupled receptors self-assemble in dynamics simulations of model
bilayers. J. Am. Chem. Soc. 129, 10126–10132 (2007). https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 | Competing interests h g
The authors declare no competing interests. Additional informationh Additional information
Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-021-83374-y. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1038/s41598-021-83374-y. Correspondence and requests for materials should be addressed to X.P. or B.S. Reprints and permissions information is available at www.nature.com/reprints. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
License, which permits use, sharing, adaptation, distribution and reproduction in any medium or
format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the
material. If material is not included in the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2021 https://doi.org/10.1038/s41598-021-83374-y Scientific Reports | (2021) 11:4164 |
|
https://openalex.org/W2948771469
|
https://www.int-arch-photogramm-remote-sens-spatial-inf-sci.net/XLII-2-W13/1709/2019/isprs-archives-XLII-2-W13-1709-2019.pdf
|
English
| null |
PROGRESS ON ISPRS BENCHMARK ON MULTISENSORY INDOOR MAPPING AND POSITIONING
|
The international archives of the photogrammetry, remote sensing and spatial information sciences/International archives of the photogrammetry, remote sensing and spatial information sciences
| 2,019
|
cc-by
| 4,142
|
ABSTRACT: This paper presents the design of the benchmark dataset on multisensory indoor mapping and position (MIMAP) which is sponsored
by ISPRS scientific initiatives. The benchmark dataset including point clouds captured by indoor mobile laser scanning system (IMLS)
in indoor environments of various complexity. The benchmark aims to stimulate and promote research in the following three fields:
(1) SLAM-based indoor point cloud generation; (2) automated BIM feature extraction from point clouds, with an emphasis on the
elements, such as floors, walls, ceilings, doors, windows, stairs, lamps, switches, air outlets, that are involved in building management
and navigation tasks ; and (3) low-cost multisensory indoor positioning, focusing on the smartphone platform solution. MIMAP
provides a common framework for the evaluation and comparison of LiDAR-based SLAM, BIM feature extraction, and smartphone
indoor positioning methods. PROGRESS ON ISPRS BENCHMARK ON MULTISENSORY INDOOR MAPPING AND
POSITIONING Wang1 *, Yudi Dai1, Naser El-Sheimy2, Chenglu Wen1, Guenther Retscher3, Zhizhong Kang4, and Andrea Lingua5 Cheng Wang1 *, Yudi Dai1, Naser El-Sheimy2, Chenglu Wen1, Guenther Retscher3, Zhizhong Kang4, and Andrea Lingua5 Fujian Key Laboratory of Sensing and Computing, School of Informatics, Xiamen University, 422 Siming Road South, Xiamen
361005, China - (cwang, clwen@xmu.edu.cn), (daiyudi@stu.xmu.edu.cn)
2 University of Calgary, Canada - elsheimy@ucalgary.ca
3 TU Wien - Vienna University of Technology, Austria - guenther.retscher@geo.tuwien.ac.at
4 China University of Geosciences, Beijing - zzkang@cugb.edu.cn
5 Polytechnic University of Turin, Italy - andrea.lingua@polito.it 1 Fujian Key Laboratory of Sensing and Computing, School of Informatics, Xiamen University, 422 Siming Road South, Xiamen
361005, China - (cwang, clwen@xmu.edu.cn), (daiyudi@stu.xmu.edu.cn) 361005, China - (cwang, clwen@xmu.edu.cn), (daiyudi@stu.xmu.edu.cn)
2 University of Calgary, Canada - elsheimy@ucalgary.ca
3 TU Wien - Vienna University of Technology, Austria - guenther.retscher@geo.tuwien.ac.at
4 China University of Geosciences, Beijing - zzkang@cugb.edu.cn
5 Polytechnic University of Turin Italy
andrea lingua@polito it 2 University of Calgary, Canada - elsheimy@ucalgary.ca
3 TU Wien - Vienna University of Technology, Austria - guenther.retscher@geo.tuwien.ac.at
4 China University of Geosciences, Beijing - zzkang@cugb.edu.cn 2 University of Calgary, Canada - elsheimy@ucalgary.ca
3 TU Wien - Vienna University of Technology, Austria - guenther.retscher@geo.tuwien.ac.at
4 China University of Geosciences, Beijing - zzkang@cugb.edu.cn 5 Polytechnic University of Turin, Italy - andrea.lingua@polito.it KEY WORDS: Multi-sensor, Indoor, Mapping, Positioning, Benchmark Dataset, Design 1.
INTRODUCTION package of XBeibao. Also, the Rigel VZ 1000 (Figure 1. (c)) can
provide a high accuracy point cloud as ground-truth for the
indoor mapping. Indoor environments are essential to people’s daily life. Indoor
mapping and positioning technologies have become in high
demand in recent years. Visualization, positioning, and location-
based services (LBS), routing and navigation in large public
buildings, navigational assistance for disabled or aged people and
evacuation under different emergency conditions are just a few
examples of the emerging applications that require 3D mapping
and positioning of indoor environments. SLAM-based indoor
mobile laser scanning systems (IMLS) like provide an effective
tool for indoor applications. During the IMLS procedure, 3D
point clouds and high accuracy trajectories with position and
orientation are acquired. Many efforts have been made in the last
few years to improve the SLAM algorithms (Zhang & singh,
2014a) and the geometric/semantic information extraction from
point clouds and images (Armeni et al., 2016a). There are still
some challenges as follows: first, lack of efficient or real-time 3D
point cloud generation methods of as-built 3D indoor
environment; second, difficulties of building information model
(BIM) features extraction in the clustered and occluded indoor
environment. Also, given the relatively high accuracy, the IMLS
trajectory provides a good reference or ground-truth for the low-
cost indoor positioning solutions. 2.1 Sensor setup Our sensors are listed as below: Our sensors are listed as below:
2×Velodyne VLP-16L rotating 3D laser scanner. 20Hz, 16
beams, 0.1° ~ 0.4° horizontal angle resolution, 3cm accuracy,
collecting 0.3 million points/second, field of view: 360°
horizontal, ±15° vertical, range: 100m.
1×Mi Sphere Camera. Lens components: 2 × (5 pieces
spherical glass lens + 2 pieces aspheric glass lens + 2 pieces
right angle glass prism), 3456*1728 @ 30fps video
resolution, the field of view: 2×190°.
1×Mi 6 smartphone. Sensors: gyroscope, accelerometer,
barometer, electronic compass, WiFi sensor, magnetometer,
GPS.
1×Rigel VZ 1000 scanner (www.riegl.com/datasheet_vz-
1000). Range from 1.5m up to 1200m, 5mm precision, 8mm
accuracy, collecting 0.3 million points/second, with field of
view of 100° vertical ×360° horizontal. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands 2.2 Dataset We collect the raw data in three different scenes; each scene is
recorded more than three times with a different route. Each round
of the data consisting of three parts, the raw data, the benchmarks
data, and the calibration files. Only half of the complete version
of the overall dataset was released for the purpose of applying in
different tests. No benchmark releases for indoor LiDAR-based
SLAM test and BIM feature extraction methods test. For
smartphone indoor positioning methods test, there are only raw
smartphone data and the calibration files. The “dataformat.txt” details the format of each file in
“/phone/unixtime_data/”. The three kinds of benchmarks are saved in the corresponding
zip file. Files’ format and detailed description are all included in
the zip file. The benchmark will be discussed in subsection 3.3. The
calibration
files
are
saved
in
the
subdirectory
“date_calaibration/." Note that camera's intrinsic matrix,
extrinsic matrix and distortion coefficients are all saved in
"calib_cam_x_to_LiDAR_A.txt”, where “cam_x” refers to the
two cameras (the camera close to left hand is left camera), and
the extrinsic matrix is used to convert the camera’s coordinate
system to LiDAR A’s coordinate system. The 4×4 calibration
matrix converting the LiDAR B’s coordinate system to LiDAR
A’s coordinate system is saved in “calib_lidar_B_to_ lidar
_A.txt." For each scene, we manually select a coordinate system
and origin(Figure 3) in the real world, and the 4 × 4
transformation matrix from LiDAR A’s coordinate system to the
world
coordinate
system
is
saved
in
“calib_LiDAR_A_to_scene.txt." We also provide each scene's
architectural plan in "scene_architectural_plan.png." 2.2.1
Data description: A sequence of data is compressed
into a file with the name format “date _number_ type.zip," where
“date” is the placeholder for recording date and “number”
represents the serial number of this day’s recording round. The
“type” has four values——00, 01, 03 and 04, representing the
complete data, the SLAM test data, the BIM feature extraction
test data and the indoor positioning test data, respectively. The
directory structure is shown in Figure 2. Figure 2. Structure of the dataset. Here, ‘date’, ‘number’,
‘unixtime’, ‘sensor_name’, ‘scene’ and ‘type’ are placeholders. The ‘date_x. pacp’ refers to the two LiDAR streams, and
‘date_x.mp4' refers to the two video camera streams. Figure 3. An example of a scene's architectural plan, the red dot
on the picture is the origin we select, which is on the ground. 2.
SENSORS AND DATA ACQUISITION Standard datasets are critical for evaluating and comparing
indoor mapping and positioning methodologies. In this project,
The XBeibao II system (Wen et. al., 2016a) shown in Figure 1. (a) , which was developed by SCSC Lab in Xiamen University is
used to collect the multi-sensory indoor data. The system
includes two Velodyne multi-beam laser scanners, fisheye lens
camera (Figure 1. (b)). Also, the navigation-related data from
smart-phone built-in sensors, such as barometer, magnetometer,
six degrees of freedom MEMS IMU data and Wifi information
can be collected. The SLAM-based 3D point cloud of the indoor
environment can also be provided using the processing software 1709 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands (a) (b)
(c)
Figure 1. (a) The XBeibao II Multi-sensor system. (b)
Multi-sensor coordinates system. (c) Riegl VZ-1000. (b) (a) The raw data is saved in the subdirectory “date_rawdata/,” there
are three kinds of sensor mentioned above: LiDAR, camera, and
smartphone. Velodyne LiDAR: To separate the Velodyne readings from
LiDAR sensor A and LiDAR B, we name the LiDAR scans
“date_A.pcap” or “date_B.pcap”, where ‘date’ is the date that
collecting these data. Each point is stored with its (x, y, z)
coordinate and its reflectance intensity value (r). The
“unixtime_start.txt” records the starting time of this record. (b) Camera: We convert the videos captured by the cameras into
images according to the frame rate of the video. We name the
images as “date_x_frameN.png," where "x" refers to the left
or the right camera and “frameN” represents the serial frame
number of this image in the raw video. The starting recording
time
of
the
first
frame
is
saved
in
"camera/unixtime_start.txt." Figure 1. (a) The XBeibao II Multi-sensor system. (b)
Multi-sensor coordinates system. (c) Riegl VZ-1000. When collecting the data, we placed the smartphone facing up on
the top of the upper LiDAR sensor. A laptop is used to control
the camera and LiDARs. Also, it is used as a hotspot to connect
with the smartphone to synchronize the sensors and used to store
the incoming LiDAR data streams. A system operator needs to
carry the laptop during the collection process. 2.
SENSORS AND DATA ACQUISITION Smartphone: Each sensor’s recording data is saved in the file
“unixtime_data/sensor_name.txt," where "unixtime” and
“sensor_name” are the placeholders of the starting time of
this record and this sensor’s abbreviation name, respectively. For each piece of data of different sensors, we record the
Unix-timestamp. The “timeOffset.txt” records the time
offsets from the phone to a local NTP server at different time. The “dataformat.txt” details the format of each file in
“/phone/unixtime_data/”. This contribution has been peer reviewed.
prs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License.
1710 This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. 2.2 Dataset Also, the blue arrows point the direction of X-axis and Y-axis. Figure 2. Structure of the dataset. Here, ‘date’, ‘number’,
‘unixtime’, ‘sensor_name’, ‘scene’ and ‘type’ are placeholders. The ‘date_x. pacp’ refers to the two LiDAR streams, and
‘date_x.mp4' refers to the two video camera streams. Figure 3. An example of a scene's architectural plan, the red dot
on the picture is the origin we select, which is on the ground. Also, the blue arrows point the direction of X-axis and Y-axis. This contribution has been peer-reviewed. This contribution has been peer-reviewed. https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. 1710 𝑃
The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands the sub-map for the nearest neighbour point set, 𝑃𝑃𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛
𝑛𝑛
. Lastly, an
environmental consistency constraint is introduced to obtain
𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐. 𝑓𝑐 The Z-axis is perpendicular to the X and Y axis and the direction
is from the ground to the ceiling. 3.1 Time synchronization In order to synchronize all the sensors, our laptop is set as a local
NTP (Network Time Protocol) server, then all the sensors are
connected to it to synchronize the time. The LiDAR is connected
to the laptop through a network cable; the smartphone and
camera's connections are through WiFi. For the LiDAR, we only
get the start Unix-timestamp of the data collection. The
timestamp of every point or frame is a relative time to the start
Unix-timestamp. As for the Camera, we also could only get the
start Unix-timestamp of the videos. The good news is that every
frame's time can be obtained via interpolation according to the
frame rate. However, unfortunately, frame loss sometimes
happens. For the smartphone, the time can synchronize to the
local NTP server during the recording, so the Unix-timestamp in
every piece of data is relatively accurate. Since all data’s
timestamps are acquired, we can obtain the position at any time
by interpolation and also can use the LiDAR’s positioning result
as the smartphone’ positioning ground-truth. We utilize a TLS (e.g., Riegl VZ 1000) to bridge the calibration
between LiDAR sensors and cameras. By manually selected
matching points between them, we can acquire the camera’s
extrinsic transformation [𝑅𝑅, 𝑇𝑇], where 𝑅𝑅 is the 3×3 rotation
matrix, and 𝑇𝑇 is the 1×3 translation vector. We utilize a TLS (e.g., Riegl VZ 1000) to bridge the calibration
between LiDAR sensors and cameras. By manually selected
matching points between them, we can acquire the camera’s
extrinsic transformation [𝑅𝑅, 𝑇𝑇], where 𝑅𝑅 is the 3×3 rotation
matrix, and 𝑇𝑇 is the 1×3 translation vector. 3.2.3
Phone-to-LiDAR
calibration:
We
place
the
smartphone face up on the LiDAR A (Figure 5), and making the
Y-axis parrallel to the laser beam scanning direction. Thus, the
phone’s coordinate system and the LiDAR’s coordinate system
have the same XYZ-axis direction. Then we use Rigel VZ 1000
TLS to scan the XBeibao II system and calibrate the translation
(𝑋𝑋, 𝑌𝑌, 𝑍𝑍) to LiDAR by manually picking the points in the high
accuracy 3-D point cloud. 3.2 Multi-Sensors Calibration 𝑋𝑌 In this system, LiDAR sensor A ( 𝑋𝑋𝑙𝑙1, 𝑌𝑌𝑙𝑙1, 𝑍𝑍𝑙𝑙1 ) is mounted
horizontally; LiDAR sensor B (𝑋𝑋𝑙𝑙2, 𝑌𝑌𝑙𝑙2, 𝑍𝑍𝑙𝑙2) is mounted 45°
below the LiDAR sensor A (Figure 1 (b)). Based on our previous
work (Gong et al., 2018a), point cloud data of LiDAR sensor A,
(𝑃𝑃𝐴𝐴), and point cloud data of LiDAR sensor B, (𝑃𝑃𝐵𝐵), are fused
into 𝑃𝑃𝑓𝑓 by the 4 × 4 transform matrix between the two LiDAR
sensors (𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐). (Eq. (2)). Additionally, Terrestrial Laser Scanning
(TLS) data is introduced to bridge the calibration between
LiDAR sensors and cameras. The calibration process is shown in
Figure 4. 𝑃𝑃𝑇𝑃 Figure 5. The smart phone’s position and coordinate. 𝑃𝑃𝑓𝑓= 𝑃𝑃𝐴𝐴+ 𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐∗𝑃𝑃𝐵𝐵
(1)
Figure 4. Flowchart of the calibration process. (1) Figure 5. The smart phone’s position and coordinate. 3. CHALLENGES AND METHODOLOGY 3.2.2
Camera -to-LiDAR calibration: The camera Intrinsic
calibration matrix is given by
𝑓𝑓𝑥𝑥
0
𝑐𝑐𝑥𝑥
0
𝑓𝑓𝑦𝑦
𝑐𝑐𝑦𝑦
0
0
1
൩ and (𝑘𝑘1, 𝑘𝑘2, 𝑘𝑘3),
where (𝑓𝑓𝑥𝑥, 𝑓𝑓𝑦𝑦) is the focal length of the camera, (𝑐𝑐𝑥𝑥, 𝑐𝑐𝑦𝑦) is the
position of the of the camera and (𝑘𝑘1, 𝑘𝑘2, 𝑘𝑘3) is the factors of
radial distortion. Also, Scaramuzza’s the camera calibration
method (Scaramuzza et al., 2006a) is used to determine the
internal parameters and distortion factors of the camera and
obtain the camera internal reference model. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. 3.3 Reference data generation For benchmark evaluation, we generated reference data from a
subset of the raw data and introduced other high accuracy data. Figure 4. Flowchart of the calibration process. 3.2.1
LiDAR-to-LiDAR calibration: The calibration of the
multi-LIDAR sensor is calculated recursively in the construction
of the sub-map and its isomorphism constraint (Gong et al.,
2018a). Assuming 𝑇𝑇𝐴𝐴
𝑛𝑛 is the trajectory of LIDAR sensor A at a
time (0~n) in the mapping algorithm, 𝑃𝑃𝐵𝐵
𝑛𝑛 is the point cloud of
LIDAR sensor B at time n. 𝑇𝑇𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 is the initial coordinate system
transformation between the LIDAR sensors. Calibration is the
calculation of the exact calibration matrix 𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐 by: 𝑃𝑛𝑛𝑛𝑛𝑛𝑁𝑁𝑀𝑇𝐴𝑛𝑃𝐵𝑛𝑇𝑖𝑖𝑖𝑖 For SLAM-based indoor point cloud evaluation, we built a high
accuracy 3-D reference map via the data collected by Rigel VZ
1000. Firstly, we placed many high-reflection rectangle stickers
on the wall and ground. Then we scanned the scene in a different
position and ensured there is an overlap between adjacent sub-
maps. Finally, the sub-maps were manually calibrated by picking
the same sticker and other feature points via the software named
RiSCAN PRO. 𝑃𝑃𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛
𝑛𝑛
= 𝑁𝑁𝑁𝑁(𝑀𝑀, 𝑇𝑇𝐴𝐴
𝑛𝑛 , 𝑃𝑃𝐵𝐵
𝑛𝑛, 𝑇𝑇𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖)
(2)
𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐= 𝑎𝑎𝑎𝑎𝑎𝑎𝑚𝑚𝑚𝑚𝑚𝑚
𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐
∑𝑇𝑇𝑐𝑐𝑐𝑐𝑐𝑐∗𝑃𝑃𝐵𝐵
𝑛𝑛 −
𝑛𝑛
𝑃𝑃𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛
𝑛𝑛
(3) 𝑁𝑁𝑇𝑇𝑃 (2)
(3) For BIM feature benchmark, we used the building line
framework exacted by the wang’s method (Wang et al. 2018a)
and the semantic objects labeled via our manually work. We
selected the building lines with their length greater than 0.1 m in ( )
(3) where 𝑁𝑁𝑁𝑁(·) is the nearest neighbour point search algorithm. Using 𝑇𝑇1
𝑛𝑛 and 𝑇𝑇𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖, 𝑃𝑃𝐵𝐵
𝑛𝑛 is first transformed to its location at time
n in the sub-map M. Then the 𝑁𝑁𝑁𝑁(·) algorithm is used to search 1711 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands ⎩
⎪
⎨
⎪
⎧𝜃𝜃= arccos (
𝑣𝑣𝑔𝑔∙𝑣𝑣𝑒𝑒
ห𝑣𝑣𝑔𝑔ห|𝑣𝑣𝑒𝑒|)
𝑑𝑑= ฮ𝑝𝑝𝑔𝑔−𝑝𝑝𝑒𝑒ฮ2
Δ𝑙𝑙= ฮ𝑙𝑙𝑔𝑔−𝑙𝑙𝑒𝑒ฮ1
𝑎𝑎𝑎𝑎𝑎𝑎=
𝑁𝑁𝑇𝑇
𝑁𝑁
(6) 𝑁 structured indoor building and saved their own two endpoints’
coordinates. Fig.6 gives an example of BIM features. (6) Figure 6. BIM feature examples. (a) Point cloud data. (b) BIM
structure model. The green lines are doors and pillars. The red
lines are ceilings. 3.5 Examples of dataset Fig. 7 shows some examples of this dataset. The Fig 7. (a) is a
frame of the Velodyne VLP-16L LiDAR data. Different color
represents the intensity of every point, the brighter color means
the stronger intensity. The Fig 7. (b) shows the high accuracy data
from Rigel VZ 1000, which is used as Indoor LiDAR SLAM
ground truth. The (c) and (d) in Figure 7 show the BIM
benchmark, and (e) and (d) show the Indoor positioning
benchmark. The blue dots in (d) are trajectories generated from
LiDAR SLAM method, and the yellow dots are trajectories
generated by the smartphone sensor data. ℰ𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡(δ) =
1
𝑁𝑁∑
𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡(δ𝑖𝑖,𝑗𝑗⊖𝛿𝛿𝑖𝑖,𝑗𝑗
∗)2
𝑖𝑖,𝑗𝑗
(4)
ℰ𝑟𝑟𝑟𝑟𝑟𝑟(δ) =
1
𝑁𝑁∑
𝑟𝑟𝑟𝑟𝑟𝑟(δ𝑖𝑖,𝑗𝑗⊖𝛿𝛿𝑖𝑖,𝑗𝑗
∗)2
𝑖𝑖,𝑗𝑗
(5) (4) (5) where N is the number of relative relations, and ⊖ is the inverse
of a standard motion composition operator. Let δ𝑖𝑖,𝑗𝑗 be the
relative transformation from pose j to pose i and 𝛿𝛿𝑖𝑖,𝑗𝑗
∗ be the
reference relative relation. 𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡(·) and 𝑟𝑟𝑟𝑟𝑟𝑟(·) are used to
separate the translation and rotation error. (a)
(b)
(c)
(d)
(e)
(f)
Figure 7. (a)A single frame from LiDAR stream. (b) An indoor
view of Rigel VZ 1000 data. (c) BIM structure model of a circular
corridor. (d) BIM structure model with its point cloud. (e) An
example of the indoor positioning benchmark. (f) The ground-
truth trajectory with the corresponding point cloud. (a) (b) However, for indoor environments, it is hard to get the reference
for the trajectory poses. However, based on K¨ummerle’s method,
we can apply the metric operating on the landmark locations
instead of based on the trajectory poses. In this way, the relations
can be determined by measuring the relative distances between
landmarks. (b) (a) (a)
(c) ( )
(d) 3.4.2
BIM feature: We propose a method to evaluate the BIM
feature extraction method. Here, we assume that we have the
ground truth line 𝐿𝐿𝑔𝑔 and the evaluation line 𝐿𝐿𝑒𝑒(the nearest
midpoint to 𝐿𝐿𝑔𝑔’ midpoint). For both lines, we calculate the
corresponding direction vector 𝑣𝑣𝑔𝑔 and 𝑣𝑣𝑒𝑒, midpoint 𝑝𝑝𝑔𝑔 and 𝑝𝑝𝑒𝑒,
and length 𝑙𝑙𝑔𝑔 and 𝑙𝑙𝑒𝑒. Based on the above information, we can get
the angle 𝜃𝜃 between the two lines, the distance 𝑑𝑑 between the
two midpoints, and the length difference Δ𝑙𝑙 by Eq. (6). Then we
set three thresholds 𝜃𝜃𝑡𝑡ℎ𝑟𝑟, 𝑑𝑑𝑡𝑡ℎ𝑟𝑟 and Δ𝑙𝑙𝑡𝑡ℎ𝑟𝑟. 3.4 Evaluation Metrics where 𝑡𝑡 falls within the interval ( 𝑡𝑡𝑠𝑠, 𝑡𝑡𝑒𝑒) which are two
timestamps of the trajectory from the benchmark. 𝐩𝐩𝒔𝒔 and 𝐩𝐩𝒆𝒆
represents the ground-truth positions at time 𝑡𝑡𝑠𝑠 and 𝑡𝑡𝑒𝑒
respectively. And ⊕ denotes a compositional operator. 3.4.1
SLAM-based
indoor
point
cloud:
Kümmerle
(K¨ummerle at al., 2009a) proposed a metric for measuring the
performance of a SLAM algorithm by considering poses of a
robot during data acquisition. It is not based on the error of the
trajectory end-point, but the average of all relations between
poses. Geiger (Geiger at al., 2012a) extended the metric by treat
the rotation and translation errors separately. Here, we do similar
operation as 𝑡𝑡𝑡𝑡𝑡𝛿 3.4.3
Indoor positioning: The approach of evaluating indoor Figure 6. BIM feature examples. (a) Point cloud data. (b) BIM
structure model. The green lines are doors and pillars. The red
lines are ceilings. The blue lines represent the ground. For our Indoor positioning evaluation, we used the LiDAR’s
trajectory generated by a SLAM method (Zhang & singh, 2014a)
with loop closure as the reference. 𝐩𝐩𝑡𝑡=
𝑡𝑡−𝑡𝑡𝑠𝑠
𝑡𝑡𝑒𝑒−𝑡𝑡𝑠𝑠𝐩𝐩𝑒𝑒⊕
𝑡𝑡𝑒𝑒−𝑡𝑡
𝑡𝑡𝑒𝑒−𝑡𝑡𝑠𝑠𝐩𝐩𝑠𝑠
(7) 𝑡𝑠𝑡𝑒𝐩𝐩 (7) This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. p
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. 3.3 Reference data generation The blue lines represent the ground. (a) Point cloud data. (b) BIM where 𝑁𝑁𝑇𝑇 is the true line number and 𝑁𝑁 is the all ground-
truth line number. 3.4.3
Indoor positioning: The approach of evaluating indoor
positioning is the same as the translation evaluating in subsection
3.4.1. However, there exists a problem that the frequency of
positions output by mobile phones varies with the ground-truth's
frequency generated by SLAM. To solve this problem, we
generate position at a time by a linear interpolation according to
the timestamp. Formally, the ground truth position at time 𝑡𝑡 is
calculated by: 𝐩𝑡𝑡𝑠𝐩𝑡𝑒𝑡𝐩 3.5 Examples of dataset We consider the
evaluation line 𝐿𝐿𝑒𝑒 is valid only if three conditions are all met: (1)
𝜃𝜃≤𝜃𝜃𝑡𝑡ℎ𝑟𝑟, (2) 𝑑𝑑≤𝑑𝑑𝑡𝑡ℎ𝑟𝑟, (3) Δ𝑙𝑙≤Δ𝑙𝑙𝑡𝑡ℎ𝑟𝑟. Finally, we can calculate
the accuracy acc by Eq. (6). (d) (c) (e) (f) (f) (e) Figure 7. (a)A single frame from LiDAR stream. (b) An indoor
view of Rigel VZ 1000 data. (c) BIM structure model of a circular
corridor. (d) BIM structure model with its point cloud. (e) An
example of the indoor positioning benchmark. (f) The ground-
truth trajectory with the corresponding point cloud. 1712 The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands 4. CONCLUSION This paper presents the design of the benchmark dataset on
multisensory indoor mapping and position (MIMAP). Each scene
in the dataset contains the point clouds from the multi-beam laser
scanner, the images from fisheye lens camera, the signals from
MIMU and the records from the attached smartphone sensors. The benchmark dataset can be used to evaluate algorithms on: (1)
SLAM-based indoor point cloud generation; (2) automated BIM
feature extraction from point clouds; and (3) low-cost
multisensory indoor positioning, focusing on the smartphone
platform solution. This paper presents the design of the benchmark dataset on
multisensory indoor mapping and position (MIMAP). Each scene
in the dataset contains the point clouds from the multi-beam laser
scanner, the images from fisheye lens camera, the signals from
MIMU and the records from the attached smartphone sensors. This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. The International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume XLII-2/W13, 2019
ISPRS Geospatial Week 2019, 10–14 June 2019, Enschede, The Netherlands This contribution has been peer-reviewed.
https://doi.org/10.5194/isprs-archives-XLII-2-W13-1709-2019 | © Authors 2019. CC BY 4.0 License. This contribution has been peer-reviewed. 5. REFERENCES Armeni, I., Sener, O., Zamir, A. R., Jiang, H., Brilakis, I., Fischer,
M., & Savarese, S., 2016a. 3d semantic parsing of large-scale
indoor spaces. In Proceedings of the IEEE Conference on
Computer Vision and Pattern Recognition (pp. 1534-1543). Geiger, A., Lenz, P., Urtasun, R., 2012a. Are we ready for
autonomous driving? the kitti vision benchmark suite. In 2012
IEEE
Conference
on
Computer
Vision
and
Pattern
Recognition (pp. 3354-3361). Gong, Z., Wen, C., Wang, C., Li, J., 2018a. A target-free
automatic self-calibration approach for multibeam laser
scanners. IEEE Trans. Instrum. Meas, 67(1), 238-240. Kümmerle, R., Steder, B., Dornhege, C., Ruhnke, M., Grisetti,
G., Stachniss, C., Kleiner, A., 2009a. On measuring the accuracy
of SLAM algorithms. Auton. Robots, 27(4), 387. Scaramuzza, D., Martinelli, A., Siegwart, R., 2006a. A toolbox
for easily calibrating omnidirectional cameras. In 2006
IEEE/RSJ International Conference on Intelligent Robots and
Systems (pp. 5695-5701). Wen, C., Pan, S., Wang, C., Li, J., 2016a. An indoor backpack
system for 2-D and 3-D mapping of building interiors. IEEE
Geosci. Rem. Sens. Lett., 13(7), 992-996. Wang, C., Hou, S., Wen, C., Gong, Z., Li, Q., Sun, X., Li, J.,
2018a. Semantic line framework-based indoor building modeling
using backpacked laser scanning point cloud. ISPRS J. Photogramm. Rem. Sens., 143, 150-166. Zhang, J., Singh, S., 2014a. LOAM: Lidar Odometry and
Mapping in Real-time. In Robotics: Science and Systems (Vol. 2,
p. 9). 1713
|
https://openalex.org/W2899682789
|
https://curis.ku.dk/ws/files/239163844/d90fba35_ce36_45b4_9a54_688335056f2a_16642_bennedikte_madsen_v2.pdf
|
English
| null |
Adverse reactions of dimethyl sulfoxide in humans: a systematic review
|
F1000Research
| 2,019
|
cc-by
| 15,019
|
university of copenhagen
Adverse reactions of dimethyl sulfoxide in humans
A systematic review
Madsen, Bennedikte Kollerup; Hilscher, Maria; Zetner, Dennis; Rosenberg, Jacob
Published in:
F1000Research
DOI:
10.12688/f1000research.16642.2
Publication date:
2019
Document version
Publisher's PDF, also known as Version of record
Document license:
CC BY
Citation for published version (APA):
Madsen, B. K., Hilscher, M., Zetner, D., & Rosenberg, J. (2019). Adverse reactions of dimethyl sulfoxide in
humans: A systematic review. F1000Research, 7, [1746]. https://doi.org/10.12688/f1000research.16642.2 university of copenhagen Adverse reactions of dimethyl sulfoxide in humans
A systematic review Document version
Publisher's PDF, also known as Version of record Document version
Publisher's PDF, also known as Version of record Download date: 24. Oct. 2024 SYSTEMATIC REVIEW
Adverse reactions of dimethyl sulfoxide in humans: a
systematic review [version 2; peer review: 2 approved]
Bennedikte Kollerup Madsen
,
Maria Hilscher, Dennis Zetner
, Jacob Rosenberg
Department of Surgery, Center for Perioperative Optimization (CPO), Herlev Hospital, Herlev, 2730, Denmark 05 Nov 2018, :1746 (
First published:
7
)
https://doi.org/10.12688/f1000research.16642.1
06 Aug 2019, :1746 (
Latest published:
7
)
https://doi.org/10.12688/f1000research.16642.2
v2 Document license:
CC BY Citation for published version (APA):
Madsen, B. K., Hilscher, M., Zetner, D., & Rosenberg, J. (2019). Adverse reactions of dimethyl sulfoxide in
humans: A systematic review. F1000Research, 7, [1746]. https://doi.org/10.12688/f1000research.16642.2 Citation for published version (APA):
Madsen, B. K., Hilscher, M., Zetner, D., & Rosenberg, J. (2019). Adverse reactions of dimethyl sulfoxide in
humans: A systematic review. F1000Research, 7, [1746]. https://doi.org/10.12688/f1000research.16642.2 Download date: 24. Oct. 2024 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Open Peer Review
Any reports and responses or comments on the
article can be found at the end of the article. SYSTEMATIC REVIEW
Adverse reactions of dimethyl sulfoxide in humans: a
systematic review [version 2; peer review: 2 approved]
Bennedikte Kollerup Madsen
,
Maria Hilscher, Dennis Zetner
, Jacob Rosenberg
Department of Surgery, Center for Perioperative Optimization (CPO), Herlev Hospital, Herlev, 2730, Denmark
Abstract
Dimethyl sulfoxide (DMSO) has been used for medical
Background:
treatment and as a pharmacological agent in humans since the 1960s. Today, DMSO is used mostly for cryopreservation of stem cells, treatment
of interstitial cystitis, and as a penetrating vehicle for various drugs. Many
adverse reactions have been described in relation to the use of DMSO, but
to our knowledge, no overview of the existing literature has been made. Our
aim was to conduct a systematic review describing the adverse reactions
observed in humans in relation to the use of DMSO. This systematic review was reported according to the
Methods:
PRISMA-harms (Preferred Reporting Items for Systematic reviews and
Meta-Analysis) guidelines. The primary outcome was any adverse
reactions occurring in humans in relation to the use of DMSO. We included
all original studies that reported adverse events due to the administration of
DMSO, and that had a population of five or more. We included a total of 109 studies. Gastrointestinal and skin
Results:
reactions were the commonest reported adverse reactions to DMSO. Most
reactions were transient without need for intervention. A relationship
between the dose of DMSO given and the occurrence of adverse reactions
was seen. DMSO may cause a variety of adverse reactions that are
Conclusions:
mostly transient and mild. The dose of DMSO plays an important role in the
occurrence of adverse reactions. Document license:
CC BY DMSO seems to be safe to use in small
doses
Reviewer Status
Invited Reviewers
version 2
published
06 Aug 2019
version 1
published
05 Nov 2018
1
2
report
report
report
report
, Queen's University Belfast,
Curly Morris
Belfast, UK
1
, University of Oxford,
Igho J. Onakpoya
Oxford, UK
2
05 Nov 2018, :1746 (
First published:
7
)
https://doi.org/10.12688/f1000research.16642.1
06 Aug 2019, :1746 (
Latest published:
7
)
https://doi.org/10.12688/f1000research.16642.2
v2 Abstract Dimethyl sulfoxide (DMSO) has been used for medical
Background:
treatment and as a pharmacological agent in humans since the 1960s. Today, DMSO is used mostly for cryopreservation of stem cells, treatment
of interstitial cystitis, and as a penetrating vehicle for various drugs. Many
adverse reactions have been described in relation to the use of DMSO, but
to our knowledge, no overview of the existing literature has been made. Our
aim was to conduct a systematic review describing the adverse reactions
observed in humans in relation to the use of DMSO. This systematic review was reported according to the
Methods:
PRISMA-harms (Preferred Reporting Items for Systematic reviews and
Meta-Analysis) guidelines. The primary outcome was any adverse
reactions occurring in humans in relation to the use of DMSO. We included
all original studies that reported adverse events due to the administration of
DMSO, and that had a population of five or more. We included a total of 109 studies. Gastrointestinal and skin
Results:
reactions were the commonest reported adverse reactions to DMSO. Most
reactions were transient without need for intervention. A relationship
between the dose of DMSO given and the occurrence of adverse reactions
was seen. DMSO may cause a variety of adverse reactions that are
Conclusions:
mostly transient and mild. The dose of DMSO plays an important role in the
occurrence of adverse reactions. DMSO seems to be safe to use in small
doses. PROSPERO
. Registration:
CRD42018096117 Keywords
Dimethyl Sulfoxide, DMSO, Adverse reactions, Toxicology Keywords Keywords
Dimethyl Sulfoxide, DMSO, Adverse reactions, Toxicology Page 1 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Bennedikte Kollerup Madsen (
)
Corresponding author:
Benedikte.kollerupmadsen@gmail.com
: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration,
Author roles: Kollerup Madsen B
Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Data Curation, Formal Analysis,
Hilscher M
Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Formal Analysis,
Zetner D
Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
:
Rosenberg J
Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft
Preparation, Writing – Review & Editing
No competing interests were disclosed. Competing interests:
The author(s) declared that no grants were involved in supporting this work. Grant information:
© 2019 Kollerup Madsen B
. This is an open access article distributed under the terms of the
Copyright:
et al
Creative Commons Attribution
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Licence
Kollerup Madsen B, Hilscher M, Zetner D and Rosenberg J. How to cite this article:
Adverse reactions of dimethyl sulfoxide in humans: a
F1000Research 2019, :1746 (
)
systematic review [version 2; peer review: 2 approved]
7
https://doi.org/10.12688/f1000research.16642.2
05 Nov 2018, :1746 (
)
First published:
7
https://doi.org/10.12688/f1000research.16642.1 Bennedikte Kollerup Madsen (
)
Corresponding author:
Benedikte.kollerupmadsen@gmail.com
: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration,
Author roles: Kollerup Madsen B
Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Data Curation, Formal Analysis,
Hilscher M
Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Formal Analysis,
Zetner D
Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
:
Rosenberg J
Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft
Preparation, Writing – Review & Editing
No competing interests were disclosed. Competing interests:
The author(s) declared that no grants were involved in supporting this work. Grant information:
© 2019 Kollerup Madsen B
. This is an open access article distributed under the terms of the
Copyright:
et al
Creative Commons Attribution
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Licence
Kollerup Madsen B, Hilscher M, Zetner D and Rosenberg J. Keywords How to cite this article:
Adverse reactions of dimethyl sulfoxide in humans: a
F1000Research 2019, :1746 (
)
systematic review [version 2; peer review: 2 approved]
7
https://doi.org/10.12688/f1000research.16642.2
05 Nov 2018, :1746 (
)
First published:
7
https://doi.org/10.12688/f1000research.16642.1 Bennedikte Kollerup Madsen (
)
Corresponding author:
Benedikte.kollerupmadsen@gmail.com
: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration,
Author roles: Kollerup Madsen B
Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Data Curation, Formal Analysis,
Hilscher M
Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Formal Analysis,
Zetner D
Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
:
Rosenberg J
Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft
Preparation, Writing – Review & Editing
No competing interests were disclosed. Competing interests:
The author(s) declared that no grants were involved in supporting this work. Grant information:
© 2019 Kollerup Madsen B
. This is an open access article distributed under the terms of the
Copyright:
et al
Creative Commons Attribution
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Licence
Kollerup Madsen B, Hilscher M, Zetner D and Rosenberg J. How to cite this article:
Adverse reactions of dimethyl sulfoxide in humans: a
F1000Research 2019, :1746 (
)
systematic review [version 2; peer review: 2 approved]
7
https://doi.org/10.12688/f1000research.16642.2
05 Nov 2018, :1746 (
)
First published:
7
https://doi.org/10.12688/f1000research.16642.1 Bennedikte Kollerup Madsen (
)
rresponding author:
Benedikte.kollerupmadsen@gmail.com Bennedikte Kollerup Madsen (
)
Corresponding author:
Benedikte.kollerupmadsen@gmail.com : Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Project Administration,
Author roles: Kollerup Madsen B
Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Data Curation, Formal Analysis,
Hilscher M
Investigation, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Formal Analysis,
Zetner D
Investigation, Methodology, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing;
:
Rosenberg J
Conceptualization, Formal Analysis, Investigation, Methodology, Project Administration, Supervision, Validation, Writing – Original Draft
Preparation, Writing – Review & Editing The author(s) declared that no grants were involved in supporting this work. Grant information: Page 2 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 searched on February 23, 2018. Our search strategy was formulated
with the help of a medical research librarian. Amendments from Version 1
REVISED In this updated version an additional table (Table 9) has been
added with an overview of the different administration routes of
DMSO used in the included studies. The search string used in PubMed was: ((dimethyl sulfoxide)
OR DMSO) AND ((((((administration and dosage) OR adverse
reactions) OR alternate effects) OR secondary response) OR
toxicology) OR side effects)). The search was restricted to
humans. The search string was adapted to EMBASE and Cochrane
Library using the same search-words as abovementioned. See referee reports Study selection and data extraction Two authors (B.K.M. and D.Z.) independently screened title
and abstract according to the eligibility criteria using www. covidence.org. Discrepancies were resolved by discussion. One
author screened the full-text articles (B.K.M.). Russian articles
were screened by an author fluent in Russian (M.H.). If M.H. was in doubt regarding inclusion of a study the results were
presented to B.K.M. and then discussed until a mutual decision
was made. After the screening process was finished, all included studies
were imported to an Excel sheet (Microsoft Excel 2016). Data
extraction was performed by two authors (M.H. extracted from
the Russian articles and B.K.M. extracted from the rest). Data
extracted were: author, publication year, country, study charac-
teristics (study design, sample size, size of comparison group
if present, time to follow-up), use of DMSO (reason for use,
treatment duration, administration route, dose of DMSO), and
adverse reactions observed (number of persons experiencing an
adverse reaction, method of assessing, and duration of adverse
reaction). To our knowledge, no systematic reviews have been performed
on the adverse reactions of DMSO. Our aim was therefore to
provide an extensive overview of the suspected adverse reactions to
DMSO in humans. Methods Protocol and eligibility criteria Protocol and eligibility criteria
Our study-protocol is registered at PROSPERO (Registration
number:
CRD42018096117). The
systematic
review
was
performed according to PRISMA-harms (Preferred Reporting
Items for Systematic Reviews and Meta-Analyses) guidelines13. g
y
Our study-protocol is registered at PROSPERO (Registration
number:
CRD42018096117). The
systematic
review
was
performed according to PRISMA-harms (Preferred Reporting
Items for Systematic Reviews and Meta-Analyses) guidelines13. Introductioni The first medical report on the use of dimethyl sulfoxide (DMSO)
as a pharmacological agent was published in 19641. A year later,
the use of DMSO in humans was terminated because experi-
mental studies had shown refractive index changes to the lens
of the eye1,2. Years later, DMSO was again approved for use in
humans since this side effect was only proven in animal
studies2. DMSO has since been used for a variety of purposes,
such as treatment of musculoskeletal and dermatological diseases,
cryopreservation of stem cells, treatment of interstitial cystitis,
treatment of increased intracranial pressure, and many more3–9. The search string used in EMBASE was: ((dmso or dimethyl
sulfoxide) and ((side effect or toxicology or secondary response
or alternate effects or alternate reactions or (administration and
dosage)) and (dmso or dimethyl sulfoxide))).mp. The search
was restricted to humans, articles and Medline journals were
excluded. The search string used in Cochrane was: (adverse drug events and
dimethyl sulfoxide). The search was restricted to trials. DMSO is a colourless liquid, which is rapidly absorbed
when administered dermally or orally10,11. DMSO is used as a
cryoprotectant because it decreases osmotic stress and cellular
dehydration, and thereby enables stem cells to be stored for
several years12. DMSO is mostly excreted through the kidneys,
but a small part is excreted through the lungs and liver10. Part of the DMSO is transformed to the volatile metabolite
dimethyl sulfide, which gives a characteristic garlic- or
oyster-like smell when excreted through the lungs10. DMSO may
induce histamine release, which can be the reason for adverse
reactions such as flushing, dyspnoea, abdominal cramps, and
cardiovascular reactions11. Study selection and data extraction Analysis y
The Newcastle-Ottawa-Scale was used to assess the risk of
bias in non-randomized observational studies14. Risk of bias in
randomized controlled trials was assessed using the Cochrane
Handbook “Risk of Bias” assessment tool15. Risk of bias was
assessed at the outcome level. No limitations were set on the date of publication. The language
was restricted to English, Danish, Swedish, Norwegian, and
Russian. We included all original studies that administered
DMSO to humans and included five or more participants. There
was no gender or age restriction. For a study to be included,
the authors had to suspect that an observed adverse reaction
could be caused by DMSO. The primary summary measure was percentage of persons
experiencing an adverse reaction, as well as the range in which a
reaction occurred in the studies included. No meta-analysis and
further summery measures were planned due to the expected
large heterogeneity of the studies. Primary outcome Primary outcome The primary outcome was any adverse reaction seen in relation to
the use of DMSO in humans. Literature search Our primary search identified 2599 studies (Figure 1). After
the evaluation process, 109 studies were included in the final
review2,4,6–9,16–118. The search was performed in PubMed (1966-present), EMBASE
(1980-present), and the Cochrane Library. The databases were last Page 3 of 21 Page 3 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Figure 1. PRISMA flow diagram. Figure 1. PRISMA flow diagram. Figure 1. PRISMA flow diagram. Gastrointestinal reactions
Gastrointestinal adverse reactions were reported in 61 stud-
ies. Of
these,
10
studies
were
randomized
controlled
trials16,30,33,55,57,59,67,79,93,95, 49 were cohort studies2,4,7,9,18,19,23,25–27,
29,35,38–43,45,46,48,50–54,58,60,66,68,69,71,73,83,85–88,90,94,97,98,101,104,105,112,113,115,118,
and 2 were case series84,109. Most studies reported the number of
patients experiencing an adverse reaction (Table 1). Other stud-
ies reported adverse reactions observed in relation to the number
of treatments given (Table 2). Gastrointestinal reactions Adverse reaction
Studies
Total
patients, n
Patients
with adverse
reaction, n (%)
(%, min-max) †
Nausea (overall incidence)
[2,18,27,33,45,46,48,53,55,57,59,60,67,84,90,93,109,118] 2214
257 (12)
(2–41) [55] - [48]
Intravenous administration
[18,27,33,46,48,53,59,90,118]
1154
199 (17)
(2–41) [59] - [48]
Transdermal application
[2,45,55,57,67,93,109]
1039
51 (5)
(2–32) [55] - [2]
>1 administration route
[60,84]
21
7 (33)
(29–36) [84] - [60]
Vomiting (overall incidence) [2,18,27,33,46,48,55,57,59,118]
1611
115 (7)
(0–64) [55] - [48]
Intravenous administration
[18,27,33,46,48,59,118]
972
108 (11)
(2–64) [59] - [48]
Transdermal application
[2,55,57]
639
7 (1)
(0–6) [55] - [2]
Nausea and vomiting‡
[7,38,41,54,66,69,73,85,87,115]
4529
591 (13)
(0–46) [66] - [73]
Abdominal cramps/stomach
ache (overall incidence)
[18,26,27,39,41,54,55,59,73,85,87,93,115]
1629
88 (5)
(1–52) [117] - [116]
Intravenous administration
[18,26,27,39,41,54,59,73,85,87,115]
1253
72 (6)
(1–52) [18] - [26]
Transdermal application
[55,93]
376
16 (4)
(2–16) [55] - [93]
Halitosis/garlic-like breath
(overall incidence)
[4,9,16,19,29,30,35,42,43,45,50,52,55,57,58,66–68,79,83,
85,88,94,95,97,98,109,112,113]
5782
607 (11)
(0–100) [30] -
[19,43,45,83,98]
Intravenous administration
[16,85,94,98]
239
14 (6)
(1–100) [85] - [98]
Transdermal application
[4,19,29,30,42,45,50,52,55,57,58,66,67,79,83,88,95,109,
112,113]
5333
556 (10)
(0–100) [30]
- [19,45,83]
Intravesical administration
[35,43,97]
165
33 (20)
(1–100) [35] - [43]
Oral administration
[9]
15
4 (27)
Diarrhea (overall incidence) [2,18,41,54,57,85,93]
1107
27 (2)
(1–6) [85] - [93]
Intravenous administration
[18,41,54,85]
744
15 (2)
(1–6) [85] - [41]
Transdermal application
[2,57,93]
363
12 (3)
(2–6) [57] - [93]
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an Table 1. Gastrointestinal adverse reactions observed per number of patients. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. ‡ Nausea and vomiting are reported as one combined adverse reaction in some studies. ‡ Nausea and vomiting are reported as one combined adverse reaction in some studies. Table 2. Gastrointestinal adverse reactions observed per number of treatments. Gastrointestinal reactions Adverse reaction
Studies
Total
patients, n
Patients
with adverse
reaction, n (%)
(%, min-max) †
Nausea (overall incidence)
[2,18,27,33,45,46,48,53,55,57,59,60,67,84,90,93,109,118] 2214
257 (12)
(2–41) [55] - [48]
Intravenous administration
[18,27,33,46,48,53,59,90,118]
1154
199 (17)
(2–41) [59] - [48]
Transdermal application
[2,45,55,57,67,93,109]
1039
51 (5)
(2–32) [55] - [2]
>1 administration route
[60,84]
21
7 (33)
(29–36) [84] - [60]
Vomiting (overall incidence) [2,18,27,33,46,48,55,57,59,118]
1611
115 (7)
(0–64) [55] - [48]
Intravenous administration
[18,27,33,46,48,59,118]
972
108 (11)
(2–64) [59] - [48]
Transdermal application
[2,55,57]
639
7 (1)
(0–6) [55] - [2]
Nausea and vomiting‡
[7,38,41,54,66,69,73,85,87,115]
4529
591 (13)
(0–46) [66] - [73]
Abdominal cramps/stomach
ache (overall incidence)
[18,26,27,39,41,54,55,59,73,85,87,93,115]
1629
88 (5)
(1–52) [117] - [116]
Intravenous administration
[18,26,27,39,41,54,59,73,85,87,115]
1253
72 (6)
(1–52) [18] - [26]
Transdermal application
[55,93]
376
16 (4)
(2–16) [55] - [93]
Halitosis/garlic-like breath
(overall incidence)
[4,9,16,19,29,30,35,42,43,45,50,52,55,57,58,66–68,79,83,
85,88,94,95,97,98,109,112,113]
5782
607 (11)
(0–100) [30] -
[19,43,45,83,98]
Intravenous administration
[16,85,94,98]
239
14 (6)
(1–100) [85] - [98]
Transdermal application
[4,19,29,30,42,45,50,52,55,57,58,66,67,79,83,88,95,109,
112,113]
5333
556 (10)
(0–100) [30]
- [19,45,83]
Intravesical administration
[35,43,97]
165
33 (20)
(1–100) [35] - [43]
Oral administration
[9]
15
4 (27)
Diarrhea (overall incidence) [2,18,41,54,57,85,93]
1107
27 (2)
(1–6) [85] - [93]
Intravenous administration
[18,41,54,85]
744
15 (2)
(1–6) [85] - [41]
Transdermal application
[2,57,93]
363
12 (3)
(2–6) [57] - [93]
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. ‡ N
d
iti
t d
bi
d
d
ti
i
t di Table 1. Gastrointestinal adverse reactions observed per number of patients. Gastrointestinal reactions Gastrointestinal reactions be less common with the transdermal administration of DMSO
compared with intravenous administration. The majority of
studies reported an incidence of nausea between 2–14%, with the
exception of one study, reporting an incidence of 32%2. In one
study that failed to specify the dose, 8 of 42 patients reported
nausea and anorexia, but the symptoms disappeared in five of
the eight patients when the dose of DMSO was reduced45. Gastrointestinal adverse reactions were reported in 61 stud-
ies. Of
these,
10
studies
were
randomized
controlled
trials16,30,33,55,57,59,67,79,93,95, 49 were cohort studies2,4,7,9,18,19,23,25–27,
29,35,38–43,45,46,48,50–54,58,60,66,68,69,71,73,83,85–88,90,94,97,98,101,104,105,112,113,115,118, and 2 were case series84,109. Most studies reported the number of
patients experiencing an adverse reaction (Table 1). Other stud-
ies reported adverse reactions observed in relation to the number
of treatments given (Table 2). Often the studies had short follow-up periods (less than 24 hours),
especially when DMSO was used as a cryoprotectant. The study
reporting the highest incidence of nausea had a follow-up period
of 5 days48, and the authors concluded that the high incidence The most commonly reported gastrointestinal adverse reactions
were nausea and vomiting. The incidence of nausea seems to Page 4 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Table 1. Gastrointestinal adverse reactions observed per number of patients. Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highe
bserved incidence of an adverse reaction. g
p
‡ Nausea and vomiting are reported as one combined adverse reaction in some studies. Gastrointestinal reactions Halitosis was reported in 29 studies4,9,16,19,29,30,35,42,43,45,50,52,55,57,58,
66–68 79 83 85 88 94 95 97 98 109 112 113
I
fi
t di
ti
t
di
ti In some studies, hypertension did not require intervention61,102,
but cases where medication was needed to control the hyper-
tension, or where treatment was stopped due to hyperten-
sion, are described41,54,85. Hypotension was also described as
transient most of the time18,23,68,87,104, with some cases needing
intervention40,51,54. One study reported a severe case of nausea, vomiting, and
abdominal cramps in one patient with an acute allergic reaction59. However, in most studies the reported gastrointestinal reactions
were transient and mild, often lasting only minutes to a couple
of hours16,38,41,68,85,87,90. Several studies reported a relationship
between the dose of DMSO and the occurrence of gastrointestinal
adverse reactions26,33,53,73,83,85. between the dose of DMSO and the occurrence of gastrointestinal
adverse reactions26,33,53,73,83,85. One study reported 11 cases of transient extrasystoles in
22 patients receiving cryopreserved autologous blood stem
cells, monitored with Holter during infusion73. There were two
studies reporting cases of asystole during embolization of dural
arteriovenous fistulas with a substance called Onyx, a non-
adhesive liquid embolic agent dissolved in DMSO91,100. Gastrointestinal reactions Adverse reaction
Studies
Total
treatments, n
Adverse reactions
observed, n (%)
(min%–max%) †
Nausea (overall incidence)
[40,51,68,84,105]
474
161 (34)
(16–57) [105] - [40]
Intravenous administration
[40,51,68]
323
137 (42)
(41–57) [68] - [40]
Intravesical administration
[105]
151
24 (16)
Vomiting ‡
[51,68]
316
112 (35)
(29 - 71) [68] - [51]
Nausea and/or vomiting ‡
[25,74,101]
1557
220 (14)
(8–17) [25] - [101]
Abdominal cramps/stomach ache ‡
[51,68,101]
495
16 (5)
(1–19) [68] - [51]
Halitosis ‡
[68]
262
4 (2)
Diarrhea ‡
[51,101]
233
2 (1)
(1–2) [101] - [51]
† Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest
observed incidence of an adverse reaction. † Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest
observed incidence of an adverse reaction. Page 5 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 trials33,59, 30 were cohort studies7,18,23,25–27,36,39–41,51,54,61,65,66,68,73,74,80,
85 87 90 100 102 104 115 117
d
li i
91
f trials33,59, 30 were cohort studies7,18,23,25–27,36,39–41,51,54,61,65,66,68,73,74,80,
85–87,90,100–102,104,115,117, and one was a preliminary report91. Except for
one study66, all studies reporting cardiovascular and respiratory
reactions administered DMSO intravenously (Table 3 and
Table 4). of nausea observed might be due to the long follow-up period48. In another article using the same data119, it was suggested that the
delayed nausea was due to gastrointestinal mucosal damage, and
only the initial nausea could be related to DMSO, and therefore
we decided only to include the data from the first 2 days after
infusion48. ,
85–87,90,100–102,104,115,117, and one was a preliminary report91. Except for
one study66, all studies reporting cardiovascular and respiratory
reactions administered DMSO intravenously (Table 3 and
Table 4). Bradycardia was defined as a heart rate less than 60 beats per
minute41,61 and was often transient23,61,90,115, but cases where
atropine was needed are described49,96. A lowered heart rate
not enough to be considered bradycardia was observed in four
studies39,41,54,61. Halitosis was reported in 29 studies4,9,16,19,29,30,35,42,43,45,50,52,55,57,58,
66–68,79,83,85,88,94,95,97,98,109,112,113. In five studies, patients discontin-
ued treatment due to halitosis9,45,83,94. In five studies, all patients
experienced halitosis9,45,83,94. Unlike halitosis, other gastrointes-
tinal side effects were reported more often when DMSO was
administered intravenously, than transdermally or intravesically. Cardiovascular and respiratory reactions Cardiovascular and respiratory adverse reactions were reported
in 33 studies. Of these, two were randomized controlled Table 3. Cardiovascular and respiratory adverse reactions observed per number of patients. Adverse reaction
Studies
Total
patients, n
Patients with adverse
reactions, n (%)
(min%–max%) †
Cardiac
Hypotension ‡
[7,18,23,33,71,73,87,104,115]
2752
115 (4)
(1–14) [18,71] - [87]
Hypertension§
[7,18,23,33,41,54,61,73,85,87,102]
2998
385 (13)
(2–95) [85] - [61]
Bradycardia (mild and severe) ‡ [23,36,54,61,65,85,90,91,115,117]
882
94 (11)
(0–49) [36] - [61]
Decrease in heart rate ‡
[41,54,61,80]
193
152 (79)
(11–94) [80] - [41]
Tachycardia ‡
[23,27,36]
565
13 (2)
(0–6) [36] - [23]
Ventricular extrasystoles ‡
[73]
22
11 (50)
Cardiac event, unspecified ‡
[26,86]
165
18 (11)
(5–12) [26] - [86]
Asystole ¶
[91,100]
45
3 (7)
(3–20) [100] - [91]
Left cardiac insufficiency
[85]
194
1 (1)
Chest discomfort/tightness ‡
[18,27,54,73,87,91,115]
901
22 (2)
(1–10) [27] - [54]
Respiratory
Unspecified respiratory
symptoms ‡
[26,86]
165
43 (26)
(21–62) [86] - [26]
Dyspnead
[18,27,54,66,85]
2748
26 (1)
(0–10) [66] - [54]
Cough
[85,101]
373
52 (14)
(5–22) [101] [85]
Lung edema ‡
[59,85]
241
3 (1)
(1–2) [85] - [59]
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence
of an adverse reaction. ‡ DMSO
as administered intra eno sl in all st dies Table 3. Cardiovascular and respiratory adverse reactions observed per number of patients. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence
of an adverse reaction. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence
of an adverse reaction. ¶ In both studies, asystole occurred because of DMSO effect on the trigeminal nerve and activation of the trigeminal cardiac reflex. d) in one study
DMSO was administered transdermally F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Table 4. Cardiovascular and respiratory adverse reactions observed per number of treatments. Dermatological reactions Dermatological side effects are common when DMSO is admin-
istered transdermally. Skin reactions or allergic reactions were
reported in 58 studies. DMSO was applied transdermally in
43 studies2,4,6,17,19–22,24,28–32,37,44,45,52,55,57,63,64,66,67,69,72,75,76,78,79,82,83,88,89,93,95,96,
106,108,109,111–113, intravenously in 14 studies25,40,41,51,59,73,74,77,85,86,92,98,101,110
and intraarticular in one103 (Table 5). Flushing was regarded as an allergic reaction in this review
and was only reported when DMSO was administered intra-
venously25,40,41,51,54,73,74. A total of four studies, not depicted in
Table 5, reported 204 cases of flushing during 1439 stem
cell infusions25,40,51,74. Several studies observed a relationship
between the dose of DMSO and the occurrence of adverse
reactions67,75,78,83,88,93. Dermatological side effects are common when DMSO is admin-
istered transdermally. Skin reactions or allergic reactions were
reported in 58 studies. DMSO was applied transdermally in
43 studies2,4,6,17,19–22,24,28–32,37,44,45,52,55,57,63,64,66,67,69,72,75,76,78,79,82,83,88,89,93,95,96, 106,108,109,111–113, intravenously in 14 studies25,40,41,51,59,73,74,77,85,86,92,98,101,110
and intraarticular in one103 (Table 5). and intraarticular in one103 (Table 5). The most common skin reaction was a local burning sensation
reported in 13 studies17,21,24,28,30,45,55,57,67,69,79,93,106. In one study, all
participants experienced this burning sensation45. In the same
study, four participants experienced a transient peripheral
edema associated with itching and erythema45. A single study
described a burning sensation in four of 669 patients when
DMSO was given as a local injection92; another study described
burning in two out of 17 patients when DMSO was injected
intraarticularly103. Cardiovascular and respiratory reactions Adverse reaction
Studies
Total number of
treatments
Adverse reactions
observed, n (%)
(min%–max%) †
Cardiac
Hypotension ‡
[40,51,68]
323
10 (3)
(2–14) [68] - [40]
Hypertension ‡
[25,51,68]
425
60 (14)
(3–21) [25] - [68]
Bradycardia (mild and severe) ‡ [51]
54
4 (7)
Decrease in heartrate ‡
[39]
32
30 (94)
Tachycardia ‡
[51]
54
4 (7)
Cardiac event, unspecified ‡
[74]
1269
35 (3)
Chest discomfort/tightness ‡
[25,68,74]
1640
83 (5)
(0–6) [68] - [74]
Respiratory
Dyspnea
[25,68]
371
3 (1)
(0–2) [68] - [25]
Shortness of breath ‡
[74]
1269
40 (3)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max) are the lowest and
highest observed incidence of an adverse reaction observed in the group of studies included. ‡ DMSO was administered intravenously. Table 4. Cardiovascular and respiratory adverse reactions observed per number of treatments. Table 4. Cardiovascular and respiratory adverse reactions observed per number of treatments. †Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max) are the lowest and
highest observed incidence of an adverse reaction observed in the group of studies included. ‡ DMSO was administered intravenously. Dyspnea was reported in seven studies18,25,27,54,66,68,85. A single
study reported eight patients with transient shock after stem cell
transfusion51. Some of these patients developed loss of conscious-
ness and cyanosis but recovered promptly and had no need for
additional therapy, whereas the rest of the patients developed
severe hypotension or transient dyspnea, which was described
as the reason for the transient shock. Further description of the
condition was not provided. describing that skin reactions to DMSO would disappear after
days of continuous treatment45,83. Another study reported that 1
of 18 patients treated for psoriasis with DMSO was hospitalized
due to exfoliative erythroderma63. In another study, two patients,
diagnosed with dermographia developed prominent areas of weals
after DMSO application95. Acute allergic reactions due to use of DMSO were reported
in six studies37,44,59,86,98,110. One study reported that 63 of
144 patients experienced allergic reactions, which was not
described as serious adverse events (bronchospasms, facial
flushing, rash)86. In two other studies, acute allergic reactions were
characterized as serious adverse events59,110. Several of the studies found a correlation between the dose
of DMSO used and the incidence of cardiovascular adverse
reactions41,67,71,75,78,85,86,93,101,115. Dermatological reactions Urogenital reactions Few urogenital reactions were described (Table 6 and Table 7). Hemoglobinuria was described as an adverse reaction seen
after transfusion of stem cell products39,51,56,73. However, hemo-
globinuria is often attributed to erythrocyte debris in the trans-
plant material and has thus not been interpreted as being caused
by DMSO39,73. The other urogenital reactions (Table 6 and
Table 7) all occurred after DMSO instillation in the bladder38,49,97. Overall, most studies administered DMSO intravenously or
transdermally (Table 9) Risk of bias within studies In this review, we included 76 cohort studies, of which 64 were
prospective2,4,6,7,20,22,24–27,29,31,32,34–38,40–45,48,50–54,56,58,60,63,65,66,68–70,72,73,
77,80,81,83,85,88,90,92,94,97,98,101–104,107,108,110,112,115,117,118 and 13 were retro-
spective9,18,23,39,46,47,61,71,74,86,87,100,105. Bias was assessed using The
Newcastle-Ottawa-Scale14. Using this scale, studies were given
zero to nine stars. A high number of stars equals low risk of bias
and vice versa. The studies in this review had a median value of
5 stars, with a range of 2–8. No studies received the highest
possible value of nine stars. Very few studies had a comparison
group that did not receive DMSO, and often the occurrence
of adverse reactions was poorly described. There were 24
randomized controlled trials (Figure 2). Many studies received
an unclear risk of bias because often it was vaguely described
how adverse reactions were reported. Neurological reactions (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. § One study administered DMSO through intraarticular injection [38]. ¶ DMSO was administered intravenously in all studies. ¶ DMSO was administered intravenously in all studies. Very few cases of serious adverse reactions associated with
DMSO have been described18,36,51,59. Very few cases of serious adverse reactions associated with
DMSO have been described18,36,51,59. Urogenital reactions Neurological reactions Headache is the most common neurological adverse reaction
reported. In one study, headache was the reason for withdrawal
of 2 out of 21 patients being treated with DMSO116. Three studies using DMSO as a cryoprotectant in stem cell
transfusions
described
seizures
after
administration18,36,47. Severe encephalopathy was observed in one patient99, and
transient cranial nerve III and IV palsy was observed in one
patient after Onyx embolization34. One study described neuro-
logical symptoms occurring during and after transfusion, but
they did not define neurological symptoms in detail86. Most skin reactions were transient, only lasting minutes17,24,32,67,72,
but some studies reported cases described as serious, causing
discontinuation of treatment2,6,52,63,78,96. There were two studies Page 7 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Table 5. Dermatological and allergic adverse reactions observed per number of patients. Adverse reactions
Studies
Total
patients, n
Patients
with adverse
reactions, n (%)
(%, min-max) †
Skin reactions
Erythema ‡
[19,32,64,66,82,95]
2352
201 (9)
(3–95) [95] - [82]
Itching/Pruritus ‡
[6,55,57,64,66,72,82,93]
3421
215 (6)
(0–70) [55] - [82]
Urticaria ‡
[24,31,83]
58
9 (16)
(4–59) [24] - [83]
Rash
[29,30,55,57,64,93,101,111]
2682
121 (5)
(1–40) [30] - [93]
Paresthesia/burning or
stinging sensation§‡
[17,21,24,28,30,44,45,55,57,67,69,79,91,93,106] 2141
335 (16)
(0–100) [30] - [45]
Scaling of skin/desquamation/
dry skin/local irritant ‡
[22,29,30,37,52,55,57,64,66,69,75,82,88,89,106] 4739
731 (15)
(1–96) [66] - [52]
Blistering ‡
[31,32,66,69,93,112]
2038
79 (4)
(3–20) [66] - [112]
Roughness and/or thickening
of skin ‡
[66,82,93]
1986
191 (10)
(6–10) [93] - [82]
Bullous dermatitis/dermatitis
with vesicles ‡
[20,29,64]
1116
79 (7)
(1–9) [64] - [29]
Contact dermatitis ‡
[6,20,28–30,64,111]
2587
161 (6)
(1–13) [28] - [29]
Skin reaction, unspecified ‡
[2,78,96,113]
457
159 (35)
(4–48) [96] - [113]
Increase in skin pigmentation ‡
[6]
548
28 (5)
Peripheral edema ‡
[45,55,66,109]
2291
22 (0)
(1–14) [66] - [109]
Allergic reactions
[37,44,59,86,98,110]
309
75 (24)
(3–55) [44,110] - [86]
Intravenous administration
[59,86,98,110]
229
66 (29)
(2–55) [59] - [86]
Transdermal application
[37,44]
86
9 (10)
(3–19) [44] - [37]
Flushing ¶
[41,54,73]
292
34 (12)
(2–9) [54] - [73]
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. ‡ Transdermal application only. Table 5. Dermatological and allergic adverse reactions observed per number of patients. †Incidences of the adverse reactions have been calculated for all the individual studies. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. Other reactions Only one study in this review administered DMSO as eye-
drops114. In this study, two patients experienced severe conjunc-
tival hyperemia due to allergic reactions, and 25% of patients
experienced a stinging sensation when eye-drops were applied114. Other studies performed eye examinations to determine whether
DMSO caused changes in the lens; however, no such cases were
observed2,45. Hyponatremia occurred in six patients after they received large
doses of DMSO as treatment for cranial hypertension62. This
adverse reaction was not reported in other studies (Table 8). Page 8 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Table 6. Neurological and urogenital adverse reactions observed per number of patients. Adverse reaction
Studies
Total
patients, n
Patients
with adverse
reactions, n (%)
(min%–max%)†
Neurological
Headache
[2,18,29,33,38,41,55,59,70,71,81,84,85,98,101,104,116]
2516
150 (6)
(1–50) [101] - [70]
Intravenous administration
[18,33,41,59,70,71,81,85,98,101,104]
1271
42 (3)
(1–50) [101] - [70]
Transdermal application
[2,29,55]
1197
102 (8)
(5–35) [55] - [2]
Intravesical administration
[38]
20
1 (5)
Rectal administration
[116]
21
3 (14)
>1 administration route
[84]
7
2 (29)
Seizures
[18,36,47]
301
2 (1)
(0–2) [18] - [47]
Neurological symptoms,
unspecified
[86]
144
5 (3)
Transient CN III and IV
palsy
[34]
12
1 (8)
Severe encephalopathy
[99]
124
1 (1)
Urogenital
Pelvic discomfort/pain/
irritation
[38,49,97]
107
10 (9)
(6–30) [49] - [38]
Dysuria/strangury
[49]
36
6 (17)
Renal and urinary disorder [49]
36
8 (22)
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. Table 6. Neurological and urogenital adverse reactions observed per number of patients. Other reactions Adverse reaction
Studies
Total
patients, n
Patients
with adverse
reactions, n (%)
(min%–max%)†
Neurological
Headache
[2,18,29,33,38,41,55,59,70,71,81,84,85,98,101,104,116]
2516
150 (6)
(1–50) [101] - [70]
Intravenous administration
[18,33,41,59,70,71,81,85,98,101,104]
1271
42 (3)
(1–50) [101] - [70]
Transdermal application
[2,29,55]
1197
102 (8)
(5–35) [55] - [2]
Intravesical administration
[38]
20
1 (5)
Rectal administration
[116]
21
3 (14)
>1 administration route
[84]
7
2 (29)
Seizures
[18,36,47]
301
2 (1)
(0–2) [18] - [47]
Neurological symptoms,
unspecified
[86]
144
5 (3)
Transient CN III and IV
palsy
[34]
12
1 (8)
Severe encephalopathy
[99]
124
1 (1)
Urogenital
Pelvic discomfort/pain/
irritation
[38,49,97]
107
10 (9)
(6–30) [49] - [38]
Dysuria/strangury
[49]
36
6 (17)
Renal and urinary disorder [49]
36
8 (22)
†Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. †Incidences of the adverse reactions have been calculated for all the individual studies. (min%–max%) are the lowest and highest observed incidence of an
adverse reaction observed in the group of studies included. Table 8. Other adverse reactions observed per number of patients. Adverse
reaction
Studies
Total
patients, n
Patients with
reaction, n (%)
(min%–max%)†
Fever
[27,71,73,77,101]
547
44 (8)
(2–19) [27] - [77]
Chills
[27,33,70,71,81,85,101]
852
60 (7)
(1–31) [101] - [71]
Dizziness
[2,46,55,85,101]
885
18 (2)
(1–15) [55] - [2]
Weakness
[33,45,46]
293
19 (6)
(1–29) [46] - [45]
Sedation
[2]
78
34 (44)
Hyponatremia
[62]
6
6 (100)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of studies
included. Table 7. Neurological and urogenital adverse reactions observed per number of
treatments. Adverse reaction
Studies
Total
treatments, n
Adverse reactions
observed, n (%)
(min%–max%) †
Neurological
Headache
[39,51]
86
40 (47)
(6 - 73) [39] - [51]
Urogenital
Urethral irritation
[73]
151
110 (73)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of
studies included. Page 9 of 21 Table 7. Neurological and urogenital adverse reactions observed per number of
treatments. †Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of studies
included. Other reactions Adverse reaction
Studies
Total
treatments, n
Adverse reactions
observed, n (%)
(min%–max%) †
Neurological
Headache
[39,51]
86
40 (47)
(6 - 73) [39] - [51]
Urogenital
Urethral irritation
[73]
151
110 (73)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of
studies included. Table 7. Neurological and urogenital adverse reactions observed per number of
treatments. Adverse reaction
Studies
Total
treatments, n
Adverse reactions
observed, n (%)
(min%–max%) †
Neurological
Headache
[39,51]
86
40 (47)
(6 - 73) [39] - [51]
Urogenital
Urethral irritation
[73]
151
110 (73)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of
studies included. Table 7. Neurological and urogenital adverse reactions observed per number of
treatments. †Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of
studies included. Table 8. Other adverse reactions observed per number of patients. Adverse
reaction
Studies
Total
patients, n
Patients with
reaction, n (%)
(min%–max%)†
Fever
[27,71,73,77,101]
547
44 (8)
(2–19) [27] - [77]
Chills
[27,33,70,71,81,85,101]
852
60 (7)
(1–31) [101] - [71]
Dizziness
[2,46,55,85,101]
885
18 (2)
(1–15) [55] - [2]
Weakness
[33,45,46]
293
19 (6)
(1–29) [46] - [45]
Sedation
[2]
78
34 (44)
Hyponatremia
[62]
6
6 (100)
†Incidences of the adverse reactions have been calculated for all the individual studies. (%, min-max)
are the lowest and highest observed incidence of an adverse reaction observed in the group of studies
included. Page 9 of 21 Table 8. Other adverse reactions observed per number of patients. Table 8. Other adverse reactions observed per number of patients. Page 9 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Table 9. Way of administration of DMSO in included studies. Administration
Number
of studies
References
Intravenous
49
[7,16,18,23,25,26,33,34,36,39–41,46–48,51,53,54,56,59,61,62,65,68,70–74,
77,80,81,84–87,90,91,94,98–102,104,110,115,117,118]
Transdermal
48
[2,4,6,17,19–22,24,28–32,37,42,44,45,50,52,55,57,58,63,64,66,67,69,72,75,76,78,79,
82–84,88,89,93,95,96,106–109,111–113]
Intravesical
7
[8,35,38,43,49,97,105]
Oral
2
[9,60]
Eye-drops
1
[114]
Local injection
1
[92]
Intra-articular
1
[103]
Rectal
1
[116] Table 9. Way of administration of DMSO in included studies. Table 9. Way of administration of DMSO in included studies. Table 9. Way of administration of DMSO in included studies. Figure 2. Grant information The author(s) declared that no grants were involved in supporting
this work. Supplementary material
Supplementary File 1. Completed PRISMA harms checklist. Click here to access the data Supplementary material
Supplementary File 1. Completed PRISMA harms checklist. Click here to access the data 5.
Kulah A, Akar M, Baykut L: Dimethyl sulfoxide in the management of patient
with brain swelling and increased intracranial pressure after severe closed
head injury. Neurochirurgia (Stuttg). 1990; 33(6): 177–80.
PubMed Abstract | Publisher Full Text
6.
Rosenbaum EE, Herschler RJ, Jacob SW: Dimethyl Sulfoxide in Musculoskeletal
Disorders. JAMA. 1965; 192: 309–13.
PubMed Abstract | Publisher Full Text
7.
Morris C, de Wreede L, Scholten M, et al.: Should the standard dimethyl
sulfoxide concentration be reduced? Results of a European Group for Blood
and Marrow Transplantation prospective noninterventional study on usage
and side effects of dimethyl sulfoxide. Transfusion. 2014; 54(10): 2514–22.
PubMed Abstract | Publisher Full Text Discussion Gastrointestinal and dermatological adverse reactions were the
most commonly reported in the included studies. Cardiac
adverse reactions only occurred when DMSO was administered
intravenously, whereas dermatological reactions mostly occurred
when DMSO was administered on the skin. Serious neurologi-
cal and cardiac reactions were rare and only described in few
studies. There seems to be a dose-response relationship between
DMSO and adverse reactions with no or mild reactions in low
doses. Many studies on the use of DMSO have been performed in
Russia. These studies have not been readily accessible to the
global community due to the language barrier. In this review, we
have included not only studies dating back almost 50 years, but
also articles written in Russian, which is an important strength
of the review. This study has several limitations: 1) Some stud-
ies used the NCI-CTC (National Cancer Institute’s Common
Terminology Criteria for adverse events), but often no scale
was used, and the occurrence of adverse reactions were poorly
reported. 2) It was difficult to make conclusions on the fre-
quency of a specific adverse reaction, because the exact number
of patients experiencing a reaction was often not stated. 3) Sev-
eral studies using DMSO as a cryoprotectant concluded that other
factors affected the occurrence of adverse reactions7,85,86. One
study prospectively looked at the adverse reactions observed in
relation to autologous transplantation in 64 European Blood and
Marrow Transplant Group centers7. They had difficulties isolat-
ing the effects of DMSO from confounding factors such as cell
breakdown products and conditioning chemotherapy. Factors In conclusion, adverse reactions due to DMSO are often mild
and transient and do not qualify as serious adverse events. Cardiovascular and respiratory adverse reactions occur mostly
when DMSO is administered intravenously, whereas derma-
tological reactions have a higher incidence when DMSO is
administered transdermally. An important finding is that the
occurrence of adverse reactions seems to be related to the dose
of DMSO, and it therefore seems safe to continue the use of
DMSO in small doses. F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Overall, there was a high risk of bias when assessing the descrip-
tion of adverse reactions. Some studies were not assessed
for bias due to being case-reports, preliminary trials, or because
they included more than one study design17,19,28,62,84,91,99,109,111,113. Overall, there was a high risk of bias when assessing the descrip-
tion of adverse reactions. Some studies were not assessed
for bias due to being case-reports, preliminary trials, or because
they included more than one study design17,19,28,62,84,91,99,109,111,113. such as age, gender, volume transfused, granulocyte concentra-
tion, clumping of transplant material, and amount of red blood
cells played a role in the occurrence of adverse reactions61,86,120–122. Another study believed that acute volume expansion, electro-
lyte imbalance and vagal responses to the coldness of the freshly
thawed infusate were more likely reasons for cardiac arrhyth-
mias during stem cell transfusions than the DMSO infused123. This differs from other studies, which found a clear connec-
tion between dose of DMSO and occurrence of cardiac adverse
reactions41,67,71,75,78,85,86,93,101,115. Therefore, it is possible that
some adverse reactions are more or less common than found
in this review. The rarer side effects are often reported in case
reports, which often did not meet the eligibility criteria in this
review. However, we have included several larger studies in this
review, and they found a very small occurrence of serious adverse
events7,55,66,74. Data availability All data underlying the results are available as part of the article
and no additional source data are required. 1.
Brown JH: Dimethyl sulfoxide (DMSO)--a unique therapeutic entity. Aviat Space
Environ Med. 1982; 53(1): 82–8.
PubMed Abstract
2.
Brobyn RD: The human toxicology of dimethyl sulfoxide. Ann N Y Acad Sci.
1975; 243: 497–506.
PubMed Abstract | Publisher Full Text
3.
Swanson BN: Medical use of dimethyl sulfoxide (DMSO). Rev Clin Basic Pharm.
1985; 5(1–2): 1–33.
PubMed Abstract
4.
Paul MM: Interval therapy with dimethyl sulfoxide. Ann N Y Acad Sci. 1967;
141(1): 586–98.
PubMed Abstract | Publisher Full Text Other reactions Risk of bias in randomized controlled trials. Figure 2. Risk of bias in randomized controlled trials. Page 10 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 ( )
PubMed Abstract | Publisher Full Text 34. Elhammady MS, Wolfe SQ, Farhat H, et al.: Onyx embolization of carotid-
cavernous fistulas. J Neurosurg. 2010; 112(3): 589–94. PubMed Abstract | Publisher Full Text 14. Wells G, Shea B, O’Connell D, et al.: The Newcastle-Ottawa Scale (NOS) for
assessing the quality of nonrandomised studies in meta-analyses. Reference Source 35. Hung MJ, Chen YT, Shen PS, et al.: Risk factors that affect the treatment
of interstitial cystitis using intravesical therapy with a dimethyl sulfoxide
cocktail. Int Urogynecol J. 2012; 23(11): 1533–9. PubMed Abstract | Publisher Full Text 15. Higgins JPT, Altman DG, Sterne JAC: Chapter 8: Assessing risk of bias in
included studies. In: Higgins JPT GS. Cochrane Handbook for Systematic Reviews
of Interventions Version 5.1.0 (updated March 2011). The Cochrane Collaboration. 2008. 36. Lemarie C, Camels B, Malenfant C, et al.: Clinical experience with the delivery
of thawed and washed autologous blood cells, with an automated closed fluid
management device: CytoMate. Transfusion. 2005; 45(5): 737–42. PubMed Abstract | Publisher Full Text ,
PubMed Abstract | Publisher Full Text 9. Amemori S, Iwakiri R, Endo H, et al.: Oral dimethyl sulfoxide for systemic
amyloid A amyloidosis complication in chronic inflammatory disease: a
retrospective patient chart review. J Gastroenterol. 2006; 41(5): 444–9. PubMed Abstract | Publisher Full Text p
p
y
PubMed Abstract | Publisher Full Text 43. Barker SB, Matthews PN, Philip PF, et al.: Prospective study of intravesical
dimethyl sulphoxide in the treatment of chronic inflammatory bladder disease. Br J Urol. 1987; 59(2): 142–4. PubMed Abstract | Publisher Full Text y
py
;
( )
PubMed Abstract | Publisher Full Text 41. Davis J, Rowley SD, Santos GW: Toxicity of autologous bone marrow graft
infusion. Prog Clin Biol Res. 1990; 333: 531–40. PubMed Abstract 20. Ivanov OL, Potekaev NS, Aliab’eva AP: [Dimethyl sulfoxide applications in the
treatment of erythema nodosum]. Ter Arkh. 1983; 55(9): 104–7. PubMed Abstract 42. Bertelli G, Gozza A, Forno GB, et al.: Topical dimethylsulfoxide for the
prevention of soft tissue injury after extravasation of vesicant cytotoxic drugs:
a prospective clinical study. J Clin Oncol. 1995; 13(11): 2851–5. PubMed Abstract | Publisher Full Text 21. Vuopala U, Isomäki H, Kaipainen WJ: Dimethyl sulfoxide (DMSO) ointment in the
treatment of rheumatoid arthritis. A double blind study. Acta Rheumatol Scand. 1969; 15(2): 139–44. PubMed Abstract | Publisher Full Text References 1. Brown JH: Dimethyl sulfoxide (DMSO)--a unique therapeutic entity. Aviat Space
Environ Med. 1982; 53(1): 82–8. PubMed Abstract
2. Brobyn RD: The human toxicology of dimethyl sulfoxide. Ann N Y Acad Sci. 1975; 243: 497–506. PubMed Abstract | Publisher Full Text
3. Swanson BN: Medical use of dimethyl sulfoxide (DMSO). Rev Clin Basic Pharm. 1985; 5(1–2): 1–33. PubMed Abstract
4. Paul MM: Interval therapy with dimethyl sulfoxide. Ann N Y Acad Sci. 1967;
141(1): 586–98. PubMed Abstract | Publisher Full Text Page 11 of 21 Page 11 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 8. Peeker R, Haghsheno MA, Holmäng S, et al.: Intravesical bacillus Calmette-
Guerin and dimethyl sulfoxide for treatment of classic and nonulcer interstitial
cystitis: a prospective, randomized double-blind study. J Urol. 2000; 164(6):
1912–5, discussion 1915–6. the safety of topical diclofenac solution containing dimethyl sulfoxide in the
treatment of the osteoarthritic knee. Am J Ther. 2010; 17(6): 566–76. PubMed Abstract | Publisher Full Text 30. Simon LS, Grierson LM, Naseer Z, et al.: Efficacy and safety of topical diclofenac
containing dimethyl sulfoxide (DMSO) compared with those of topical placebo,
DMSO vehicle and oral diclofenac for knee osteoarthritis. Pain. 2009; 143(3):
238–45. PubMed Abstract | Publisher Full Text 31. Engel MF: Dimethyl sulfoxide in the treatment of scleroderma. South Med J. 1972; 65(1): 71–3. PubMed Abstract 10. Hucker HB, Miller JK, Hochberg A, et al.: Studies on the absorption, excretion
and metabolism of dimethylsulfoxide (DMSO) in man. J Pharmacol Exp Ther. 1967; 155(2): 309–17. PubMed Abstract 32. Ludwig CU, Stoll HR, Obrist R, et al.: Prevention of cytotoxic drug induced skin
ulcers with dimethyl sulfoxide (DMSO) and alpha-tocopherole. Eur J Cancer
Clin Oncol. 1987; 23(3): 327–9. PubMed Abstract | Publisher Full Text 11. Kligman AM: Topical Pharmacology and Toxicology of Dimethyl Sulfoxide. 1. JAMA. 1965; 193: 796–804. PubMed Abstract | Publisher Full Text 33. Mitrus I, Smagur A, Fidyk W, et al.: Reduction of DMSO concentration in
cryopreservation mixture from 10% to 7.5% and 5% has no impact on
engraftment after autologous peripheral blood stem cell transplantation:
results of a prospective, randomized study. Bone Marrow Transplant. 2018;
53(3): 274–280. PubMed Abstract | Publisher Full Text 12. Lovelock JE, Bishop MW: Prevention of freezing damage to living cells by
dimethyl sulphoxide. Nature. 1959; 183(4672): 1394–5. PubMed Abstract | Publisher Full Text 13. Zorzela L, Loke YK, Ioannidis JP, et al.: PRISMA harms checklist: improving
harms reporting in systematic reviews. BMJ. 2016; 352: i157. PubMed Abstract | Publisher Full Text ;
( )
PubMed Abstract | Publisher Full Text 47. Mueller LP, Theurich S, Christopeit M, et al.: Neurotoxicity upon infusion of
dimethylsulfoxide-cryopreserved peripheral blood stem cells in patients with
and without pre-existing cerebral disease. Eur J Haematol. 2007; 78(6): 527–31. PubMed Abstract | Publisher Full Text 26. Syme R, Bewick M, Stewart D, et al.: The role of depletion of dimethyl sulfoxide
before autografting: on hematologic recovery, side effects, and toxicity. Biol
Blood Marrow Transplant. 2004; 10(2): 135–41. PubMed Abstract | Publisher Full Text 48. Gonella S, Di Giulio P: Delayed chemotherapy-induced nausea and vomiting in
autologous hematopoietic cell transplant patients: an exploratory analysis. Tumori. 2015; 101(6): e154–9. PubMed Abstract | Publisher Full Text 27. Ozdemir E, Akgedik K, Akdogan S, et al.: The lollipop with strawberry aroma
may be promising in reduction of infusion-related nausea and vomiting during
the infusion of cryopreserved peripheral blood stem cells. Biol Blood Marrow
Transplant. 2008; 14(12): 1425–8. PubMed Abstract | Publisher Full Text ;
( )
PubMed Abstract | Publisher Full Text 44. Tseraidis GS, Popova SS, Zykova NIa: [Use of a progesterone ointment in
hirsutism]. Vestn Dermatol Venerol. 1986; (7): 14–7. PubMed Abstract 23. Castillo N, Garcia-Cadenas I, Garcia O, et al.: Few and nonsevere adverse
infusion events using an automated method for diluting and washing before
unrelated single cord blood transplantation. Biol Blood Marrow Transplant. 2015;
21(4): 682–7. PubMed Abstract | Publisher Full Text 45. Scherbel AL, McCormack LJ, Layle JK: Further observations on the effect of
dimethyl sulfoxide in patients with generalized scleroderma. (Progressive
systemic sclerosis). Ann N Y Acad Sci. 1967; 141(1): 613–29. PubMed Abstract | Publisher Full Text ( )
PubMed Abstract | Publisher Full Text 24. Pandhi R, Kaur I, Kumar B: Lack of effect of dim
amyloidosis. J Dermatolog Treat. 2002; 13(1): 1
PubMed Abstract | Publisher Full Text 24. Pandhi R, Kaur I, Kumar B: Lack of effect of dimethylsulphoxide in cutaneous
amyloidosis. J Dermatolog Treat. 2002; 13(1): 11–4. PubMed Abstract | Publisher Full Text 46. Mitrus I, Smagur A, Giebel S, et al.: A faster reconstitution of hematopoiesis
after autologous transplantation of hematopoietic cells cryopreserved in 7.5%
dimethyl sulfoxide if compared to 10% dimethyl sulfoxide containing medium. Cryobiology. 2013; 67(3): 327–31. PubMed Abstract | Publisher Full Text 25. Halle P, Tournilhac O, Knopinska-Posluszny W, et al.: Uncontrolled-rate freezing
and storage at -80 degrees C, with only 3.5-percent DMSO in cryoprotective
solution for 109 autologous peripheral blood progenitor cell transplantations. Transfusion. 2001; 41(5): 667–73. PubMed Abstract | Publisher Full Text ( )
PubMed Abstract | Publisher Full Text 22. Menne T: Nickel allergy--reliability of patch test. Evaluated in female twins. Derm Beruf Umwelt. 1981; 29(6): 156–60. PubMed Abstract Publisher Full Text 16. Vadhan-Raj S, Kavanagh JJ, Freedman RS, et al.: Safety and efficacy of
transfusions of autologous cryopreserved platelets derived from recombinant
human thrombopoietin to support chemotherapy-associated severe
thrombocytopenia: a randomised cross-over study. Lancet. 2002; 359(9324):
2145–52. P bMed Abstract | P blisher F ll Te t 37. Mendelow AY, Forsyth A, Florence AT, et al.: Patch testing for nickel allergy. The influence of the vehicle on the response rate to topical nickel sulphate. Contact Dermatitis. 1985; 13(1): 29–33. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 38. Fowler JE Jr: Prospective study of intravesical dimethyl sulfoxide in treatment
of suspected early interstitial cystitis. Urology. 1981; 18(1): 21–6. PubMed Abstract | Publisher Full Text 17. Ogden HD: Experiences with DMSO in treatment of headache. Ann N Y Acad
Sci. 1967; 141(1): 646–8. PubMed Abstract | Publisher Full Text 39. Perseghin P, Balduzzi A, Bonanomi S, et al.: Infusion-related side-effects in
children undergoing autologous hematopoietic stem cell transplantation for
acute leukemia. Bone Marrow Transplant. England; 2000; 26(1): 116–8. PubMed Abstract | Publisher Full Text 18. Martin-Henao GA, Resano PM, Villegas JM, et al.: Adverse reactions during
transfusion of thawed haematopoietic progenitor cells from apheresis are
closely related to the number of granulocyte cells in the leukapheresis
product. Vox Sang. 2010; 99(3): 267–73. PubMed Abstract | Publisher Full Text 40. Rowley SD, Feng Z, Yadock D, et al.: Post-thaw removal of DMSO does not
completely abrogate infusional toxicity or the need for pre-infusion histamine
blockade. Cytotherapy. 1999; 1(6): 439–46. PubMed Abstract | Publisher Full Text 19. Savastano AA: Clinical experiences with dimethyl sulfoxide (DMSO) in
human subjects. Approval must be withheld until safety in extended use is
established. R I Med J. 1984; 67(3): 119–21. PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 54. Davis JM, Rowley SD, Braine HG, et al.: Clinical toxicity of cryopreserved bone
marrow graft infusion. Blood. 1990; 75(3): 781–6. PubMed Abstract 76. Melikhova NI, Murav’ev IuV, Sigidin IaA, et al.: [Effectiveness of the treatment of
juvenile rheumatoid arthritis with dimethyl sulfoxide gel]. Pediatriia. 1986; (6):
53–4. PubMed Abstract 55. Roth SH, Shainhouse JZ: Efficacy and safety of a topical diclofenac solution
(pennsaid) in the treatment of primary osteoarthritis of the knee: a
randomized, double-blind, vehicle-controlled clinical trial. Arch Intern Med. 2004; 164(18): 2017–23. 77. Holbro A, Graf L, Topalidou M, et al.: Cryopreserved stem cell products
containing dimethyl sulfoxide lead to activation of the coagulation system
without any impact on engraftment. Transfusion. 2014; 54(6): 1508–14. PubMed Abstract | Publisher Full Text ( )
PubMed Abstract | Publisher Full Text 68. Bojanic I, Cepulic BG, Mazic S, et al.: Toxicity related to autologous peripheral
blood haematopoietic progenitor cell infusion is associated with number of
granulocytes in graft, gender and diagnosis of multiple myeloma. Vox Sang. 2008; 95(1): 70–5. P bM d Ab t
t | P bli h
F ll T
t 90. Galmés A, Besalduch J, Bargay J, et al.: Cryopreservation of hematopoietic
progenitor cells with 5-percent dimethyl sulfoxide at -80 degrees C without
rate-controlled freezing. Transfusion. 1996; 36(9): 794–7. PubMed Abstract | Publisher Full Text PubMed Abstract | Publisher Full Text 84. Vinnik CA, Jacob SW: Dimethylsulfoxide (DMSO) for human single-stage
intraoperative tissue expansion and circulatory enhancement. Aesthetic Plast
Surg. 1991; 15(4): 327–37. PubMed Abstract | Publisher Full Text ;
( )
PubMed Abstract | Publisher Full Text 82. Bosso JA, Spruance SL, Wenerstrom G: Tolerance and percutaneous absorption
of topically applied arildone. J Clin Pharmacol. 1985; 25(2): 95–9. PubMed Abstract | Publisher Full Text 60. Fuks JZ, Egorin MJ, Aisner J, et al.: Cyclophosphamide and dimethylsulfoxide in
the treatment of squamous carcinoma of the lung. Therapeutic efficacy, toxicity,
and pharmacokinetics. Cancer Chemother Pharmacol. 1981; 6(2): 117–20. PubMed Abstract | Publisher Full Text 83. Ozkaya-Bayazit E, Kavak A, Güngör H, et al.: Intermittent use of topical dimethyl
sulfoxide in macular and papular amyloidosis. Int J Dermatol. 1998; 37(12):
949–54. PubMed Abstract | Publisher Full Text 61. Styler MJ, Topolsky DL, Crilley PA, et al.: Transient high grade heart block
following autologous bone marrow infusion. Bone Marrow Transplant. 1992;
10(5): 435–8. PubMed Abstract | Publisher Full Text 86. Cordoba R, Arrieta R, Kerguelen A, et al.: The occurrence of adverse events
during the infusion of autologous peripheral blood stem cells is related to the
number of granulocytes in the leukapheresis product. Bone Marrow Transplant. 2007; 40(11): 1063–7. PubMed Abstract | Publisher Full Text 64. Roth SH, Fuller P: Pooled safety analysis of diclofenac sodium topical solution
1.5% (w/w) in the treatment of osteoarthritis in patients aged 75 years or older. Clin Interv Aging. 2012; 7: 127–37. PubMed Abstract | Publisher Full Text | Free Full Text ;
(
)
PubMed Abstract | Publisher Full Text 56. Galmés A, Besalduch J, Bargay J, et al.: A simplified method for
cryopreservation of hematopoietic stem cells with -80 degrees C mechanical
freezer with dimethyl sulfoxide as the sole cryoprotectant. Leuk Lymphoma. 1995; 17(1–2): 181–4. PubMed Abstract | Publisher Full Text 78. Williams HJ, Furst DE, Dahl SL, et al.: Double-blind, multicenter controlled trial
comparing topical dimethyl sulfoxide and normal saline for treatment of hand
ulcers in patients with systemic sclerosis. Arthritis Rheum. 1985; 28(3): 308–14. PubMed Abstract | Publisher Full Text ;
(
)
PubMed Abstract | Publisher Full Text 57. Bookman AA, Williams KS, Shainhouse JZ: Effect of a topical diclofenac solution
for relieving symptoms of primary osteoarthritis of the knee: a randomized
controlled trial. CMAJ. 2004; 171(4): 333–8. PubMed Abstract | Publisher Full Text | Free Full Text 79. Simpson JR: Idoxuridine in the treatment of herpes zoster. Practitioner. 1975;
215(1286): 226–9. PubMed Abstract 80. Lv X, Li Y, Jiang C, et al.: The incidence of trigeminocardiac reflex in
endovascular treatment of dural arteriovenous fistula with onyx. Interv
Neuroradiol. 2010; 16(1): 59–63. PubMed Abstract | Publisher Full Text | Free Full Text 58. Aliab’eva AP, Melikhova NI, Murav’ev IuV: [Use of heparin applications in a
dimethyl sulfoxide medium in the overall treatment of rheumatoid arthritis in
children]. Pediatriia. 1980; (9): 50–1. PubMed Abstract 81. Hoang BX, Tran DM, Tran HQ, et al.: Dimethyl sulfoxide and sodium bicarbonate
in the treatment of refractory cancer pain. J Pain Palliat Care Pharmacother. 2011; 25(1): 19–24. PubMed Abstract | Publisher Full Text 59. Shpall EJ, LeMaistre CF, Holland K, et al.: A prospective randomized trial of
buffy coat versus CD34-selected autologous bone marrow support in high-risk
breast cancer patients receiving high-dose chemotherapy. Blood. 1997; 90(11):
4313–20. PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 91. Amiridze N, Darwish R: Hemodynamic instability during treatment of
intracranial dural arteriovenous fistula and carotid cavernous fistula with
Onyx: preliminary results and anesthesia considerations. J Neurointerv Surg. 2009; 1(2): 146–50. PubMed Abstract | Publisher Full Text 69. Blumenthal LS, Fuchs M: The clinical use of dimethyl sulfoxide on various
headaches, musculoskeletal, and other general medical disorders. Ann N Y
Acad Sci. 1967; 141(1): 572–85. PubMed Abstract | Publisher Full Text p
PubMed Abstract | Publisher Full Text 66. Demos CH, Beckloff GL, Donin MN, et al.: Dimethyl sulfoxide in musculoskeletal
disorders. Ann N Y Acad Sci. 1967; 141(1): 517–23. PubMed Abstract | Publisher Full Text 88. Binnick SA, Shore SS, Corman A, et al.: Failure of dimethyl sulfoxide in the
treatment of scleroderma. Arch Dermatol. 1977; 113(10): 1398–402. PubMed Abstract | Publisher Full Text 67. Spruance SL, Stewart JC, Freeman DJ, et al.: Early application of topical 15%
idoxuridine in dimethyl sulfoxide shortens the course of herpes simplex
labialis: a multicenter placebo-controlled trial. J Infect Dis. 1990; 161(2): 191–7. PubMed Abstract | Publisher Full Text 89. Zuurmond WW, Langendijk PN, Bezemer PD, et al.: Treatment of acute reflex
sympathetic dystrophy with DMSO 50% in a fatty cream. Acta Anaesthesiol
Scand. 1996; 40(3): 364–7. PubMed Abstract | Publisher Full Text ;
(
)
PubMed Abstract | Publisher Full Text 65. Lv X, Jiang C, Li Y, et al.: Percutaneous transvenous packing of cavernous
sinus with Onyx for cavernous dural arteriovenous fistula. Eur J Radiol. 2009;
71(2): 356–62. PubMed Abstract | Publisher Full Text 87. Alessandrino P, Bernasconi P, Caldera D, et al.: Adverse events occurring during
bone marrow or peripheral blood progenitor cell infusion: analysis of 126
cases. Bone Marrow Transplant. 1999; 23(6): 533–7. PubMed Abstract | Publisher Full Text ;
( )
PubMed Abstract | Publisher Full Text 49. Cervigni M, Sommariva M, Tenaglia R, et al.: A randomized, open-label,
multicenter study of the efficacy and safety of intravesical hyaluronic acid
and chondroitin sulfate versus dimethyl sulfoxide in women with bladder pain
syndrome/interstitial cystitis. Neurourol Urodyn. 2017; 36(4): 1178–86. PubMed Abstract | Publisher Full Text 28. Silvestri DL, Corey L, Holmes KK: Ineffectiveness of topical idoxuridine in
dimethyl sulfoxide for therapy for genital herpes. JAMA. 1982; 248(8): 953–9. PubMed Abstract | Publisher Full Text 50. 50. Vuopala U, Kaipainen WJ: DMOS in the treatment of Dupuytren’s contracture. A 29. Shainhouse JZ, Grierson LM, Naseer Z: A long-term, open-label study to confirm Page 12 of 21 Page 12 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 71. Stroncek DF, Fautsch SK, Lasky LC, et al.: Adverse reactions in patients
transfused with cryopreserved marrow. Transfusion. 1991; 31(6): 521–6. PubMed Abstract | Publisher Full Text therapeutic experiment. Acta Rheumatol Scand. 1971; 17(1): 61–2. PubMed Abstract | Publisher Full Text 51. Okamoto Y, Takaue Y, Saito S, et al.: Toxicities associated with cryopreserved
and thawed peripheral blood stem cell autografts in children with active
cancer. Transfusion. 1993; 33(7): 578–81. PubMed Abstract | Publisher Full Text 72. Kaidbey KH: Therapy of resistant psoriasis with topical corticosteroids and
dimethylsulfoxide. Dermatologica. 1976; 152(5): 316–20. PubMed Abstract | Publisher Full Text 52. Zuckner J, Uddin J, Gantner GE Jr: Local application of dimethyl sulfoxide and
DMSO combined with triamcinolone acetonide in rheumatoid arthritis. Ann N Y
Acad Sci. 1967; 141(1): 555–9. PubMed Abstract | Publisher Full Text 73. Zambelli A, Poggi G, Da Prada G, et al.: Clinical toxicity of cryopreserved
circulating progenitor cells infusion. Anticancer Res. 1998; 18(6B): 4705–8. PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 74. Otrock ZK, Sempek DS, Carey S, et al.: Adverse events of cryopreserved
hematopoietic stem cell infusions in adults: a single-center observational
study. Transfusion. 2017; 57(6): 1522–6. PubMed Abstract | Publisher Full Text 53. Akkök CA, Liseth K, Nesthus I, et al.: Autologous peripheral blood progenitor
cells cryopreserved with 5 and 10 percent dimethyl sulfoxide alone give
comparable hematopoietic reconstitution after transplantation. Transfusion. 2008; 48(5): 877–83. PubMed Abstract | Publisher Full Text 75. Matsumoto J: Clinical trials of dimethyl sulfoxide in rheumatoid arthritis
patients in Japan. Ann N Y Acad Sci. 1967; 141(1): 560–8. PubMed Abstract | Publisher Full Text ( )
PubMed Abstract 62. Marshall LF, Camp PE, Bowers SA: Dimethyl sulfoxide for the treatment of
intracranial hypertension: a preliminary trial. Neurosurgery. 1984; 14(6): 659–63. PubMed Abstract | Publisher Full Text 85. Donmez A, Tombuloglu M, Gungor A, et al.: Clinical side effects during
peripheral blood progenitor cell infusion. Transfus Apher Sci. 2007; 36(1):
95–101. PubMed Abstract | Publisher Full Text 63. Engel MF: Dimethyl sulfoxide (DMSO) in clinical dermatology. South Med J. 1966; 59(11): 1318–9. PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 98. Duijvestein M, Vos AC, Roelofs H, et al.: Autologous bone marrow-derived
mesenchymal stromal cell treatment for refractory luminal Crohn’s disease:
results of a phase I study. Gut. 2010; 59(12): 1662–9. PubMed Abstract | Publisher Full Text 114. Garcia CA: Ocular toxicology of dimethyl sulfoxide and effects on retinitis
pigmentosa. Ann N Y Acad Sci. 1983; 411: 48–51. PubMed Abstract | Publisher Full Text 115. Kim DH, Jamal N, Saragosa R, et al.: Similar outcomes of cryopreserved
allogeneic peripheral stem cell transplants (PBSCT) compared to fresh
allografts. Biol Blood Marrow Transplant. 2007; 13(10): 1233–43. PubMed Abstract | Publisher Full Text 99. Marcacci G, Corazzelli G, Becchimanzi C, et al.: DMSO-associated
encephalopathy during autologous peripheral stem cell infusion: a
predisposing role of preconditioning exposure to CNS-penetrating agents? Bone Marrow Transplant. England, 2009; 44(2): 133–5. PubMed Abstract | Publisher Full Text 116. Salim AS: Role of oxygen-derived free radical scavengers in the treatment of
recurrent pain produced by chronic pancreatitis. A new approach. Arch Surg. 1991; 126(9): 1109–14. PubMed Abstract | Publisher Full Text 100. Rabinov JD, Yoo AJ, Ogilvy CS, et al.: ONYX versus n-BCA for embolization
of cranial dural arteriovenous fistulas. J Neurointerv Surg. 2013; 5(4): 306–10. PubMed Abstract | Publisher Full Text py
p
PubMed Abstract | Publisher Full Text 111. Stewart BH: Dimethyl sulfoxide (DMSO) in the treatment of troublesome
genitourinary disorders: a preliminary report. Cleve Clin Q. 1966; 33(2): 81–4. PubMed Abstract 96. Perez RS, Zuurmond WW, Bezemer PD, et al.: The treatment of complex
regional pain syndrome type I with free radical scavengers: a randomized
controlled study. Pain. 2003; 102(3): 297–307. PubMed Abstract | Publisher Full Text 112. Olver IN, Aisner J, Hament A, et al.: A prospective study of topical dimethyl
sulfoxide for treating anthracycline extravasation. J Clin Oncol. 1988; 6(11): 1732–5. PubMed Abstract | Publisher Full Text 97. Stav K, Beberashvili I, Lindner A, et al.: Predictors of response to intravesical
dimethyl-sulfoxide cocktail in patients with interstitial cystitis. Urology. 2012;
80(1): 61–5. PubMed Abstract | Publisher Full Text 113. Brown JH: Clinical experience with DMSO in acute musculoskeletal conditions
comparing a noncontrolled series with a controlled double blind study. Ann N
Y Acad Sci. 1967; 141(1): 496–505. PubMed Abstract | Publisher Full Text 123. Keung YK, Lau S, Elkayam U, et al.: Cardiac arrhythmia after infusion of
cryopreserved stem cells. Bone Marrow Transplant. 1994; 14(3): 363–7.
PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 117. Martino M, Morabito F, Messina G, et al.: Fractionated infusions of
cryopreserved stem cells may prevent DMSO-induced major cardiac
complications in graft recipients. Haematologica. 1996; 81(1): 59–61. PubMed Abstract 101. Milone G, Mercurio S, Strano A, et al.: Adverse events after infusions of
cryopreserved hematopoietic stem cells depend on non-mononuclear cells
in the infused suspension and patient age. Cytotherapy. 2007; 9(4): 348–55. PubMed Abstract | Publisher Full Text 118. Eisenberg S, Wickline M, Linenberger M, et al.: Prevention of dimethylsulfoxide-
related nausea and vomiting by prophylactic administration of ondansetron
for patients receiving autologous cryopreserved peripheral blood stem cells. Oncol Nurs Forum. 2013; 40(3): 285–92. PubMed Abstract | Publisher Full Text 102. Horacek JM, Jebavy L, Jakl M, et al.: Cardiovascular changes associated with
infusion of hematopoietic cell grafts in oncohematological patients -- impact
of cryopreservation with dimethylsulfoxide. Exp Oncol. 2009; 31(2): 121–2. PubMed Abstract 103. Murav’ev IuV: [Treatment of rheumatoid synovitis by intra-articular administration
of dimethyl sulfoxide and corticosteroids]. Ter Arkh. 1986; 58(7): 104–5. PubMed Abstract 119. Gonella S, Berchialla P, Bruno B, et al.: Are orange lollies effective in preventing
nausea and vomiting related to dimethyl sulfoxide? A multicenter randomized
trial. Support Care Cancer. 2014; 22(9): 2417–24. PubMed Abstract | Publisher Full Text 104. Akkok CA, Holte MR, Tangen JM, et al.: Hematopoietic engraftment of dimethyl
sulfoxide-depleted autologous peripheral blood progenitor cells. Transfusion. 2009; 49(2): 354–61. PubMed Abstract | Publisher Full Text 120. Kessinger A, Schmit-Pokorny K, Smith D, et al.: Cryopreservation and infusion of
autologous peripheral blood stem cells. Bone Marrow Transplant. 1990; 5 Suppl 1:
25–7. PubMed Abstract 105. Rössberger J, Fall M, Peeker R: Critical appraisal of dimethyl sulfoxide
treatment for interstitial cystitis: discomfort, side-effects and treatment
outcome. Scand J Urol Nephrol. 2005; 39(1): 73–7. PubMed Abstract | Publisher Full Text 121. Foïs E, Desmartin M, Benhamida S, et al.: Recovery, viability and clinical
toxicity of thawed and washed haematopoietic progenitor cells: analysis of
952 autologous peripheral blood stem cell transplantations. Bone Marrow
Transplant. 2007; 40(9): 831–5. PubMed Abstract | Publisher Full Text 106. Burton WJ, Gould PW, Hursthouse MW, et al.: A multicentre trial of Zostrum (5
percent idoxuridine in dimethyl sulphoxide) in herpes zoster. N Z Med J. 1981;
94(696): 384–6. PubMed Abstract ;
( )
PubMed Abstract | Publisher Full Text 70. Hoang BX, Le BT, Tran HD, et al.: Dimethyl sulfoxide-sodium bicarbonate
infusion for palliative care and pain relief in patients with metastatic prostate
cancer. J Pain Palliat Care Pharmacother. 2011; 25(4): 350–5. PubMed Abstract | Publisher Full Text 92. Vinokurov VL, Zharinov GM, Val’kovich AA, et al.: [The prevention of radiation
injuries to the rectum and bladder in cervical cancer patients]. Vopr Onkol. 1990; 36(9): 1119–20. PubMed Abstract ;
( )
PubMed Abstract Page 13 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 93. Percy EC, Carson JD: The use of DMSO in tennis elbow and rotator cuff
tendonitis: a double-blind study. Med Sci Sports Exerc. 1981; 13(4): 215–9. PubMed Abstract treating psoriasis]. Vestn Dermatol Venerol. 1989; (9): 71–2. PubMed Abstract treating psoriasis]. Vestn Dermatol Venerol. 1989; (9): 71–2. PubMed Abstract 109. Parsons JL, Shepard WL, Fosdick WM: DMSO an adjutant to physical therapy in
the chronic frozen shoulder. Ann N Y Acad Sci. 1967; 141(1): 569–71. PubMed Abstract | Publisher Full Text 94. Weigel BJ, Blaney SM, Reid JM, et al.: A phase I study of 17-
allylaminogeldanamycin in relapsed/refractory pediatric patients with solid
tumors: a Children’s Oncology Group study. Clin Cancer Res. 2007; 13(6):
1789–93. PubMed Abstract | Publisher Full Text 110. Delaney C, Milano F, Cicconi L, et al.: Infusion of a non-HLA-matched ex-vivo
expanded cord blood progenitor cell product after intensive acute myeloid
leukaemia chemotherapy: a phase 1 trial. Lancet Haematol. 2016; 3(7): e330–9. PubMed Abstract | Publisher Full Text 95. Dawber R: Idoxuridine in herpes zoster: further evaluation of intermittent
topical therapy. Br Med J. 1974; 2(5918): 526–7. PubMed Abstract | Publisher Full Text | Free Full Text Open Peer Review Current Peer Review Status: p
;
( )
PubMed Abstract | Publisher Full Text 122. Calmels B, Lemarié C, Esterni B, et al.: Occurrence and severity of adverse events
after autologous hematopoietic progenitor cell infusion are related to the amount
of granulocytes in the apheresis product. Transfusion. 2007; 47(7): 1268–75. PubMed Abstract | Publisher Full Text 107. Lockie LM, Norcross BM: A clinical study on the effects of dimethyl sulfoxide
in 103 patients with acute and chronic musculoskeletal injuries and
inflammations. Ann N Y Acad Sci. 1967; 141(1): 599–602. PubMed Abstract | Publisher Full Text 123. Keung YK, Lau S, Elkayam U, et al.: Cardiac arrhythmia after infusion of
cryopreserved stem cells. Bone Marrow Transplant. 1994; 14(3): 363–7. PubMed Abstract 108. Evstaf’ev VV: [The use of a heparin ointment in combination with dimexide in Page 14 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Open Peer Review
Current Peer Review Status:
Version 2
20 August 2019
Reviewer Report
https://doi.org/10.5256/f1000research.22126.r52126
© 2019 Morris C. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Curly Morris
Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, Belfast, UK
The manuscript is now suitable for indexing. No competing interests were disclosed. Competing Interests:
I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard. 13 August 2019
Reviewer Report
https://doi.org/10.5256/f1000research.22126.r52125
© 2019 Onakpoya I. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Igho J. Onakpoya
Centre for Evidence-based Medicine, Nuffield Department of Primary Care Health Sciences, University of
Oxford, Oxford, UK
The authors have adequately addressed my concerns. No competing interests were disclosed. Competing Interests:
Reviewer Expertise: Adverse drug reactions; systematic reviews
I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard. Page 15 of 21 https://doi.org/10.5256/f1000research.22126.r52126 © 2019 Morris C. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Version 2 20 August 2019
Reviewer Report Curly Morris 13 August 2019
Reviewer Report https://doi.org/10.5256/f1000research.22126.r52125 Igho J. Onakpoya g
p y
Centre for Evidence-based Medicine, Nuffield Department of Primary Care Health Sciences, University of
Oxford, Oxford, UK The authors have conducted a systematic review assessing reports of adverse reactions attributed to
DMSO. The topic is interesting, and the authors have conducted their searches in a reasonable way. However, there are several flaws in this manuscript that need to be addressed: The authors have conducted a systematic review assessing reports of adverse reactions attributed to
DMSO. The topic is interesting, and the authors have conducted their searches in a reasonable way. However, there are several flaws in this manuscript that need to be addressed: https://doi.org/10.5256/f1000research.22126.r52125 © 2019 Onakpoya I. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Igho J. Onakpoya
Centre for Evidence-based Medicine, Nuffield Department of Primary Care Health Sciences, University of
Oxford, Oxford, UK
The authors have adequately addressed my concerns. No competing interests were disclosed. Competing Interests:
Reviewer Expertise: Adverse drug reactions; systematic reviews
I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard. The authors have adequately addressed my concerns. Page 15 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 https://doi.org/10.5256/f1000research.18188.r45643 © 2019 Onakpoya I. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Version 1 21 March 2019
Reviewer Report https://doi.org/10.5256/f1000research.18188.r45643 Reviewer Expertise: Adverse drug reactions; systematic reviews Reviewer Expertise: Adverse drug reactions; systematic reviews I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard, however I have significant
reservations, as outlined above. , Herlev Hospital, Herlev, Denmark
bennedikte Madsen , Herlev Hospital, Herlev, Denmark
bennedikte Madsen , Herlev Hospital, Herlev, Denmark
bennedikte Madsen Dear Igho J. Onakpoya, Dear Igho J. Onakpoya,
Thank your for reviewing our manuscript “Adverse reactions of dimethyl sulfoxide in humans: a
systematic review”. Your comments were very helpful and we appreciate the effort you put in to
reviewing our manuscript. We have addressed the individual questions in the section below. We
hope your find our replies satisfactory. Questions are written in
and answers in plain. italic Q1: The term “possible adverse reactions” is incorrect. Suspected adverse reactions is more
reasonable. A1: We have changed the paragraph in the introduction section to “suspected adverse rea Q2: If Russian articles were screened by only one author, how were discrepancies resolved in
these cases? Specify which authors extracted the data, and whether this was done independently. A1: We have clarified in the manuscript how the screening process was performed
Two authors
: “
(B.K.M. and D.Z.) independently screened title and abstract according to the eligibility criteria
using www.covidence.org. Discrepancies were resolved by discussion. One author screened the
full-text articles (B.K.M.). Russian articles were screened by an author fluent in Russian (M.H.). If
M.H was in doubt regarding inclusion of a study the results were presented to B.K.M and then
discussed until a mutual decision was made. After the screening process was finished, all included
studies were imported to an Excel sheet (Microsoft Excel 2016). Data extraction was performed by
two authors (M.H. extracted from the Russian articles and B.K.M. extracted from the rest).” Q3: The term “possibly due” is incorrect. There are 4 levels in describing associations between
. medicines and suspected adverse reactions. The authors should revise their terminology
A3: We have rewritten the paragraph so it now states: “Gastrointestinal adverse reactions were
reported in 61 studies. Of these, 10 studies were randomized controlled trials.” Q4: You state “in some studies patients discontinued treatments due to halitosis”; however, you
have provided references for 5 studies – the report can be more precise
A4: We have made our report more precise and it now states: “In five studies, patients
discontinued treatment due to halitosis.” Q5: How does “including Russian studies” strengthen the review? What about several other
? languages that have been omitted
A5: A Russian Chemist, Dr. Alexander Saytzeff, identified DMSO in 1866, however it was not used
for medical use at the time . Introduction The term “possible adverse reactions” is incorrect. Suspected adverse reactions is more
reasonable
Methods If Russian articles were screened by only one author, how were discrepancies resolved in these
cases? Specify which authors extracted the data, and whether this was done independently. Results You state “in some studies patients discontinued treatments due to halitosis”; however, you have
provided references for 5 studies – the report can be more precise. Discussion You state that there seems to be a dose-response relationship, and have drawn similar
conclusions. However, at no point in the results do you report data to support this claim. You state
that studies reported associations between dose and the occurrence of adverse reactions, but fail
to report the doses in question. Please enumerate the limitations of your review. Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Are sufficient details of the methods and analysis provided to allow replication by others? Partly Is the statistical analysis and its interpretation appropriate? Partly Page 16 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard, however I have significant
reservations, as outlined above. Author Response 31 Jul 2019 , Herlev Hospital, Herlev, Denmark
bennedikte Madsen But the fact that he was Russian might have been the reason why
R
i
i
ti t h
d
t di
i
DMSO W th
f
th
ht it
ld b
1 Page 17 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Russian scientists have made numerous studies using DMSO. We therefore thought it would be
valuable to include Russian articles since many of these studies have never been translated, and
therefore are not available to the international scientific society. Of course, we could have included
many other languages, but we thought it most relevant to include Russian since we observed a
large amount of articles in Russian during our initial examination of the subject. Q6: You state that there seems to be a dose-response relationship and have drawn similar
conclusions. However, at no point in the results do you report data to support this claim. You state
that studies reported associations between dose and the occurrence of adverse reactions but fail
to report the doses in question. A6: As mentioned in our study several studies described a dose-response relationship between
the amount of DMSO and the occurrence of adverse reactions (
101
26,33,41,53,67,71,73,75,78,83,85,86,93,
101 115 A6: As mentioned in our study several studies described a dose-response relationship between
the amount of DMSO and the occurrence of adverse reactions (
101
26,33,41,53,67,71,73,75,78,83,85,86,93,
101 115 ). However, since the doses of DMSO and the route of administration differ between the
101
studies, we were not able to give an exact dose. We can only say that an association seems likely. 101,115 ). However, since the doses of DMSO and the route of administration differ between the
101
studies, we were not able to give an exact dose. We can only say that an association seems likely. 101,115 Q7: Please enumerate the limitations of your review Q7: Please enumerate the limitations of your review
A7: We have enumerated the limitations listed in the discussion in the manuscript. Q7: Please enumerate the limitations of your review
A7: We have enumerated the limitations listed in the discussion in the manuscript. y
A7: We have enumerated the limitations listed in the discussion in the manuscript. No competing interests were disclosed. Competing Interests: 05 February 2019
Reviewer Report https://doi.org/10.5256/f1000research.18188.r40223 © 2019 Morris C. This is an open access peer review report distributed under the terms of the Creative Commons
, which permits unrestricted use, distribution, and reproduction in any medium, provided the original
Attribution Licence
work is properly cited. Curly Morris Centre for Cancer Research and Cell Biology (CCRCB), Queen's University Belfast, Belfast, UK With well over 20,000 patients receiving DMSO based autologous transplants annually in Europe alone,
this is a timely review of the toxic effects of this valuable agent. It has been performed in an appropriate
and scholarly manner and brings added value by including the Russian literature not easily accessible to
the average English-speaking reader. However there are ways in which the review might be improved and give added value to th It is not easy to ascertain the number of patients receiving DMSO intravenously and those receiving it by
other routes. A small table could clarify this. It is not easy to ascertain the number of patients receiving DMSO intravenously and those receiving it by
other routes. A small table could clarify this. The side effect tables either as numbers of patients or numbers of treatments. If they cannot be presented
as one combined set of data then some explanation of the two separate tables would be beneficial. The side effect tables either as numbers of patients or numbers of treatments. If they cannot be presented
as one combined set of data then some explanation of the two separate tables would be beneficial. There seems to have been no attempt to quantify the dose of DMSO which patients have received or to
characterize the severity of the reactions and relate these. Furthermore DMSO is usually a vehicle to
facilitate giving the patient some other treatment e.g. a stem cell transplant or drug so the reasons for the Page 18 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 use of DMSO are not clear. This also means there are side effects from the drug or treatment facilitated
by the DMSO; is it possible to separate these effects in any way? Do the authors of the many papers
selected for analysis recommend an upper limit to the amount of DMSO given or have a strategy for
minimising the dose? In their final paragraph the authors suggest that "reactions due to DMSO are often mild and transient". In
their previous paragraph they admit that the case reports the less common and more severe side effects
which did not meet the eligibility criteria of this review. Curly Morris However as long ago as 2005 it was possible to
identify severe side effects in an appreciable number of cases (Windrum
, 2005 ). Furthermore
et al. although they do not separate the factors responsible the authors of reference 7 record a SAE (Grades 3,
4 and 5) profile in excess of 3%. The authors should possibly be a little more circumspect in this
paragraph particularly as they recommend the use of DMSO in (unspecified) small doses. 1 Are the rationale for, and objectives of, the Systematic Review clearly stated?
Yes Are the rationale for, and objectives of, the Systematic Review clearly stated? Yes Yes
Are sufficient details of the methods and analysis provided to allow replication by others? Yes
Is the statistical analysis and its interpretation appropriate? Not applicable
Are the conclusions drawn adequately supported by the results presented in the review? Yes
No competing interests were disclosed. Competing Interests:
Reviewer Expertise: haematology, myeloma, autologous transplantation
I confirm that I have read this submission and believe that I have an appropriate level of
expertise to confirm that it is of an acceptable scientific standard, however I have significant
reservations, as outlined above. Author Response 31 Jul 2019
, Herlev Hospital, Herlev, Denmark
bennedikte Madsen
Dear Curly Morris,
Thank for reviewing our manuscript: “Adverse reactions of dimethyl sulfoxide in humans: a
systematic review”. We appreciate the effort put into reviewing our manuscript, and we have tried
our best to use your comments to improve our manuscript. We have addressed the individual
questions in the section below. We hope your find our replies satisfactory. Questions are written in
and answers in plain. italic References 1. Windrum P, Morris TC, Drake MB, Niederwieser D, Ruutu T, EBMT Chronic Leukaemia Working Party
Complications Subcommittee: Variation in dimethyl sulfoxide use in stem cell transplantation: a survey of
EBMT centres. . 2005;
(7): 601-3
|
Bone Marrow Transplant
36
PubMed Abstract Publisher Full Text Are the conclusions drawn adequately supported by the results presented in the re
Yes Page 19 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 Q1: It is not easy to ascertain the number of patients receiving DMSO intravenously and those
receiving it by other routes. A small table could clarify this. A1: We have added a table (Table 9) to our manuscript describing the route of administration of
DMSO. Q2 & 3: There seems to have been no attempt to quantify the dose of DMSO which patients have
received or to characterize the severity of the reactions and relate these. Furthermore DMSO is
usually a vehicle to facilitate giving the patient some other treatment e.g. a stem cell transplant or
drug so the reasons for the use of DMSO are not clear. This also means there are side effects from
the drug or treatment facilitated by the DMSO; is it possible to separate these effects in any way? Do the authors of the many papers selected for analysis recommend an upper limit to the amount
? of DMSO given or have a strategy for minimising the dose In their final paragraph the authors suggest that "reactions due to DMSO are often mild and
transient". In their previous paragraph they admit that the case reports the less common and more
severe side effects which did not meet the eligibility criteria of this review. However as long ago as
2005 it was possible to identify severe side effects in an appreciable number of cases (Windrum et
al., 2005 ). Furthermore although they do not separate the factors responsible the authors of
reference 7 record a SAE (Grades 3, 4 and 5) profile in excess of 3%. The authors should possibly
be a little more circumspect in this paragraph particularly as they recommend the use of DMSO in
. (unspecified) small doses
1 A2 & 3: DMSO is most often used as a vehicle in combination with other drugs. Therefore, it is not
possible to separate completely the adverse reactions related to the use of DMSO and the adverse
reactions related to other drugs, since adverse reactions such as nausea, vomiting, headache etc. are not specific for solely DMSO. As described by the authors of reference 7 , it was difficult to isolate the effect of DMSO from side
effects related to conditioning chemotherapy. The only adverse effect that can solely be attributed
to DMSO is halitosis. No competing interests were disclosed.
Competing Interests: Are the conclusions drawn adequately supported by the results presented in the re
Yes Therefore, we could not conclude that DMSO was the cause of SAE’s in
reference 7 and have not included it in our study. Correctly, Windrum et al. describes several
adverse reactions which may be contributed to DMSO. However, the study does not describe the
seriousness of the adverse reactions. 7
1 As described in our study, it is very possible that some events are underrepresented in our study,
which is a limitation. The upper limit was not described by any studies; on the other hand several studies evaluated
different doses of DMSO and found that a lesser amount of DMSO created fewer adverse
reactions (
.). Based on this observation, we feel confident that the use of small
amounts of DMSO is recommendable, since DMSO works well as a vehicle. However, limiting the
amount would always be desirable. 61,86,120–122 No competing interests were disclosed. Competing Interests: Page 20 of 21 F1000Research 2019, 7:1746 Last updated: 23 AUG 2019 The benefits of publishing with F1000Research:
Your article is published within days, with no editorial bias
You can publish traditional articles, null/negative results, case reports, data notes and more
The peer review process is transparent and collaborative
Your article is indexed in PubMed after passing peer review
Dedicated customer support at every stage
For pre-submission enquiries, contact
research@f1000.com The benefits of publishing with F1000Research:
Your article is published within days, with no editorial bias
You can publish traditional articles, null/negative results, case reports, data notes and more
The peer review process is transparent and collaborative
Your article is indexed in PubMed after passing peer review
Dedicated customer support at every stage
For pre-submission enquiries, contact
research@f1000.com Page 21 of 21
|
https://openalex.org/W2565254008
|
http://www.journalijar.com/uploads/450_IJAR-13718.pdf
|
English
| null |
EVALUATION OF MORPHOLOGICAL AND CYTOLOGICAL VARIATIONS INDUCED BY ETHYL METHANE SULPHONATE IN CAPSICUM ANNUM.L
|
International journal of advanced research
| 2,016
|
cc-by
| 4,473
|
ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Journal Homepage: - www.journalijar.com
Article DOI: 10.21474/IJAR01/2350
DOI URL: http://dx.doi.org/10.21474/IJAR01/2350 EVALUATION OF MORPHOLOGICAL AND CYTOLOGICAL VARIATIONS INDUCED BY ETHYL
METHANE SULFONATE IN CAPSICUM ANNUUM L. Rajender Vadluri1,2*, Murali Krishna Thupurani2 , Phanikanth Jogam1 and Sadanandam Abbagani1. 1. Department of Biotechnology, Kakatiya University, Warangal Urban, Telangana. 506009- India
2. Department of Biotechnology, Chaitanya Postgraduate College (Autonomous), Kishanpura, Hanamkonda,
Warangal Urban, Telangana 506001-India ………………………………………………………………
Capsicum genus being the family of Solanaceae comprises of
approximately 20 species gained immense momentum towards the
genetic mutational growing at tropical and subtropical regions of the
World. The present research work was framed out to evaluate the
morphological and cytological variations in Chilli plant. During these
studies we have screened various parameters such as, seed
germination, plant height, number of fruits per plant, fruit length,
number of branches, number of leaves etc,. Using single concentration
of EMS (0.1%) the seeds are treated at various time periods viz., 6, 12,
24hrs. The healthy seed germination and overall morphological and
cytological variations were observed with seeds treated for 6hrs time
period. The results are compared with healthy control plant. According to our results, every parameter that we have tested
exhibited grater variations in 6 hrs treated plants compared with that
from 12 and 24 hrs and control plants. Copy Right, IJAR, 2016,. All rights reserved. Introduction Capsicum genus being the family of Solanaceae comprises of approximately 20 species gained immense momentum
towards the genetic mutational growing at tropical and subtropical regions of the World (Basu and De, 2003). Capsicum genus being the family of Solanaceae comprises of approximately 20 species gained immense momentum
towards the genetic mutational growing at tropical and subtropical regions of the World (Basu and De, 2003). Mutation breeding is a method of inducing mutations at loci control economically important traits and/or eliminates
undesirable genes from elite breeding lines (Lippert et al., 1964). These may arise spontaneously or they may be
induced using radioactive or chemical mutagen. Chemical mutagenic agents are commonly used to cause mutations
in plants. Among the chemical mutagens, EMS induces vastly higher proportion of point mutations by loss of
complete chromosome or its segment (Minocha & Arnason, 1962; Hajra, 1979). It has been reported that EMS is
highly potential to cause mutations in plants leads to produce plants with different and new characteristics such as
early flowering in spring rape, male sterility in wheat, herbicide tolerance in soybean (Thurling and Depittayanan
1992; Sebastian et al., 1989; Maan & Williams, 1984). The present investigation was under taken to induce viable mutations in capsicum annum,
morphological and cytological variations in that caused by the EMS. 2398 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Int. J. Adv. Res. 4(11), 2398-2405 Parameters studied:- In the current investigation we have studied various parameters in treated and untreated plants. Initially, all the
plants were self fertilized in first generation (M1) and were observed routinely for any sort of physical or genetical
changes in second generation in them. Morphological traits such as seed germination, chlorophyll mutants, days
taken to flowering, plant height, number of branches, number of leaves, days taken to fruiting, number of fruits. Seed germination:- Plants were cultivated on a well-prepared growth media using farmyard manure, soil and sand with a ratio of 1:1:1
respectively. Approximately, 100 seeds of each treatment as well as control were sown in four separate pots (three
for treated seeds and one for untreated seeds). These pots were irrigated well in spraying manner. Plants were
irrigated on alternate days with 5 g of the mixture of Urea and Potash in the ratio of 1:1 up to two months. To avoid
aphids, plants were sprayed twice with 0.2% solution of Monitor (insecticide) at 30 and 40 days stage. Plants were
irrigated on alternate days with 5 g of the mixture of Urea and Potash in the ratio of 1:1 up to two months. To avoid
aphids, plants were sprayed twice with 0.2% solution of Monitor (insecticide) at 30 and 40 days stage. Material and Methods:- Plant material and treatments applied:- pp
Seeds of Capsicum annuum L.of various varieties were subjected to different treatment levels with ethyl methane
sulfonate (0.1 %EMS). Treatment with EMS carried out at varied treatment time periods. Amount of 5g seeds approximately 800-1000 were soaked in distilled water for 12-16hrs at room temperature and
dried on filter paper. All seeds were uniformly treated with freshly prepared EMS solution for about 6, 12 and 24hrs. Seeds are rinsed thoroughly with distilled water, air-dried and transferred to pots for seed germination and
subsequent mutagenic studies. Statistical Analysis of Data:- lyses were performed using one-way ANOVA using NPRC software package at 0.05 and 0.01 leve Statistical analyses were performed using one-way ANOVA using NPRC software package at 0. Cytogenetical preparations for mitotic abnormal studies in M1 generation:- For the cytogenetical analysis, root meristems of chilli (2n = 24) were used. The chilli root tips about 3 cm in length
were excised, fixed in glacial acetic acid: alcohol (1:3) solution for 48hr. Then root tip squashes were made by using
iron alum, haematoxylin squash technique. Cell divisions and cytogenetical abnormalities were observed and
photographed under a Nikon image capturing system. The various types of cells with normal and abnormal
chromosomal behavior at various stages were observed and counted. Cytological preparations for meiotic abnormal chromosomal studies in selected plants:- For meiotic studies young flower buds from 30–40 Morphologically selected from M2 plants were kept in a freshly
prepared fixative solution containing glacial acetic acid and absolute alcohol (1:3) for 24 h. flower buds are washed
and then preserved in 70% alcohol at 40C. Squashing was done in 2% acetocarmine and the slides were made
permanent by dehydration in butyl alcohol series followed by mounting on Canada balsam. Pollen sterility was
assessed by staining pollen in 2% acetocarmine where, stained pollen grains with regular outline were considered as
fertile. Statistical Analysis of Data:- Seed germination:- Seed germination:- g
Germination initiation was observed from 7th day of inoculation. The healthy germination was noticed after 14 days. After passing 25-30 days the seedlings were (4-leaf stage) were shifted to new pots and as well in the field
(Department of Biotechnology Kakatiya University). Among treated seeds maximum germination percentage was observed in 6hrs of treatment seeds with 68 %. The
seeds treated with 12 and 24hrs showed low germination percentage 56 and 48 respectively. In case of control,
germination percentage was 52%. EMS is a toxic compound and its treatment has an adverse effect on seed
germination. Increase in treatment time with EMS resulted in gradual decrease in percentage germination of
seedlings. 2399 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Chlorophyll mutants:- p y
Mutagenic effectiveness reflects rate of mutation in relation to mutagenic dose, whereas, mutagenic efficiency is the
mutagenic rate in relation to leathlity or biological injury. Leathlity based on germination, increased with EMS
treatment time. Mutagenesis at 24 hrs time was found high cytotoxic on the chlorophyll mutations comparing to
other treatments. 6 hr treatment was resulted low levels of changes in chlorophyll mutations. During these studies,
we observed that the increased treatment time with EMS leads to high level of chlorophyll mutations (The results
are presented in table 1.1). Frequency and spectrum of chlorophyll mutations were observed to be time dependent of EMS treatment. In
Accordance to the results obtained (Table 1.1) the frequency of chlorophyll mutation was directly proportional to the
duration of EMS treatment (Table 1.1). ISSN: 2320-5407 ISSN: 2320-5407 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Int. J. Adv. Res. 4(11), 2398-2405 Int. J. Adv. Res. 4(11), 2398-2405 Out of these 9 tall mutants, 4 were fertile and late maturing (mutant 6P18 time taken to maturity was 130 days,
mutant 6P7 time taken to maturity was 123 days, mutant 6P39 time taken to maturity was 121 days, mutant 6P46
time taken to maturity was 118 days) while in controls time taken to maturity ranged from 92-105 days. Some of these tall mutants such as 6P7, 6P18, 12P36 showed spinach like leaves These types of mutants with
variable leaves were also reported earlier in Capsicum annuum L. cultivar Keystone Resistant Giant no 3 (Alcantara
et al., 1996). Leaf variegation is a common mutation which can be either a nuclear or cytoplasmic mutation EMS
may have a high specificity for mitochondrial and plastid genomes (Miller et al., 1984). Table 1.0 Individual mutants and the changes observed Table 1.0 Individual mutants and the changes observed Table 1.0 Individual mutants and the changes observed
6, 12 and 24 indicates the treatment time period and P indicates the term for plant and the number followed by this
indicates allotted number for plant identification. Plant
Morphological changes
6P7
Tall, late maturing, prolific with more number of leaves and six petal flower
6P18
Tall, late maturing, prolific with more number of leaves and branches, flower smaller in size. 6P39
Tall, late maturing with more number of leaves and branches, sterile flower buds. 6P46
Dwarf, fertile, more number of leaves, 7 petals. 12P2
Early maturing, more number of leaves, different shape of leaves, 7 petals. 12P17
Late maturing with more number of leaves and different branching pattern, prolific, petaloid
flower buds
12P36
Tall, more number of leaves, originally stem was divided into two branches of equal size each
was acting as main stem. 24P41
More number of leaves, more number of branches, bushy habit, prolific with 7 petal flower
24P86
Tall, more number of leaves, originally stem was divided into two branches of equal size each
was acting as main stem. Number of branches:- In the study we observed that there was more variation in the number of branches of the treated plants compared
with control plants. The maximum variation in the number of branches was observed in plants subjected to EMS
treatment for 6 hrs. The highest number of branches generated from the mutant plant 6P46. The average branches
were noticed from mutant plants 6P18, 12P17. The results shown in standard error of the mean and are presented in
table 1.1 There was more variation in the days taken to flowering of the treated plants as compare with control plants. Maximum variants were observed in plants treated with 24 hrs. (106.6 ± 0 .33) and minimum was observed in plants
treated for 6hrs 96.88 ± 0.43. In case of control group average days taken to flowering were 101.3 ±0.21. The results
are shown in table 1.1. Plant height:- g
Mutants were characterized on morphological basis. Overall 9 mutants were isolated. Four (6P7, 6P18, 6P39, 6P46)
from 6hrs EMS treated plants, Three (12P2, 12P17, 12P36) from 12hrs and Two from 24hrs (24P41, 24P86). Individual mutants and the changes observed are represented in table 1.0 and 1.1. The morphological mutants
isolated were categorized as tall, dwarf and of normal height. Among the individual mutants, 6P18 mutant was found to be highest in height with 128 cm. 6P7 noticed (Fig 1.0
and 1.1) second largest height with 121 cm. Following this 12P41 and 12P17 showed moderate height with 98 and
72 cm respectively (Fig Fig 1.0 and 1.1). During our studies, we observed that mutants 6P46 and 24P86 were
exhibited dwarf characters (Fig 1.0 and 1.1) with less height 43 and 41 cm respectively. While in control height
ranged from 65- 72 cm. Fig. 1.0 A&B- Plants (6P7,6P18) showing the maximum height (121cm,128cm) in treated with 0.1% EMS for 6
hrs. C-plant (6P46) with short height – dwarf (43cm). D- Control plant (69cm) Fig. 1.0 A&B- Plants (6P7,6P18) showing the maximum height (121cm,128cm) in treated with 0.1% EMS for 6
hrs. C-plant (6P46) with short height – dwarf (43cm). D- Control plant (69cm) Fig. 1.1 A&B- Plants showing the maximum height (72cm,98cm) in treated with 0.1% EMS for 12 & 24 hrs. C-
plant with short height – dwarf (41cm). D- Control plant (68cm) Fig. 1.1 A&B- Plants showing the maximum height (72cm,98cm) in treated with 0.1% EMS for 12 & 24 hrs. C-
plant with short height – dwarf (41cm). D- Control plant (68cm) 2400 Flower mutations:- All these mutants had flowers with 5-lobed calyx and corolla, except from mutant 6P7 where flower had 6 petals
and 24P41 and 6P46 are with 7 petals (Fig 1.2) which are compared with control plants that exhibit only 5 petals
(Fig 1.2). Our studies are correlated with work carried out by Nyla Jabeen and Bushra Mirza (2004) where they also
reported the follower mutations with reference to change in number of petals, sepals and ovules than normal in C. annuum L. 2401 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Int. J. Adv. Res. 4(11), 2398-2405 ISSN: 2320-5407 Fig. 1.2 A – flower mutant showing 7 petals, B & C are showing 6 petals in mutant flowers, D- control flower with
5 petals. Fig. 1.2 A – flower mutant showing 7 petals, B & C are showing 6 petals in mutant flowers, D- control flower with
5 petals. In other tall mutants treated with 6hrs 6P39 exhibited sterile characteristics comparing to control were no plants
were observed as sterile.. 12P17 mutant which was treated for 12 hr resulted in the modification of anthers (2-4) to
petaloid structures (male sterile plants). In other tall mutants treated with 6hrs 6P39 exhibited sterile characteristics comparing to control were no plants
were observed as sterile.. 12P17 mutant which was treated for 12 hr resulted in the modification of anthers (2-4) to
petaloid structures (male sterile plants). These types of plants were recorded in treated populations only and were not observed in control plants. This is most
probable because of mutations in any one or more than one genes involved in flowering and subsequently fruit
development. It has been reported that there are number of genes like Leafy and Ap1 involved in flowering have
been isolated from model plants like Arabidopsis and Tomato (Leandro et al., 2001). Further studies would reveal
whether these genes mutated in these lines are comparable with the reported genes or not. However such lines can
be very helpful to understand the mechanism of flowering and fruit development and have been used to study genes
involved in crop maturation (Odeigah et al., 1996). Length of the fruit:- g
In accordance to our studies we noticed that the fruit size was found high in the plants treated with 12 hrs compared
with other treatment timings (Table 1.1). Plants treated for 12 hrs were probably undergone high genetic variation
especially that enhances the pod length and size which subsequently resulted in the increase in length of fruits (Fig
1 4) Fig. 1.4 A- Variations in length of the pods, found maximum length in 12 hrs EMS treated plants. B- Showing the
max. Length of the fruit in 12 hrs, C- control fruit with medium length. Fig. 1.4 A- Variations in length of the pods, found maximum length in 12 hrs EMS treated plants. B- Showing the
max. Length of the fruit in 12 hrs, C- control fruit with medium length. Number of fruits:- Table 1.1 represents the number of fruits of all treated and untreated plants was counted. It was found that variation
was more in the number of fruits of the treated plants compared to control plants. Maximum number of fruits
observed in plants treated for 6 hrs (120.36±0.34) and minimum number of fruits found at plants treated for 24 hrs
(56.98±0.4). In case of control the average of number of fruits are 72.4±0.8 (Fig 1.3). Fig . 1.3 A- maximum no of fruits observed (mean no. of fruits 120.36±0.34) in 0.1% EMS for 6 hrs treated plants. The observed variation in the treated population was more than that in the control population. This is the expected
result because the control plants are suppose to be genetically similar and any kind of difference observed in the Fig . 1.3 A- maximum no of fruits observed (mean no. of fruits 120.36±0.34) in 0.1% EMS for 6 hrs treated plants. The observed variation in the treated population was more than that in the control population. This is the expected
result because the control plants are suppose to be genetically similar and any kind of difference observed in the 2402 Int. J. Adv. Res. 4(11), 2398-2405 ISSN: 2320-5407 control plants is only due to environment. The results of the present study suggest that particular treatment time of
the EMS below the toxic level (24hrs) can be used to increase in the genetic variability in Capsicum annuum L. control plants is only due to environment. The results of the present study suggest that particular treatment time of
the EMS below the toxic level (24hrs) can be used to increase in the genetic variability in Capsicum annuum L. Cytological preparations for mitotic abnormal chromosomal studies in selected plants ISSN: 2320-5407 ISSN: 2320-5407 Table 1.2 Effects of EMS on mitotic index, frequency of mitotic phases and percentage of abnormalities in chilli
root tip cells. Treatment
Time. (EMS
10mM)
Total cells
divided
Total
abnormal
cells
Abnormal
cells (%)
Mitotic
index
Prophase
No. %
Metaphase
No. %
Anaphase
&
Telophase
No. %
Control
507
20
3.9
11.1
81 15.9
163 32.1
263 57.8
6hrs
739
82
11.1
16.6
172 23.3
187 25.3
380 51.4
12hrs
710
85
11.9
15.6
197 27.7
199 28.0
314 44.2
24hrs
813
146
17.9
18.0
186 22.9
200 24.6
427 52.5 ects of EMS on mitotic index, frequency of mitotic phases and percentage of abnormalities in chilli Table 1.2 Effects of EMS on mitotic index, frequency of mitotic phases and percentage of abnor
root tip cells. In this study seeds of Capsicum annuum L. treated with 0.1% EMS for 6, 12 and 24hrs. In the laboratory seed
germination test, it was observed that increase in EMS treatment time had adverse effect on seed germination. Similar results have been reported earlier (Alcantara et al., 1996). In that study seeds of Capsicum annuum L. were
treated with 0.5, 1.0 and 1.5 % EMS and exposed for 3, 6 and 9hrs. In the M1 generation, seeds treated with EMS for
24 hrs had the lowest germination percentage (48%) among all treatments. Although in this report at highest
treatment time with EMS seeds had the lowest germination percentage but was still much higher than observed in
our investigation. In our study when these treated seeds were planted in the field, at maximum concentration have
longest exposure i.e, 24hrs no viable seedlings were observed. While in another report the seeds of Capsicum
annuum L. treated with 3 concentrations (0.5, 1.0 and 5%) for 3 time durations for 3, 6 and 9 hrs were planted in the
field no detectable differences in germination percentages were observed compared to the laboratory results
(Alcantara et al., 1996). These results suggest that toxic level of EMS concentration and treatment exposure depends
on the experimental conditions and on the variety used. In most of the characters, that is plant height, number of
branches, days to flowering, number of fruits, length of the fruit, number of seeds per fruit and chlorophyll
mutations the minimum variation was observed in control plants and the maximum variation were observed in the
treated plants (Table 1.2 and Fig 1.5). Number of seeds per fruit:- p
Number of seeds per fruit was found in high in all the fruits collected from the plants treated at different time
periods with 0.1% EMS. Among the treatment timings 12 and 24hrs produced less number of seeds comparing to
the 6hrs treatment. The results are shown in table 1.1. Number of seeds per fruit was found in high in all the fruits collected from the plants treated at different time
periods with 0.1% EMS. Among the treatment timings 12 and 24hrs produced less number of seeds comparing to
the 6hrs treatment. The results are shown in table 1.1. Table 1.1 Mean performance of Capsicum annuum L. in relation to different treatment times of EMS. Treatment
Time
Chlorophyll
%
Plant
Height
No. of
Branches
Day to
Flowering
Flower
mutations
No. of
fruits/plant
Length
of fruit
No of
seeds/fruits
6
2.5
130.60
±
0.43
68.24
±
0.22
98.88
±
0.43
10.09
120.36
±
0.34
7.2
±
0.20
70.52
±
0.10
12
2.9
86.76
±
0.20
53.30
±
0.34
97.77
±
0.32
12.06
110.90
±
0.12
11.8
±
0.21
64.56
±
0.22
24
4.7
104.20
±
0.13
23.33
±
0.11
106.6
±
0.03
8.09
56.98
±
0.11
6.2
±
0.15
63.02
±
0.13
Control
----
102.00
±
0.44
25.50
±
0.25
101.3
±
0.21
----
72.4
±
0.08
6.8
±
0.11
70.40
±
0.99 Table 1.1 Mean performance of Capsicum annuum L. in relation to differen Mean performance of Capsicum annuum L. in relation to different treatment times of EMS. y
g
p
p
p
The data showed a significant increase in the percentage of prophase cells 27.7 % with using 12 hrs treatment times. All the concentrations were capable of inducing various types of chromosomal abnormalities in almost all the stages
of mitosis. Sticky chromosomes, precocious movements, bridges, micronucleus, laggards and anaphase with polar
deviation were the most common abnormalities recorded with the use of EMS in metaphase and anaphase. The
percentages of chromosomal abnormalities in different mitotic stages were significantly higher than that of the
control and calculated on mitotic index, frequency of phases and percentage of abnormalities in mitosis. 2403 Int. J. Adv. Res. 4(11), 2398-2405 ISSN: 2320-5407 The observed variation in the treated population was more than that in the control population. This is the expected
result because the control plant are suppose to be genetically similar and any kind of difference observed in the
control plants is only due to environment. The results of the present study suggest that particular treatment time of
the EMS below the toxic level (24hrs) can be used to increase in the genetic variability in Capsicum annuum L. which is the basis for any breeding program. Fig. 1.5 A- Precocious stage of 6P7, B- double bridge structure in anaphase 24P41, C- anaphase of 6P39, D-
prophase in control plant, E- sticky metaphase 24P86, F- non synchronized chromosomes in anaphase of 12P36. Fig. 1.5 A- Precocious stage of 6P7, B- double bridge structure in anaphase 24P41, C- anaphase of 6P39, D-
prophase in control plant, E- sticky metaphase 24P86, F- non synchronized chromosomes in anaphase of 12P36. 2404 ISSN: 2320-5407 Int. J. Adv. Res. 4(11), 2398-2405 Conclusion:- By our results we conclude that, using 0.1% EMS concentration for 6hrs can be used for induce morphological
mutations. Several unique and interesting mutations were induced in this study. There were some mutants that were
completely sterile and cannot be used for further studies. The fertile mutants generated in this study could be
valuable for linkage and mapping studies of Capsicum annuum L. Furthermore these mutants can also be used to
isolate genes involved at different developmental stages of plants. Mutants isolated in this study as well as in many
previous studies could serve as genetic markers. This reveals that mutation breeding is a valid and effective crop
breeding method for short genome crops like Capsicum annuum L. Cytological studies revealed that the use of the use of chemical mutagens stimulated the mitotic activity in the roots
of chilli, since the mitotic index increased with the increase in the treatment time. The mitotic index value increased
up to a certain level treatment time. However, EMS treatments induced insignificant of mitotic abnormalities
compared to control roots. Acknowledgement:- We duly thankful to Department of Biotechnology, Kakatiya Univesity, Warangal. We also sincerely, thank to Dr. CH.V. Purshotham Reddy, Chairmen of Chaitanya Colleges, to carry out my research in the college premises. References:- 1. Anonymous, the British Pharmacopoeia. Published by the Stationary Office on Behalf of the Medicines and
Health Care Products. Regulatory agency (MHRA) 2009;8: 1456-1460. 1. Anonymous, the British Pharmacopoeia. Published by the Stationary Office on Behalf of the Medicines and
Health Care Products. Regulatory agency (MHRA) 2009;8: 1456-1460. g
y g
y (
)
2. Alcantara, T.P, Bosland P.W. and Smith, D.W. Ethyl methane sulfonate induced mutagenesis of Capsicum
annuum. J. Hered., 1996;87: 239–241. g
y g
y
2. Alcantara, T.P, Bosland P.W. and Smith, D.W. Ethyl methane sulfonate induced mutagenesis of Capsicum
annuum. J. Hered., 1996;87: 239–241. 3. Basu, S.K and De A.K. Capsicum: historical and botanical perspectives. In De AK (ed).The genus Capsicum. Taylor and Francis, London, 2003:1-15 3. Basu, S.K and De A.K. Capsicum: historical and botanical perspectives. In De AK (ed).The genus Capsicum. Taylor and Francis, London, 2003:1-15 4. Hajra, N.G. Induction of mutations by chemical mutagens in tall indica rice. Indian Agric., 1979 Induction of mutations by chemical mutagens in tall indica rice. Indian Agric., 1979; 23: 67–72. 4. Hajra, N.G. Induction of mutations by chemical mutagens in tall indica rice. Indian Agric., 1979; 23: 67–72. 5. Jabeen, N and Bushra, Mirza. Ethyl Methane Sulfonate Induces Morphological Mutations in Capsicum annuum. Int. J. Agri. Biol., 2004. 6; 340-345. 5. Jabeen, N and Bushra, Mirza. Ethyl Methane Sulfonate Induces Morphological Mutations in Capsicum annuum. Int. J. Agri. Biol., 2004. 6; 340-345. g
6. Leandro, P.E, Martin, TM and Jose, J.U. Constitutive expression of Arabidopsis Leafy or Apetala1 genes in
citrus reduces their generation time. Nature., 2001 :263–5 g
6. Leandro, P.E, Martin, TM and Jose, J.U. Constitutive expression of Arabidopsis Leafy or Apetala1 genes in
citrus reduces their generation time. Nature., 2001 :263–5 g
7. Lippert L.F, Bergh, B.O and Cook, A.A. Three variegated seedling mutants in the pepper. J. Hered., 1964; 55:
78–93. 7. Lippert L.F, Bergh, B.O and Cook, A.A. Three variegated seedling mutants in the pepper. J. Hered., 1964; 55:
78–93. 8. Miller, P.D, Vaughn, K.C and Wilson, K.G. Ethyl methane sulfonate induced chloroplast mutagenesis in crops. J. Hered., 1984; 75: 86–92 8. Miller, P.D, Vaughn, K.C and Wilson, K.G. Ethyl methane sulfonate induced chloroplast mutagenesis in crops. J. Hered., 1984; 75: 86–92 9. Minocha, J.L and Arnason, T.J. Mutagenic effectiveness of ethyl methane sulfonate in barley. Nature., 1962;
196: 499-499. 9. Minocha, J.L and Arnason, T.J. References:- Mutagenic effectiveness of ethyl methane sulfonate in barley. Nature., 1962;
196: 499-499. 10. Odeigah, P.G.C, Osanyinpeju A.O and Myers, G.O. Induced male sterility in cowpea (Vigna unguiculata L. Walp). J. Genet. Breed., 1996; 50: 171–176 A, Fader, G.M, Unlrich, J.F, Forney, D.R and Chaleff, R.S. Semidominant Soybean mutations for
Sulfonyl Urea herbicides. Crop Sci., 1989; 24: 851–2. 11. Sebastian, S.A, Fader, G.M, Unlrich, J.F, Forney, D.R and Chaleff, R.S. Semidominant Soybean mutations for
resistance to Sulfonyl Urea herbicides. Crop Sci., 1989; 24: 851–2. ,
,
,
,
,
,
y,
,
y
resistance to Sulfonyl Urea herbicides. Crop Sci., 1989; 24: 851–2. 12 Thurling N and Depittayanan V EMS induction of early flowering mutants in springrape (Brassica napus) Pl resistance to Sulfonyl Urea herbicides. Crop Sci., 1989; 24: 851 2. 12. Thurling, N and Depittayanan, V. EMS induction of early flowering mutants in springrape (Brassica napus). Pl. Breed., 1992; 108: 177–184. 2405
|
https://openalex.org/W2101665744
|
https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0055462&type=printable
|
English
| null |
Improved Cardiac MRI Volume Measurements in Patients with Tetralogy of Fallot by Independent End-Systolic and End-Diastolic Phase Selection
|
PloS one
| 2,013
|
cc-by
| 5,706
|
Abstract Competing Interests: The authors have declared that no competing interests exist. * E-mail: t.p.willems@umcg.nl Improved Cardiac MRI Volume Measurements in Patients
with Tetralogy of Fallot by Independent End-Systolic and
End-Diastolic Phase Selection Hendrik G. Freling1, Petronella G. Pieper2, Karin M. Vermeulen3, Jeroen M. van Swieten1, Paul E. Sijens1,
Dirk J. van Veldhuisen2, Tineke P. Willems1* 1 Department of Radiology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands, 2 Department of Cardiology, University of
Groningen and University Medical Center Groningen, Groningen, The Netherlands, 3 Department of Epidemiology, University of Groningen and University Medical Center
Groningen, Groningen, The Netherlands Abstract Objectives: To investigate to what extent cardiac MRI derived measurements of right ventricular (RV) volumes using the left
ventricular (LV) end-systolic and end-diastolic frame misrepresent RV end-systolic and end-diastolic volumes in patients with
tetralogy of Fallot (ToF) and a right bundle branch block. Methods: Sixty-five cardiac MRI scans of patients with ToF and a right bundle branch block, and 50 cardiac MRI scans of
control subjects were analyzed. RV volumes and function using the end-systolic and end-diastolic frame of the RV were
compared to using the end-systolic and end-diastolic frame of the LV. Results: Timing of the RV end-systolic frame was delayed compared to the LV end-systolic frame in 94% of patients with ToF
and in 50% of control subjects. RV end-systolic volume using the RV end-systolic instead of LV end-systolic frame was
smaller in ToF (median 23.3 ml/m2, interquartile range 21.9 to 25.6 ml/m2; p,0.001) and close to unchanged in control
subjects. Using the RV instead of LV end-systolic and end-diastolic frame hardly affected RV end-diastolic volumes in both
groups and ejection fraction in control subjects (5464%, both methods), while increasing ejection fraction from 4567% to
4867% for patients with ToF (p,0.001). QRS duration correlated positively with the changes in the RV end-systolic volume
(p,0.001) and RV ejection fraction obtained in ToF patients when using the RV instead of the LV end-systolic and end-
diastolic frame (p = 0.004). Conclusion: For clinical decision making in ToF patients RV volumes derived from cardiac MRI should be measured in the
end-systolic frame of the RV instead of the LV. Citation: Freling HG, Pieper PG, Vermeulen KM, van Swieten JM, Sijens PE, et al. (2013) Improved Cardiac MRI Volume Measurements in Patients with Tetralogy of
Fallot by Independent End-Systolic and End-Diastolic Phase Selection. PLoS ONE 8(1): e55462. doi:10.1371/journal.pone.0055462 Editor: Marc W. Merx, University Hospital Du¨sseldorf, Germany Received August 10, 2012; Accepted December 23, 2012; Published January 31, 2013 Copyright: 2013 Freling et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by the radiology department University of Groningen. decision to publish, or preparation of the manuscript. supported by the radiology department University of Groningen. The funders had no role in study design, data collection and analysis
eparation of the manuscript. January 2013 | Volume 8 | Issue 1 | e55462 Results Image analysis was performed using commercially available
software (QMass version 7.2., Medis, Leiden, The Netherlands). The end-systolic and end-diastolic frame was defined as the frame
with the smallest and largest volume, respectively.These frames
were selected by visual assessment independently for the LV and
RV. LV and RV contours were drawn manually by tracing the
endocardial borders in every slice in the end-systolic and end-
diastolic frame of the LV. Contour tracing was aided by reviewing
the multiple phase scans in the movie mode. The papillary muscle
and trabeculae were considered part of the cavum. Additionally,
RV contours were drawn in the end-systolic and end-diastolic
frame of the RV. Study population RV contours were first drawn in the visually selected end-
systolic and end-diastolic frame of the RV. To minimize
intraobserver variability, RV contours drawn in the end-systolic
and end-diastolic frame of the RV were copied to the LV end-
systolic and end-diastolic frame and then adjusted to this frame. Our institution’s CMR database was searched to collect 65 of
the most recent CMR scans of patients with ToF and 50 normal
CMR scans performed in patients suspected for myocardial
infarction (control subjects). Normal CMR scans were defined as
normal anatomy, normal LV and RV contraction, normal LV and
RV volumes and ejection fraction with no signs of infarction, and
no valvular dysfunction [15]. Ischemia was ruled out by stress
testing. In all patients electrocardiograms performed within 6
months to the CMR date were collected to evaluate rhythm and
conductance disturbances. A RBBB was present when the longest
manually measured QRS duration$100 ms in combination with a
terminal R wave in lead V1 and V2, wide S wave in I and V6 on
the electrocardiogram [16]. To obtain intra- and interobserver reproducibility, contours
were drawn independently twice by the first observer and once by
the second observer in 25 scans of patients with ToF and 25 scans
of control subjects. The end-systolic and end-diastolic frame was
selected independently twice by the first observer and once by the
second observer. There were at least two weeks between repeated
contour drawing by the first observer. Both observers had more
than 2 years experience with RV contour drawing. RBBB is defined as complete when the QRS duration$120 ms
and defined as incomplete when the QRS duration is $100 ms
and ,120 ms [16]. CMR scans of patients with ToF were
included when RBBB was the only conductance delay and no
additional conductance delays were present. CMR scans of control
subjects were excluded when conductance delays were present on
the electrocardiogram [16]. Cardiac magnetic resonance imaging All subjects were examined on a 1.5-Tesla MRI system
(Siemens Magnetom Sonata, Erlangen, Germany or Siemens
Magnetom Avanto, Erlangen, Germany) using a 266 channel
body-coil. After single-shot localizer images, for function analysis
short axis cine loop images with breath holding in expiration were
acquired using a retrospectively gated balanced steady state free
precession sequence. Short axis slices were planned in end-diastole
from two slices above the mitral valve plane to the apex. The
following parameters were used: TR 2.7 ms, TE 1.1 ms, flip angle
80u, field of view 320 mm, matrix 1926192 mm, 25 frames per
cycle, slice thickness 6 mm, interslice gap 4 mm, voxel size
1.761.766 mm. Statistical analyses Descriptive statistics were calculated for all measurements as
mean and standard deviation for normally distributed continuous
variables, median with interquartile range (IQR) for skewed
continuous variables and absolute numbers and percentages for
dichotomous variables. Reproducibility was evaluated with the
intraclass correlation coefficient (ICC). For normally distributed
continuous variables a paired-samples Student’s t-test and for
skewed continuous variables a Wilcoxon test was used to compare
RV volumes measured in the LV end-systolic and end-diastolic
frame with RV volumes measured in the RV end-systolic and end-
diastolic frame. For normally distributed continuous variables an
independent Student’s T-test and for skewed continuous variables
a Mann-Whitney test was used to compare the difference in RV
volumes between normal scans and scans of patients with ToF
when measuring RV volumes in the end-systolic and end-diastolic
frame of the RV instead of the LV. The relation between QRS
duration and change in RV volume and function when using the
end-systolic and end-diastolic frame of the RV instead of the LV
was analyzed using linear regression. The Statistical Package for
the Social Sciences version 16.0 (SPSS Inc, Chicago, IL) was used
for all statistical analyses. All statistical tests are two-sided and a P-
value of less than .05 was considered statistically significant. This retrospective study was approved by the University
Medical Center Groningen review board. Informed consent was
not required according to the Dutch Medical Research Involving
Human Subjects act. CMR Derived Volume Measurements diastolic frame for the RV and LV, on RV volume measurements
in a large group of patients with ToF and control subjects. diastolic frame for the RV and LV, on RV volume measurements
in a large group of patients with ToF and control subjects. Stroke volume was defined as end-diastolic volume minus end-
systolic volume. Ejection fraction was defined as stroke volume
divided by end-diastolic volume. Study population Between January 2008 and January 2011, 65 CMR scans of
patients with ToF (50 with complete RBBB, 15 with incomplete
RBBB) and 50 normal CMR scans of control subjects were
collected. Patients with ToF (37 male, 28 female; median age 28
years, IQR 21 to 37 years) were younger than control subjects (33
male, 17 female; median age 56 years, IQR 41 to 65 years),
p,0.001. QRS duration was longer in patients with ToF
(145625 ms) than in control subjects (9369 ms), p,0.001. Heart
rate during the CMR scan was similar in patients with ToF
(70612 bpm) and control subjects (73615 bpm), p = NS. The basal slice was selected with aid of long-axis cine view
images. The basal slice of the LV was defined as the most basal
slice surrounded for at least 50% by the LV myocardium. When
the pulmonary valve was visible in the RV basal slice, only the
portion of the right ventricular outflow tract below the level of the
pulmonary valve was included. For the inflow part of the RV, the
blood
volume
was
included
when
the
ventricle
wall
was
trabeculated and thick compared to the right atrium wall [15]. Introduction slightly behind that of the left ventricle (LV) [8]. Most patients with
ToF have a right bundle branch block (RBBB) which leads to
intra- and interventricular dyssynchrony. This dyssynchrony
significantly extends duration of RV contraction and delays timing
of RV end-systole compared to the LV [7,9,10]. Additionally,
timing of RV ejection and end-diastole may be delayed in patients
with ToF [11,12]. In many centers the RV end-systolic and end-
diastolic frame is selected independently from the LV end-systolic
and end-diastolic frame [13,14]. However, the magnitude of the
overestimation of RV end-systolic volume and underestimation of
RV end-diastolic volume and ejection fraction is unknown. Therefore, others state that independent selection of the RV
frame is unnecessary as the magnitude of the misrepresentation of
RV volumes and function is too small to be of clinical importance. Evaluation of right ventricular (RV) volumes and function is
crucial in the management of patients with congenital heart
disease [1,2]. RV dysfunction is particularly a problem in patients
with tetralogy of Fallot (ToF) due to longstanding massive
pulmonary regurgitation. Irreversible RV dysfunction can be
prevented by pulmonary valve replacement before a certain
threshold value for RV end-systolic and end-diastolic volume is
reached [3–7]. Cardiac magnetic resonance (CMR) imaging is the
golden standard in the evaluation of RV volume and function, and
plays an important role in the decision for pulmonary valve
replacement in patients with ToF and pulmonary regurgitation
[1–7]. To acquire accurate CMR derived volume measurements,
correct selection of the RV end-systolic and end-diastolic frame
may be important. In normal hearts, contraction of the RV lags The present study is the first to quantitatively document the
influence of independent selection of the end-systolic and end- January 2013 | Volume 8 | Issue 1 | e55462 1 PLOS ONE | www.plosone.org CMR Derived Volume Measurements CMR Derived Volume Measurements Change in RV volume and function g
Table 2 shows RV volumes and function measured in the end-
systolic and end-diastolic frame of the LV and RV. Using the RV
end-systolic instead of LV end-systolic frame in patients with ToF,
mean RV end-systolic volume was reduced from 78 to 74 ml/m2
(p,0.001) while ejection fraction and stroke volume increased
from 45 to 48% (p,0.001) and from 62 to 66 ml/m2, respectively
(p,0.001). ToF patient’s changes in RV end-diastolic volume and
the changes in any of these four parameters in the controls were
very small, though still significant in paired data analysis. Figure 2
shows the difference in volumes and function when using the end-
systolic and end-diastolic frame of the RV instead of the LV. The
decrease in RV end-systolic volume was incremental when going
from controls to patients with ToF and an incomplete RBBB to
patients with ToF and a complete RBBB (p,0.001). In patients
with ToF linear regression showed a significant association
between QRS duration and change in RV end-systolic volume
(B 3.37, CI 1.62–5.13, R2 = 0.190, p,0.001), and RV ejection
fraction (B 4.25, CI 1.40–7.10, R2 = 0.124, p = 0.004) when using
the end-systolic and end-diastolic frame of the RV instead of the
LV. Figure 1. Example of the left and right end-systolic frame and
the corresponding time-volume curve. Two short axis images of
the end-systolic frame of the LV (A) and RV (B), and the corresponding
time-volume curve (C) in a patient with ToF and a complete RBBB. Timing of the RV end-systolic frame is 106 ms (3 frames) delayed
compared to LV end-systolic frame. Measuring the RV end-systolic
volume in the LV instead of the RV end-systolic frame results in a
difference of 9 ml/m2. This is visible in the short-axis image of the RV
end-systolic frame (B) in which the larger blue contour corresponds to
the RV contour of the LV end-systolic frame (A) and the yellow contour
to the RV contour of the RV end-systolic frame. Timing of the end-
diastolic frame is the same for the RV and LV. LV = left ventricle,
Max. = maximum volume, Min. = minimal volume, RBBB = right bundle
branch block, RV = right ventricle. doi:10.1371/journal.pone.0055462.g001 The increase of RV ejection fraction is mainly the result of
decrease in end-systolic volume when using the end-systolic frame
of the RV instead of the LV. Reproducibility Intra- and interobserver ICC for RV end-systolic volume, end-
diastolic volume and ejection fraction was .98, .91, .87 and .98,
.97, .95 in patients with ToF and .94, .93, .88 and .95, .97 and .89
in control subjects, respectively. Table 1. End-systolic and end-diastolic frame selection of the
right ventricle compared to the left ventricle. right ventricle compared to the left ventricle. End-systole
End-diastole
ToF
Control
ToF
Control
RV–LV frame
N (%)
N (%)
N (%)
N (%)
RV 3 frames earlier
0 (0)
0 (0)
2 (3)
0 (0)
RV 2 frames earlier
0 (0)
0 (0)
2 (3)
2 (4)
RV 1 frame earlier
0 (0)
0 (0)
14 (22)
14 (28)
No difference
4 (6)
25 (50)
38 (58)
34 (64)
RV 1 frame later
26 (40)
25 (50)
8 (12)
0 (0)
RV 2 frames later
28 (43)
0 (0)
0 (0)
0 (0)
RV 3 frames later
7 (11)
0 (0)
1 (2)
0 (0)
LV = left ventricle, RV = right ventricle, ToF = tetralogy of Fallot. doi:10.1371/journal.pone.0055462.t001 Timing of end-systole and end-diastole The difference in frame selection of end-systole and end-diastole
between the RV and LV is shown in table 1. Figure 1 shows the
time-volume curve of the RV and LV of a patient with ToF and a
complete RBBB. In almost all patients with ToF and half of
control subjects the end-systolic frame of the RV was delayed
compared to the LV. The resulting median difference in timing PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e55462 2 CMR Derived Volume Measurements Figure 1. Example of the left and right end-systolic frame and
the corresponding time-volume curve. Two short axis images of
the end-systolic frame of the LV (A) and RV (B), and the corresponding
time-volume curve (C) in a patient with ToF and a complete RBBB. Timing of the RV end-systolic frame is 106 ms (3 frames) delayed
compared to LV end-systolic frame. Measuring the RV end-systolic
volume in the LV instead of the RV end-systolic frame results in a
difference of 9 ml/m2. This is visible in the short-axis image of the RV
end-systolic frame (B) in which the larger blue contour corresponds to
the RV contour of the LV end-systolic frame (A) and the yellow contour
to the RV contour of the RV end-systolic frame. Timing of the end-
diastolic frame is the same for the RV and LV. LV = left ventricle,
Max. = maximum volume, Min. = minimal volume, RBBB = right bundle
branch block, RV = right ventricle. doi:10.1371/journal.pone.0055462.g001 between the end-systolic frame of the RV and LV was larger in
patients with ToF (median 253 ms, IQR 273 to 237 ms) than in
control subjects (median 211 ms, IQR 232 to 0 ms), p,0.001. Timing of the end-diastolic frame was not different between the
RV and LV in most patients with ToF and control subjects. Also,
the resulting median difference in timing between the end-diastolic
frame of the RV and LV frame was similar in patients with ToF
(median 0 ms, IQR 0 to 36 ms) and control subjects (median 0 ms,
IQR 0 to 32 ms), p = NS. Change in RV volume and function Using the end-systolic frame of the
RV instead of the LV resulted in a relative increase in ejection
fraction of 7%, from 4567% to 4867%, in patients with ToF and
of 1%, from 5464% to 5464%, in control subjects. The relative
increase in ejection fraction and stroke volume by using the end-
diastolic frame of the RV instead of the LV was ,1% in both
patients with ToF and control subjects. [15]. When using the end-systolic frame of the RV instead of the
LV frame, RV function changed to normal in 5 (13%) of these
patients. None of the patients with an incomplete RBBB and an
abnormal RV function showed improvement to normal values. In 17 (26%) patients with ToF the absolute increase of ejection
fraction exceeded 5% (range 5–8%), figure 2C. In 39 (60%)
patients with ToF ejection fraction fell short of the limit of 47%
indicating abnormal RV function according to reference values LV = left ventricle, RV = right ventricle, ToF = tetralogy of Fallot.
doi:10.1371/journal.pone.0055462.t001 ,
g
,
doi:10.1371/journal.pone.0055462.t001 LV = left ventricle, RV = right ventricle, ToF = tetralogy of Fallot. Discussion EDV = end-diastolic volume, EF = ejection fraction, ESV = end-systolic volume,
IQR = interquartile range, LV = left ventricle, RV = right ventricle, SD = standard
deviation, SV = stroke volume, ToF = tetralogy of Fallot. doi:10.1371/journal.pone.0055462.t002 10,17,18]. The main focus of the echocardiographic studies was to
assess intra- and interventricular dyssynchrony and their predic-
tors. They showed that in patients with ToF RV free wall
contraction lags behind LV free wall and interventricular septum
contraction. Although they did not report on timing of end-systole,
it can be expected that end-systole is also delayed. We showed that
in most scans of patients with ToF, end-systole of the RV was
more than one frame delayed compared to the LV. This was
probably largely due to the longer QRS duration in patients with
ToF and a complete RBBB. In control subjects the normal
physiologically electromechanical delay of the RV could not be
detected in every case as the delay was smaller than the time (mean
3467 ms) between two frames. Therefore, the end-systolic frame
of the RV was in the same or one frame later than the end-systolic
frame of the LV. In contrast to end-systole, timing of end-diastole
of the RV and LV was similar in patients with ToF and control
subjects. 10,17,18]. The main focus of the echocardiographic studies was to
assess intra- and interventricular dyssynchrony and their predic-
tors. They showed that in patients with ToF RV free wall
contraction lags behind LV free wall and interventricular septum
contraction. Although they did not report on timing of end-systole,
it can be expected that end-systole is also delayed. We showed that
in most scans of patients with ToF, end-systole of the RV was
more than one frame delayed compared to the LV. This was
probably largely due to the longer QRS duration in patients with
ToF and a complete RBBB. In control subjects the normal
physiologically electromechanical delay of the RV could not be
detected in every case as the delay was smaller than the time (mean
3467 ms) between two frames. Therefore, the end-systolic frame
of the RV was in the same or one frame later than the end-systolic
frame of the LV. In contrast to end-systole, timing of end-diastole
of the RV and LV was similar in patients with ToF and control
subjects. Discussion One small (N = 12) cardiac MRI study indicated that measuring
RV volumes in the two frames preceding RV end-systole causes no
clinically significant volume changes despite the observation that
end-systole of RV and LV occur in different frames [19]. Our
study has made clear that in end-systole the two previous frames
have a larger volume. When there is a difference in timing of end-
systole of the RV and LV with two or more frames, as is the case in
most patients with ToF, this leads to a significant change in
volume. When there is a difference in end-systole of the RV and
LV in control subjects, this leads to a very small volume change
only. QRS duration, unfortunately not documented in the above
study [19], appears to be an important parameter as evidenced by
the statistically significant correlation with the difference in end-
systolic volume when using the end-systolic frame of the RV
instead of the LV in patients with ToF obtained in this study. The
correlation is weak, however, probably because of our inclusion of
a group of patients who are rather homogeneous in terms of QRS
duration. In contrast to end-systole, in end-diastole the adjacent
frames had a similar volume. Figure 2. Change in right ventricular volumes and function. Scatterplots of the change in RV end-systolic volume (A), end-diastolic
volume (B) and ejection fraction (C) when using the end-systolic and
end-diastolic frame of the RV instead of the LV. EDV = end-diastolic
volume, EF = ejection fraction, ESV = end-systolic volume, LV = left
ventricular, RBBB = right bundle branch block, RV = right ventricular,
ToF = tetralogy of Fallot. doi:10.1371/journal.pone.0055462.g002 several parameters including RV function [1,2]. It is important in
this context that in 13% of the patients with ToF who were
considered to have abnormal function (ejection fraction ,47%)
[15], ejection fraction increased to normal when using the end-
systolic frame of the RV instead of the LV. The change in RV
function was mainly due to the decrease in RV end-systolic
volume when using the RV frame instead of the LV frame. Studies
comparing RV volumes and function before and after pulmonary
valve replacement have identified pre-operative threshold values
for RV volumes after which volumes can return to normal [3–6]. None of these studies describe whether they selected the end-
systolic and end-diastolic frame of the RV separately from the LV. Discussion Our study is the first to quantitatively demonstrate the
difference in RV volumes and function between using the RV
and LV end-systolic and end-diastolic frame. Performing cardiac
MRI derived measurements of RV volumes using the LV end-
systolic and end-diastolic frame, misrepresent RV end-systolic and
end-diastolic volumes in many patients with ToF, especially in the
frequent case of a complete RBBB. These findings can be of
clinical importance in the evaluation of the RV in patients with
ToF. Previous echocardiographic and cardiac MRI studies reported
on the electromechanical delay of the RV compared to the LV in
patients with congenital heart disease and normal subjects [8– January 2013 | Volume 8 | Issue 1 | e55462 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e55462 3 CMR Derived Volume Measurements Table 2. Right ventricular volumes measured in the end-
systolic and end-diastolic frame of the left and right ventricle. LV frame
RV frame
RV frame–LV
frame
mean±SD
mean±SD
median (IQR)
P
ToF
RV ESV (ml/m2)
77.8624.1
73.6623.0
23.3 (25.6 to 21.9)
,001
RV EDV (ml/m2)
139.6635.0
140.0635.0
0.0 (0.0 to 0.9)
,001
RV EF (%)
44.867.4
48.066.9
2.8 (1.8 to 4.6)
,001
RV SV (ml/m2)
61.8616.4
66.4616.7
4.1 (2.6 to 5.8)
,001
Control subjects
RV ESV (ml/m2)
35.167.6
34.967.6
0.0 (20.4 to 0.0)
003
RV EDV (ml/m2)
75.2612.4
75.4612.3
0.0 (0.0 to 0.1)
002
RV EF (%)
53.664.1
54.064.0
0.2 (0.0 to 0.7)
,001
RV SV (ml/m2)
40.166.1
40.566.1
0.2 (0.0 to 0.6)
,001
EDV = end-diastolic volume, EF = ejection fraction, ESV = end-systolic volume,
IQR = interquartile range, LV = left ventricle, RV = right ventricle, SD = standard
deviation, SV = stroke volume, ToF = tetralogy of Fallot. doi:10.1371/journal.pone.0055462.t002 Figure 2. Change in right ventricular volumes and function. Scatterplots of the change in RV end-systolic volume (A), end-diastolic
volume (B) and ejection fraction (C) when using the end-systolic and
end-diastolic frame of the RV instead of the LV. EDV = end-diastolic
volume, EF = ejection fraction, ESV = end-systolic volume, LV = left
ventricular, RBBB = right bundle branch block, RV = right ventricular,
ToF = tetralogy of Fallot. doi:10.1371/journal.pone.0055462.g002 Table 2. Right ventricular volumes measured in the end-
systolic and end-diastolic frame of the left and right ventricle. References patients with surgically repaired tetralogy of Fallot by two-dimensional speckle
tracking. Eur J Echocardiogr 11: 786–792. 1. Baumgartner H, Bonhoeffer P, De Groot NM, de Haan F, Deanfield JE, et al. (2010) ESC guidelines for the management of grown-up congenital heart disease
(new version 2010). Eur Heart J 31(23): 2915–2957. patients with surgically repaired tetralogy of Fallot by two-dimensional speckle
tracking. Eur J Echocardiogr 11: 786–792. 11. Cullen S, Shore D, Redington A (1995) Characterization of right ventricular
diastolic performance after complete repair of tetralogy of Fallot. restrictive
physiology predicts slow postoperative recovery. Circulation 91: 1782–1789. 2. Warnes CA, Williams RG, Bashore TM, Child JS, Connolly HM, et al. (2008)
ACC/AHA 2008 guidelines for the management of adults with congenital heart
disease: A report of the american college of Cardiology/American heart
association task force on practice guidelines (writing committee to develop
guidelines on the management of adults with congenital heart disease). Circulation 118: e714–833. 12. van den Berg J, Wielopolski PA, Meijboom FJ, Witsenburg M, Bogers AJ, et al. (2007) Diastolic function in repaired tetralogy of fallot at rest and during stress:
Assessment with MR imaging. Radiology 243: 212–219. 13. Maceira AM, Prasad SK, Khan M, Pennell DJ (2006) Reference right
ventricular systolic and diastolic function normalized to age, gender and body
surface area from steady-state free precession cardiovascular magnetic
resonance. Eur Heart J 27: 2879–2888. 3. Therrien J, Provost Y, Merchant N, Williams W, Colman J, et al. (2005)
Optimal timing for pulmonary valve replacement in adults after tetralogy of
Fallot repair. Am J Cardiol 95: 779–782. p
J
4. Buechel ER, Dave HH, Kellenberger CJ, Dodge-Khatami A, Pretre R, et al. (2005) Remodelling of the right ventricle after early pulmonary valve
replacement in children with repaired tetralogy of Fallot: Assessment by
cardiovascular magnetic resonance. Eur Heart J 26: 2721–2727. 14. Clarke CJ, Gurka MJ, Norton PT, Kramer CM, Hoyer AW (2012) Assessment
of the accuracy and reproducibility of RV volume measurements by CMR in
congenital heart disease. JACC Cardiovasc Imaging 5: 28–37. 15. Alfakih K, Plein S, Thiele H, Jones T, Ridgway JP, et al. (2003) Normal human
left and right ventricular dimensions for MRI as assessed by turbo gradient echo
and steady-state free precession imaging sequences. J Magn Reson Imaging 17:
323–329. 5. Oosterhof T, van Straten A, Vliegen HW, Meijboom FJ, van Dijk AP, et al. Conclusions Identifying tricuspid valve opening and closing in a 4-chamber
or RV 2-chamber view may allow for more accurate selection of
the end-systolic frame. However, in the 4-chamber view the
opening and closing of the tricuspid valve was not always clearly
visible and RV 2-chamber views were not acquired. Independent selection of the end-systolic and end-diastolic LV
and RV frame instead of using the LV end-systolic and end-
diastolic frame for RV determinations, results in more accurate
end-systolic RV volumes in patients with ToF and a RBBB. The
differences are significant and correlate with QRS duration. For
clinical decision making in patients with ToF and a RBBB, RV
volumes should be measured in the end-systolic frame of the RV
instead of the LV. There are possible confounders for the difference in timing of
end-systole between the studied groups, such as the difference in
age, pulmonary stenosis and regurgitation, RV end-systolic and
end-diastolic volume and underlying disease. However, it is
unlikely that the difference in age will have influenced our results
as age does not affect timing of contraction of the right and left
ventricle [8]. Possibly, a stronger correlation would have been
found between QRS duration and the difference in ejection
fraction and end-systolic volume when using the end-systolic and
end-diastolic frame of the RV instead of the LV when also patients Author Contributions Conceived and designed the experiments: HGF PGP TPW. Performed the
experiments: HGF KMV JMvS. Analyzed the data: HGF PGP KMV PES
DJvV TPW. Contributed reagents/materials/analysis tools: HGF PGP
JMvS TPW. Wrote the paper: HGF PGP KMV JMvS PES DJvV TPW. CMR Derived Volume Measurements CMR Derived Volume Measurements RV volume does not return to normal after PVR varies between
approximately 80 and 90 ml/m2 [3–6]. When using a threshold
for RV end-systolic volume of .85 ml/m2 [3], in our study 25
(39%) patients had volumes above this threshold when measuring
RV volumes in the end-systolic frame of the LV. When using the
end-systolic frame of the RV instead of the LV, the end-systolic
volume dropped below this threshold in 7 (28%) patients. In some
of these cases CMR measurements of RV volumes and function
may prove to be decisive when considering reoperation. There-
fore, RV volumes should be measured in end-systolic of the RV
and not of the LV. RV volume does not return to normal after PVR varies between
approximately 80 and 90 ml/m2 [3–6]. When using a threshold
for RV end-systolic volume of .85 ml/m2 [3], in our study 25
(39%) patients had volumes above this threshold when measuring
RV volumes in the end-systolic frame of the LV. When using the
end-systolic frame of the RV instead of the LV, the end-systolic
volume dropped below this threshold in 7 (28%) patients. In some
of these cases CMR measurements of RV volumes and function
may prove to be decisive when considering reoperation. There-
fore, RV volumes should be measured in end-systolic of the RV
and not of the LV. with ToF and normal QRS duration had been included in this
study. To investigate the influence of QRS duration and RBBB
more thoroughly, an additional group of patients with ToF and no
conduction delays would be useful. However, these patients are
rare and in our institution there are only three CMR scans of these
patients available over the last three years [1,20]. Although we have shown that end-systolic volume of the RV in
patients with ToF should be measured in the end-systolic frame of
the RV instead of the LV, it is uncertain whether this applies to all
patients with congenital heart diseases involving the RV and a
RBBB. Discussion The reported threshold for RV end-systolic volume above which several parameters including RV function [1,2]. It is important in
this context that in 13% of the patients with ToF who were
considered to have abnormal function (ejection fraction ,47%)
[15], ejection fraction increased to normal when using the end-
systolic frame of the RV instead of the LV. The change in RV
function was mainly due to the decrease in RV end-systolic
volume when using the RV frame instead of the LV frame. Studies
comparing RV volumes and function before and after pulmonary
valve replacement have identified pre-operative threshold values
for RV volumes after which volumes can return to normal [3–6]. None of these studies describe whether they selected the end-
systolic and end-diastolic frame of the RV separately from the LV. The reported threshold for RV end-systolic volume above which According to the guidelines of the ESC and ACC/AHA,
indication for replacement of the pulmonary valve in patients with
ToF and moderate/severe pulmonary regurgitation is based on January 2013 | Volume 8 | Issue 1 | e55462 PLOS ONE | www.plosone.org January 2013 | Volume 8 | Issue 1 | e55462 4 References (2007) Preoperative thresholds for pulmonary valve replacement in patients with
corrected tetralogy of Fallot using cardiovascular magnetic resonance. Circulation 116: 545–551. 16. Mirvis DM, Goldberger AL (2011) Electrocardiography. In: Bonow RO, Mann
DL, Zipes DP, Libby P, Braunwald E, editors. Braunwald’s Heart Disease:
Saunders Elsevier. 126–167. 6. Frigiola A, Tsang V, Bull C, Coats L, Khambadkone S, et al. (2008)
Biventricular response after pulmonary valve replacement for right ventricular
outflow tract dysfunction: Is age a predictor of outcome? Circulation 118: S182–
90. 17. Frigiola A, Redington AN, Cullen S, Vogel M (2004) Pulmonary regurgitation is
an important determinant of right ventricular contractile dysfunction in patients
with surgically repaired tetralogy of Fallot. Circulation 110: II153–157. 7. Geva T (2011) Repaired tetralogy of fallot: The roles of cardiovascular magnetic
resonance in evaluating pathophysiology and for pulmonary valve replacement
decision support. J Cardiovasc Magn Reson 13: 9. 18. Sun AM, AlHabshan F, Cheung M, Bronzetti G, Redington AN, et al. (2011)
Delayed onset of tricuspid valve flow in repaired tetralogy of Fallot: An
additional mechanism of diastolic dysfunction and interventricular dyssyn-
chrony. J Cardiovasc Magn Reson 13: 43. 8. Yu CM, Lin H, Ho PC, Yang H (2003) Assessment of left and right ventricular
systolic and diastolic synchronicity in normal subjects by tissue doppler
echocardiography and the effects of age and heart rate. Echocardiography 20:
19–27. 19. Edwards R, Shurman A, Sahn DJ, Jerosch-Herold M, Kilner PJ, et al. (2009)
Determination of right ventricular end systole by cardiovascular magnetic
resonance imaging: A standard method of selection. Int J Cardiovasc Imaging
25: 791–796. 9. D’Andrea A, Caso P, Sarubbi B, D’Alto M, Giovanna Russo M, et al. (2004)
Right ventricular myocardial activation delay in adult patients with right bundle
branch block late after repair of tetralogy of Fallot. Eur J Echocardiogr 5: 123–
131. 20. Gelband H, Waldo AL, Kaiser GA, Bowman FO, Malm JR, et al. (1971)
Etiology of right bundle-branch block in patients undergoing total correction of
tetralogy of Fallot. Circulation 44: 1022–1033. 10. Mueller M, Rentzsch A, Hoetzer K, Raedle-Hurst T, Boettler P, et al. (2010)
Assessment of interventricular and right-intraventricular dyssynchrony in January 2013 | Volume 8 | Issue 1 | e55462 PLOS ONE | www.plosone.org 5
|
https://openalex.org/W3087902378
|
https://zenodo.org/record/4432079/files/5Growth_AI-driven%205G.pdf
|
English
| null |
5Growth: AI-driven 5G for Automation in Vertical Industries
| null | 2,020
|
cc-by
| 5,483
|
II. BASELINE PLATFORM The 5Growth architecture, depicted in Fig. 1, builds onto
the 5G-TRANSFORMER [1] one, enhancing its usability,
flexibility, automation, performance and security. It enables
automated deployment and uniform operation of slices, cus-
tomized to support the requirements of the vertical industries
in the project, spanning from Industry 4.0 to Transportation
and Energy. The architecture is composed of three core
building blocks: 5Growth Vertical Slicer (5Gr-VS), 5Growth
Service Orchestrator (5Gr-SO) and 5Growth Resource Layer
(5Gr-RL), in addition to monitoring and decision automation
components supporting the former blocks. In addition to performance and radio-related enhancements,
5G has also integrated mechanisms from other technological
domains such as cloud computing, machine learning (ML)
and service-based architectures. Such integration, targets ad-
dressing the highly heterogeneous requirements posed by the
vertical industries. 5G system integration becomes paramount
to operators, manufacturers and service providers, second
only to the need for validation and experimentation along-
side the verticals themselves. Projects such as H2020 5G-
TRANSFORMER (http://5g-transformer.eu/) have provided
decisive first steps towards that direction, exploiting tech-
nologies such as Network Function Virtualization (NFV),
Software-Defined Networking (SDN) and advances in service
orchestration, to partition the network in slices addressing dif-
ferent communication needs from disparate vertical industries. Despite this important initial step, there is also the need to
assess the capability of such technologies to meet not only
key performance targets directly at verticals’ premises, but
also to support automation and optimisation of end-to-end
connectivity solutions. This is the objective of the 5Growth
project which, besides deploying such capabilities in advanced
field trials alongside verticals, adds extensions and innova-
tive capabilities to 5G platforms. In this paper, we present
design considerations and preliminary results for a key set of
such innovations and associated extensions, including network
monitoring and analytics, slicing at the radio access network,
machine-learning-based resource allocation and user profiling
supporting smart orchestration. 5Growth: AI-driven 5G for
Automation in Vertical Industries Chrysa Papagianni∗, Josep Mangues-Bafalluy†, Pedro Bermudez‡, Sokratis Barmpounakis§,
Danny De Vleeschauwer∗, Juan Brenes¶, Engin Zeydan†, Claudio Casetti∥, Carlos Guimar˜aes∗∗,
Pablo Murillo‡, Andres Garcia-Saavedra††, Daniel Corujo‡‡, Teresa Pepe
x
∗Nokia Bell Labs, †Centre Tecnol`ogic de Telecomunicacions de Catalunya, ‡Telcaria,
§National and Kapodistrian University of Athens, ¶Nextworks, ∥Politecnico di Torino, ∗∗Universidad Carlos III de Madrid,
††NEC Laboratories Europe, ‡‡Instituto de Telecomunicac¸˜oes e Universidade de Aveiro,
xEricsson Research Fig. 1: 5Growth Baseline Architecture Abstract—Spurred by a growing demand for higher-quality
mobile services in vertical industries, 5G is integrating a rich
set of technologies, traditionally alien to the telco ecosystem,
such as machine learning or cloud computing. Despite the initial
steps taken in prior research projects in Europe and beyond,
additional innovations are needed to support vertical use cases. This is the objective of the 5Growth project: automate vertical
support through (i) a portal connecting verticals to 5G platforms
(a.k.a. vertical slicer), a multi-domain service orchestrator and
a resource management layer, (ii) closed-loop machine-learning-
based Service Level Agreement (SLA) control, and (iii) end-to-
end optimization. In this paper, we introduce a set of key 5Growth
innovations supporting radio slicing, enhanced monitoring and
analytics and integration of machine learning. Fig. 1: 5Growth Baseline Architecture A. Radio Access Network support in Vertical Slicer A typical vertical service (VS) is transversal to the opera-
tor’s network since it needs to connect the end-users with the
service logic arbitrarily placed at any place of the network. For
this reason, end-to-end network slices supporting VSs typically
span from the RAN to the core. These two segments have
different characteristics and the way to model and allocate
resources is radically different. On the one side, the core seg-
ment of the network slice is usually deployed using a number
of NSs that define the Network Functions and the internal
connection among them. On the other side, a specific set of
network resources are needed at the access segment of the slice
that are tightly coupled to the mobile traffic profile of the VS. The decomposition of network slices into network services
used in the core segment has already been addressed in the 5G-
TRANSFORMER project, but the modeling and configuration
of the access segment were out of the scope of the project. In 5Growth we propose extensions to enable network slices
encompassing core and access networks to support vertical
services with end-to-end QoS and SLA guarantees. • Life-cycle management (including on-boarding, instanti-
ation, update, scaling, termination, etc.). In addition, the 5Gr-SO offers to the 5Gr-VS an integrated
view of the services, which may be running in the local or in
peer administrative domains. The 5Gr-SO receives the service requirements from the 5Gr-
VS via its northbound interface in the form of Network Service
Descriptors (NSD), expressing a NFV-NS as chains of Virtual
Network Function (VNF) components and their individual
requirements. Additional components (e.g., monitoring jobs,
scaling rules) may be included in the request. Internally,
the 5Gr-SO decides (i) the optimal service (de)composition
for the whole NFV-NS based on service availability as well
as the capabilities exposed by the local and remote peering
domains, (ii) the optimal placement of VNFs and vertical
applications (VAs) along with the optimal deployment of
virtual links connecting VNFs, through mapping operations
over the topology exposed by the local 5Gr-RL. The 5Gr-SO
is responsible for requesting network services from federated
5Gr-SOs. The 5Gr-SO works on an abstract view of the
infrastructure provided by the 5Growth Resource Layer, where
the complexity of the transport and radio mobile networks is
lightened by exposing logical links connecting the data-centers
resources dedicated for vertical applications. C. 5Growth Resource Layer (5Gr-RL) related components and underlying resource management is
handled by the lower layers of the 5Growth stack. The final
specification of the vertical service, provided by the vertical,
is formally expressed through a Vertical Service Descriptor
(VSD), which is composed of the VSB annotated with user-
defined parameters. The 5Growth Resource Layer (5Gr-RL), which is inherited
from 5G-TRANSFORMER Mobile Transport and computing
Platform (5GT-MTP), manages all the complexity of the
transport, mobile, storage and compute resources, providing,
besides a suitable abstraction, also the configuration of such
resources. Moreover, the 5Gr-RL decouples the transport,
mobile and data center resources to assure that each of them
could be owned and managed by different business actors. Such decoupling allows a single 5Gr-RL to integrate several
VIMs and WIMs from different technological domains and
exposes a unified view to the upper layers. Afterwards, the 5Gr-VS handles the requests for vertical
services by internally managing the mapping and translation
between the requested vertical services (Vertical Service In-
stances - VSIs) and a number of network slices (Network Slice
Instances – NSIs). The slices are created on-demand by the
5Growth Service Orchestrator that provisions the underlying
NFV network services (NFV-NSs). B. 5Growth Service Orchestrator (5Gr-SO) The main architectural innovations of 5Growth focus on
two main issues, namely RAN support (including the inter-
face exposed to the vertical industries and implications for
other architectural blocks) and the addition of intelligence to
support decision making, including the required monitoring
framework. The 5Gr-SO, inherited from the 5G-TRANSFORMER Ser-
vice Orchestrator, provides both network service and resource
orchestration capabilities to support: • End-to-end
orchestration
of
NFV-Network
Services
(NFV-NS), by mapping them across a single or multiple
administrative domains based on service requirements
and availability of the services/resources offered by each
of the domains; A. 5Growth Vertical Slicer (5Gr-VS) A. 5Growth Vertical Slicer (5Gr-VS) The 5Gr-VS, extending the 5G-TRANSFORMER Verti-
cal Slicer, acts as a one-stop-shop entry point for verticals
requesting the provisioning and management of services,
through a simplified and vertical-oriented northbound interface
(NBI) with the vertical operations/business support system
(OSS/BSS). Through this interface, verical service requests can be
submitted, by initially selecting a “template” from the catalog
of Vertical Service Blueprints (VSBs) to be used as the basis
for service definition. Then, verticals can complete service
specification, by providing a number of service-oriented pa-
rameters that customize the desired service instance. The goal
is to enable the verticals to focus on the requirements, the high-
level components and the logic of their service applications
and their inter-relation. The actual deployment of all network- C. 5Growth Resource Layer (5Gr-RL) A. Radio Access Network support in Vertical Slicer Additionally,
as already mentioned, the 5Gr-SO performs the life-cycle
management of the whole NFV-NS, including nested NSs and
VNFs composing the NFV-NS. Finally, it performs monitoring
tasks and SLA management, to enable the triggering of
self-adaptation actions (e.g., healing and scaling operations),
thereby preventing service performance degradation or SLA
violations. The cornerstone for all the required extensions is the inclu-
sion of mobile traffic profiles and access network information
as part of the Network Slice Template information model. This
approach is fully aligned with the 3GPP approach established
in [2]. The idea is to include the parameters that characterize
the specific service type (i.e. eMBB, URLLC, MTC) and some
common parameters such as the coverage area, the required
latency, etc. In this model, network slices are also composed
by a set of Network Slice Subnets which are in turn composed
by Network Services. This core and access combined network
slicing approach renders the demarcation border between core
and access segment functionalities more flexible, and even
allows to move the traditional 5G-Core network functions
towards the access for on-premises deployment. Fig. 2: 5Growth AI/ML workflow may need a composite of neural networks to approximate the
relationship between service and resource requirements [5] or
to forecast demands [6]. The basic workflow for both classifi-
cation/inference and reinforcement learning is the following: 0. The 5Gr-AIMLP exposes a catalog of models that can be
tuned and chained to compose more complex models. 1. The agent describes the model by selecting (a composite
of) preset models, their parameters for the problem at
hand, as well as information on how to maintain the
model and what monitoring probes are required. g p
q
2. The 5Gr-AIMLP requests 5Gr-VOMS orchestration of
monitoring probes. Fig. 2: 5Growth AI/ML workflow Fig. 2: 5Growth AI/ML workflow 3. In the case of reinforcement learning, the agent requests
5Gr-VOMS contextual information (e.g., the current num-
ber of users) and uses it as an input of the trained model. In turn, the 5Gr-AIMLP uses such contextual information
for the optimization of the model parameters. In order to profit from this enhanced network slice tem-
plates, the 5G-TRANSFORMER’s VSB and VSD information
models have also been extended. B. AI/ML Platform (5Gr-AIMLP) 3GPP has acknowledged the significance of data analytics
for future cellular systems. In particular, a new function, called
NetWork Data Analytics Function (NWDAF) has been intro-
duced in [3]. NWDAF is responsible for providing network
analysis information upon request from network functions,
e.g., assisting the Policy Control Function (PCF) in selecting
traffic steering policies. 5Growth is generalizing this idea by
extending 5G-TRANSFORMER to integrate NWDAF con-
cepts and provide an AI/ML platform for smarter control [4]. 3GPP has acknowledged the significance of data analytics
for future cellular systems. In particular, a new function, called
NetWork Data Analytics Function (NWDAF) has been intro-
duced in [3]. NWDAF is responsible for providing network
analysis information upon request from network functions,
e.g., assisting the Policy Control Function (PCF) in selecting
traffic steering policies. 5Growth is generalizing this idea by
extending 5G-TRANSFORMER to integrate NWDAF con-
cepts and provide an AI/ML platform for smarter control [4]. A workflow of the platform is depicted in Fig. 2, with
two main functional blocks assisting the 5Growth platform
(Fig. 1): the 5Gr-AIMLP—assisting in functions common to
many AI/ML schemes, such as neural network fitting—and the
5Growth Vertical-Oriented Monitoring System (5Gr-VOMS,
see subsection §III-C). This figure explains how the typical
data engineering pipeline layers have been mapped to the
5Growth architecture and provides some examples of tools
for each layer. Each decision-making entity (agent, hereafter)
in the 5Growth management platform is ultimately the one
single entity that executes the model. For instance, 5Gr-SO Architecture: Fig. 3 describes the overall 5Gr-VoMS which in-
cludes four building blocks. The Virtual Machine (VM), where
the Monitoring Agent is installed, the Kafka Message Queues
(MQ), and the Monitoring Platform itself which includes most
of the components related to the monitoring. A workflow of the platform is depicted in Fig. 2, with
two main functional blocks assisting the 5Growth platform
(Fig. 1): the 5Gr-AIMLP—assisting in functions common to
many AI/ML schemes, such as neural network fitting—and the
5Growth Vertical-Oriented Monitoring System (5Gr-VOMS,
see subsection §III-C). This figure explains how the typical
data engineering pipeline layers have been mapped to the
5Growth architecture and provides some examples of tools
for each layer. Each decision-making entity (agent, hereafter)
in the 5Growth management platform is ultimately the one
single entity that executes the model. C. Vertical-oriented Monitoring System (5Gr-VoMS) The Vertical-oriented Monitoring System (5Gr-VoMS) is
an extension of 5G-TRANSFORMER monitoring platform
(5GT-MP), designed with the objective of supporting an het-
erogeneous set of services and technological domains; and,
likewise, novel innovations devoted to enhancing end-to-end
reliability (via self-healing and auto-scaling), vertical control-
loops stability, and analytical features, such as forecasting and
anomaly detection. To this end, 5GT-MP must be extended to
include additional functionalities such as log aggregation, a
scalable data distribution system and dynamic probe reconfig-
uration [7]. Elastic stack is included in the 5Gr-VoMS archi-
tecture to support log aggregation, Kafka distributed streaming
platform as scalable data distribution system and Elastic Beats
which will, together with Prometheus node exporter, assist to
the dynamic reconfiguration of the monitoring probes. A. Radio Access Network support in Vertical Slicer On the one hand, the new
VSB shall include a parameter to establish the desired service
type and some high-level parameters to determine the default
range that shall be guaranteed by the network slice, e.g.,
service area dimension. On the other hand, the VSD shall also
be extended to allow to override the default values established
for each specific service type parameter such as, e.g., the
expected data rate. 5Gr-VS will translate these new VSBs
and VSDs into a network slice containing the access segment
resources and the network services required to support the
vertical service. From an architectural perspective, 5Gr-VS can
rely on the interface between 5GT-VS and the 5GT-SO for
life-cycle management of the network services, but extensions
are required in order to configure the radio access resources,
using the information available from the network slice. With
this regard, the interface between the 5Gr-RL and the 5Gr-SO
will be an evolution of 5G-TRANSFORMER’s 5GT-SO-MTP
interface since the 5Gr-RL should now expose an abstraction
of the RAN infrastructure and provide RAN control primitives. 4. When the conditions for collecting data samples are met,
the 5Gr-AIMLP requests and feeds the data into its fitting
function to optimize the model parameters. 5. The optimized model (i.e., its parameters) is passed
down to the agent for online execution by exploiting
performance metrics coming from 5Gr-VOMS. In the case of reinforcement learning, the agent is also re-
sponsible for integrating on-policy (e.g., SARSA) or off-policy
(e.g., Q-learning) training methods. Two specific examples
leveraging on 5Gr-AIMPL are introduced in section §IV. In the case of reinforcement learning, the agent is also re-
sponsible for integrating on-policy (e.g., SARSA) or off-policy
(e.g., Q-learning) training methods. Two specific examples
leveraging on 5Gr-AIMPL are introduced in section §IV. IV. SMART ORCHESTRATION AND CONTROL Deployment of the requested NFV-NS across a single or
multiple federated domains, is a two-step process in 5Growth. Each step bases its decisions on a different abstract view of
the underlying infrastructure. Preliminary Results: This subsection describes a set of experi-
ments that have been performed with the purpose of validating
5Gr-VoMS innovations. Specifically, they target the evaluation
of the scalability of the main component introduced to the
architecture, the Kafka MQ. The experiments are performed
instantiating the different components of the 5G-VoMS in a
Docker container. Furthermore, an external VM containing
the Berserker tool is connected to the VoMS through the
Kafka message queue. This tool allows generating monitoring
information messages at variable rates, which in this case is
used to emulate monitoring probes. The hardware equipment
is provided with 8 CPU cores, 8GB RAM and 100GB of disk. The Kafka Java VM heap memory is 4GB. 1) The 5Gr-SO, upon receiving the request from the 5Gr-VS,
decides upon the optimal NFV-NS decomposition based on
service availability as well as resource capabilities exposed at
the local and other administrative domains. Towards that end,
the 5Gr-SO builds up an abstract view (i.e. annotated topology)
of the federated infrastructure, by exchanging abstract views
(e.g., abstract topologies, computing and storage capabilities)
with other domains and consolidating them with the local view
exposed by the resource layer. The process amounts to NFV-
NS decomposition, as essentially different segments of the
initial NFV-NS graph are mapped to different domains. Fig. 4 shows the number of events received from Kafka’s
MQ by Logstash, when the Berseker tool is configured to
generate monitoring load at a rate of 102 and 105 messages/s,
respectively. The graphs are obtained using the Kibana visu-
alization tool from the VoMS. In Fig. 4a, it can be observed
that the Kafka component is able to maintain the rate of 100
messages processed per second. On the other hand, in Fig. 4b it can be appreciated that, when the number of messages
generated is 105 messages/s , the maximum number of mes- 2) The 5Gr-SOs of the selected domains within the federation
receive the aforementioned service segments from the 5Gr-SO
initiating the orchestration process, along with the parameters
needed to interconnect the segments of the composite end-
to-end NFV-NS. B. AI/ML Platform (5Gr-AIMLP) For instance, 5Gr-SO 5Gr-VoMS allows using two types of time series database
(TSDB) which are built specifically to handle metrics and
events or measurements with time stamps. It is up to the
verticals to choose which one to use, Prometheus or Elastic
Search stack. Graphana and Kibana are visualization tools that
allow the display and formatting of metric data obtained with
ElasticSearch (for Kibana) and Prometheus (for Graphana). Fig. 3: 5Growth VoMS Architecture (a) Load equal to 102 messages/s
(b) Load equal to 105 messages/s
Fig. 4: Number of Kafka MQ events as received by Logstash. Fig. 5: Latency associated to each Logstash event. (a) Load equal to 102 messages/s (b) Load equal to 105 messages/s (a) Load equal to 102 messages/s (a) Load equal to 102 messages/s (b) Load equal to 105 messages/s (b) Load equal to 105 messages/s Fig. 4: Number of Kafka MQ events as received by Logstash. Fig. 4: Number of Kafka MQ events as received by Logstash. Fig. 4: Number of Kafka MQ events as received by Logstash. Fig. 4: Number of Kafka MQ events as received by Logstash. Fig. 5: Latency associated to each Logstash event. Fig. 3: 5Growth VoMS Architecture The Monitoring Agent is responsible for collection, initial
analysis and subsequent delivery of the metrics and logs in
5Gr-RL, both network and computing resources, which could
be virtual or physical. There are several types of probes such as
Prometheus exporters, Beats monitoring probes, etc. In Fig. 3,
the Monitoring Agent collects the metrics and log data and
pushes them to the Kafka MQ. On the other side, Elastic
Search is reading the MQ using Logstash. The Prometheus MQ
agent acts as an intermediary between Kafka and Prometheus. Logs and metrics are extracted and placed in the TSDB once
they appear in the MQ. Fig. 5: Latency associated to each Logstash event. Fig. 5: Latency associated to each Logstash event. sages that Kafka is able to process oscillates around 60000
messages/s. This result demonstrates the high scalability of
the Kafka message queue, given that this scenario would be
equivalent to a scenario where 60000 probes are publishing
monitoring information at a pace of one message per second. sages that Kafka is able to process oscillates around 60000
messages/s. B. AI/ML Platform (5Gr-AIMLP) This result demonstrates the high scalability of
the Kafka message queue, given that this scenario would be
equivalent to a scenario where 60000 probes are publishing
monitoring information at a pace of one message per second. MQs are used as an interface for information exchange
between different technologies and components of the archi-
tecture. In this way, internal and external components (e.g.,
federated domains, etc.) can read/publish information in a
common way, avoiding the definition, creation, and implemen-
tation of new APIs. If needed, creating new MQs and add them
to the stack is straightforward and does not increase the com-
plexity of the architecture. Fig. 3 shows the case of VMs. The
Config Manager configures the Monitoring Agent. Finally, for
integration purposes, Prometheus and Elastic Search have an
API to provide information to other modules such as Anomaly
Detection, Forecasting and Inference or Alert Manager. Furthermore, Fig. 5 shows the latency of each event pro-
cessed by Logstash when the load is equal to 105 messages/s,
which is approximately constant at around 0.18 ms. From
this result, it can be concluded that even though the Kafka
MQ has reached its performance saturation point, the rest
of components of the architecture that process the events
generated by Kafka (in this case, Logstash), do not experiment
performance degradation, validating the platform’s scalability. A. VN-FG Embedding Preliminary Results: We compare the efficiency of the rein-
forcement learning approach, denoted as ML, to the bench-
mark baseline MILP [11] using simulations. The MILP uses
as traffic envelope the maximum inbound traffic demand
per service chain. We compare them on the basis of (i)
the VNF-FG request rejection ratio defined as the ratio of
rejected requests divided by the total number of requests,
and (ii) the resource violation ratio defined as the ratio of
the monitoring instances at which any of the resources is
violated to the total number of monitoring instances (thus
implicitly considering SLA violations). We use an event-based
simulator implemented in Java, including an SFC and DC
topology generator. The ND4J (see https://deeplearning4j.org/)
library has been adopted for tensor operations support. We use
CPLEX (branch-and-cut) for our MILP models. The VNF-FG embedding problem is often formulated as
a Mixed Integer Linear Program (MILP), tailored to the
specific objective that is pursued e.g., [9][10]. The solution
determines the placement of the VNF-FG nodes on the servers
and the mapping of the directed VNF-FG edges on substrate
paths. Since the problem is NP-hard [8], sub-optimal (meta)
heuristics and approximation algorithms have been devised to
make it computationally tractable, considering that mapping
needs to be addressed in real-time (“online problem”). Most approaches dealing with the online problem make
decisions based on a snapshot of the residual capacities in
the NFVI observed at request time, and it is usually assumed
that these capacities are known with high precision, while the
(future) evolution of the workloads for in-service (or expiring)
VNF-FGs over time is not considered. The former assumption
is unrealistic given the coarse granularity of the monitoring
information in time, e.g., to keep the corresponding network
overhead low. Moreover, making embedding decisions based
only on a snapshot of the remaining resources at request
time is not optimal over time, as it leads to fragmentation
of the physical resources. What is more, the maximum (or
average) of resources that a VNF-FG may require over its
lifetime is considered, which leads either to over-provisioning
of resources (hence under-utilizing the physical infrastructure
and rejecting incoming requests) or SLA violations. Indicatively, two simulation scenarios are evaluated. For the
first simulation scenario, we compare the efficiency of the
reinforcement learning approach. Fig. 6a depicts the evolution
of the two metrics for the different approaches. The ML-
based approach converges after approximately 1500 requests. IV. SMART ORCHESTRATION AND CONTROL For each service segment the corresponding
5Gr-SO is responsible for the placement of its constituent
VNFs at the set of interconnected PoPs within the managed
domain and in-sequence routing through them as prescribed
by the service chain segment. To facilitate this process, each 5Gr-SO retrieves from the local 5Gr-RL a uniform abstraction
of the resources (compute, storage, transport, mobile radio
resources) in the managed domain, at a different level of
abstraction compared to the initial NFV-NS decomposition. (a) Scenario 1
(b) Scenario 2
Fig. 6: Resource violation - request rejection. (b) Scenario 2 (a) Scenario 1 The resulting mappings of the virtual resources to the PoP
level topology are seamlessly pushed from the 5G-RL to
the corresponding controllers, responsible for addressing the
resource allocation problem known as VNF-Forwarding Graph
(VNF-FG) embedding [8]. In the following, we will present
our initial attempt to address the corresponding problem using
ML in the context of 5Growth. Towards intelligent resource
allocation, a Dynamic Profiling Mechanism will be used to
extract resource demands for the underlying network com-
ponents. The resource demands will be eventually used as
input for the 5Growth network optimization solutions (i.e.,
pertaining to resource allocation and scheduling). (b) Scenario 2 Fig. 6: Resource violation - request rejection. embed the VNF-FG in the NFVI. All constraints imposed
by the problem at hand (related to capacity, QoS, etc.) are
translated into rewards (made up of bonuses and penalties);
by rewarding actions that accept the requested VNF-FG and
do not violate constraints while penalizing the ones that do,
the ML-based algorithm gradually learns the best policy. REFERENCES [1] A. De la Oliva et al., “5G-TRANSFORMER: Slicing and orchestrating
transport networks for industry verticals,” IEEE Communications Mag-
azine, vol. 56, no. 8, pp. 78–84, 2018. 3) Application of a predefined set of rules in conjunc-
tion with Decision Tree Learning Algorithm 5Gr-AIMLP
models in order to extract the necessary profiles; and [2] 3GPP, “Technical Specification Group Services and System Aspects;
Management and orchestration; 5G Network Resource Model (NRM);
Stage 2 and stage 3 (Release 16),” 3rd Generation Partnership Project
(3GPP), TS 28.541, September 2019. 4) Extraction of profiles and forwarding towards the respec-
tive agents for RAN resource allocation, VNF autoscaling
and placement schemes in 5Gr-SO and 5Gr-RL. p
[3] ——, “Technical Specification Group Services and System Aspects;
System Architecture for the 5G System (Release 16),” 3rd Generation
Partnership Project (3GPP), TS 23.501, March 2019. The input data comprises diverse datasets collected from
different parts of the network and relating to user data, device
information, service levels and network resources. p
j
[4] D. M. Gutierrez-Estevez et al., “Artificial intelligence for elastic manage-
ment and orchestration of 5g networks,” IEEE Wireless Communications,
vol. 26, no. 5, pp. 134–141, 2019. We next present an initial evaluation on a RAN resource
allocation approach, where we focus on a single eMBB slice
for simplicity. Our approach extracts a first set of profiles
based on the past behavior of UEs in terms of network
service type they consume. The evaluation is done with NS-
3 network simulator. In this initial simplified evaluation, five
different UE profiles were used, consuming different network
services with different UL/DL data rate requirements. Overall,
eight scenarios were executed involving 40 UEs with different
profile distribution probabilities. The Profiling Mechanism
classified the UEs into service profiles correctly, which allows
us to proactively allocate the respective resources. [5] J. A. Ayala-Romero et al., “vrAIn: A Deep Learning Approach Tailoring
Computing and Radio Resources in Virtualized RANs,” in Proc. of ACM
MobiCom 2019. [6] J. X. Salvat et al., “Overbooking network slices through yield-driven
end-to-end orchestration,” in Proc. of ACM CoNEXT 2018. [7] Y. Wei, M. Li, and B. Xu, “Research on establish an efficient log analysis
system with kafka and elastic search,” Journal of Software Engineering
and Applications, vol. 10, no. 11, pp. 843–853, 2017. [8] J. G. Herrera and J. F. Botero, “Resource allocation in NFV: A
comprehensive survey,” IEEE Transactions on Network and Service
Management, vol. A. VN-FG Embedding (b) Downlink p
Fig. 7: Predicted vs. actual resource consumption during the UEs
activity in both uplink and downlink channels. Fig. 7: Predicted vs. actual resource consumption during the UEs
activity in both uplink and downlink channels. B. Dynamic Profiling Mechanism This paper introduced some of the innovations proposed by
the H2020 5Growth project. Specifically, we have presented
initial work and results regarding (i) architectural innovations
to apply novel AI/ML schemes into management operations,
(ii) vertical control over radio resources, (iii) enhanced
monitoring service, and (iv) automated service orchestration
mechanisms. These initial results (among others that will be
integrated in the future) make evident how 5G paves the
way to innovative use cases in vertical industries and novel
service management procedures. The project is currently on its
first year, with initial pilots under design, involving verticals
alongside the development of the identified innovations, with
first field trials projected to the end of 2020. Dynamic Profiling Mechanism (DPM) builds upon the 5Gr-
AIMLP introduced in subsection §III-B to extract network
behavior- and service usage-based UE profiles. The DPM,
which extends the functionality of the Context Extraction and
Profiling Engine (CEPE) [12], extracts a set of UE profiles,
based on past behavior in terms of UE capabilities, mobility
patterns and resource requirements and forwards them to the
resource allocation and smart orchestration layers of the NFV
Resource Orchestrator (NFV-RO) of 5Gr-SO and the 5Gr-RL. The goal of DPM is to extract UE profiles based on
UE- (user and device), network-, service- and slice-oriented
contextual information, following a step-by-step methodology. 1) Data Management/Collection: Collection of data from
multiple sources based on 5Gr-AIMLP requests to 5Gr-
VOMS, cleaning, filtering, and correlation of data; ACKNOWLEDGMENTS This work has been partially supported by EC H2020
5GPPP 5Growth project (Grant 856709). This work has been partially supported by EC H2020
5GPPP 5Growth project (Grant 856709). This work has been partially supported by EC H2020
5GPPP 5Growth project (Grant 856709). 2) Application of Divisive Hierarchical Clustering models
and fine-tuning inside the 5Gr-AIMLP platform, in order
to construct classes with similar observations; A. VN-FG Embedding In steady state there are still fluctuations due to the stochastic
nature of the requests and the exploration capability of the RL
approach. MILP has no violations by design but exhibits the
highest rejection ratio as it takes into account the maximum
inbound traffic demand per service chain. The ML-based
approach manages to keep the resource violation ratio low,
without considering capacity constraints for the embedding
problem and having limited information on the infrastructure
resources, as opposed to the MILP that is provided with the
remaining compute and transport capacity in full precision. In such an environment with uncertainty in resource de-
mands and provisioning, reinforcement learning is suited to
tackle the VNF-FG embedding problem. The reinforcement
learning based approach gradually steers the decision-making
process in the right direction based on feedback it gets on how
good the embedding decisions were. Concretely, at the end of
each episode (of e.g., 500 requests), the 5Gr-AIMLP platform
will be called upon to adapt the policy based on the (state,
action, reward) triplets that were observed over that episode. The approach can be used to address the online problem, sup-
porting decision-making in real (polynomial) time. Each time
a VNF-FG arrives at the 5Gr-RL, the reinforcement learning
agent decides if (admission control) and how (mapping) to For the second scenario, we study the ability of the re-
inforcement learning-based approach to adapt fast to chang-
ing conditions such as a surge in workload/traffic demands. To assess this aspect, we increase the requested workload
halfway through the simulation; for the 5000 remaining VNF-
FG requests the corresponding inbound traffic is increased
approximately by 30%. Fig. 6b shows that the proposed ML-
based approach adapts to this new situation by rejecting more (a) Uplink
(b) Downlink
Fig. 7: Predicted vs. actual resource consumption during the UEs
activity in both uplink and downlink channels. requests keeping the violation ratio more or less constant
because the rewards were set such that violations are expensive
and rejections are rather cheap. Convergence to the new
“steady state” is fast. The MILP approach is not able to cope
with these changing conditions: it has much more violations
while it was designed to avoid those in the first place. After
the load increase, the resource violations in the MILP case
could only be avoided by resetting the traffic envelope for the
incoming requests at the second half of the simulation. REFERENCES 13, no. 3, pp. 518–532, 2016. [9] D. Dietrich et al., “Network function placement on virtualized cellular
cores,” in Proc. of COMSNETS 2017, pp. 259–266. [10] N. Torkzaban, C. Papagianni, and J. S. Baras, “Trust-aware service chain
embedding,” in 2019 Sixth International Conference on Software Defined
Systems (SDS), June 2019, pp. 242–247. The results shown in Fig. 7 compare the predicted and actual
resources that were finally used during the UEs’ activity in the
uplink and the downlink. Although the prediction accuracy
in the UL case is clearly higher, in both cases the predicted
resources were equal or more than the ones finally used. [11] C. Papagianni, P. Papadimitriou, and J. S. Baras, “Rethinking Service
Chain Embedding for Cellular Network Slicing,” in 2018 IFIP Network-
ing Conference (IFIP Networking) and Workshops, May 2018, pp. 1–9. [12] P. Magdalinos et al., “A context extraction and profiling engine for 5G
network resource mapping,” Computer Communications, vol. 109, pp. 184–201, sep 2017.
|
W3025200532.txt
|
https://www.mdpi.com/2073-8994/12/5/817/pdf?version=1589536200
|
en
|
SU(2) × SU(2) Algebras and the Lorentz Group O(3,3)
|
Symmetry
| 2,020
|
cc-by
| 5,363
|
SS symmetry
Article
SU(2) × SU(2) Algebras and the Lorentz Group O(3,3)
Martin Walker
Independent Researcher, 3958 Grandis Place, Victoria, BC V8N 4H6, Canada; martinpwalker4691@gmail.com
Received: 24 April 2020; Accepted: 7 May 2020; Published: 15 May 2020
Abstract: The Lie algebra of the Lorentz group O(3,3) admits two types of SU(2) × SU(2) subalgebras:
a standard form based on spatial rotation generators and a second form based on temporal rotation
generators. The units of measurement for the conserved quantity due to invariance under temporal
rotations are investigated and found to be the same units of measure as the Planck constant.
The breaking of time reversal symmetry is considered and found to affect the chiral properties of a
temporal SU(2) × SU(2) algebra. Finally, the symmetry between algebras is explored and pairs of
algebras are found to be related by SU(2) × U(1) symmetry, while a group of three algebras are related
by SO(4) symmetry.
Keywords: Lie algebra; O(3,3); time rotation; Dirac; Noether
1. Introduction
Spinors were first introduced by Elli Cartan in 1913. The ideas were later adopted into quantum
mechanics to describe the intrinsic spin of a fermion and play a fundamental role in Dirac’s equation [1].
In group theory, spinors transform under the spin 12 representation of an SU(2) × SU(2) Lie algebra,
which is also the Lie algebra of the proper Lorentz group O(3,1) [2].
This article investigates some aspects of symmetry in the Lorentz group O(3,3). This Lie group can
be associated with a six-dimensional mathematical space containing three space dimensions and three
time dimensions [3]. The corresponding Lie algebra is SO(3,3) in which the symmetry of time and
the symmetry of space are isomorphic. As a result, there are two types of SU(2) × SU(2) subalgebras:
one containing spatial rotation generators and one containing temporal rotation generators.
To better understand the temporal SU(2) × SU(2) algebras, we investigate the units of measure for
the conserved quantity due to invariance under temporal rotations, for a restricted definition of action,
in an O(3,3) space. Using Noether’s theorem, it is found that the conserved quantity has the same units
of measure as the Planck constant.
We also consider the effects of breaking time reversal symmetry. For a temporal SU(2) × SU(2)
algebra, the two chiralities are related by a time reversal transformation. This suggests that breaking
time reversal symmetry affects the chiral properties of a temporal SU(2) × SU(2) algebra.
Finally, we explore symmetries between different algebras in SO(3,3). We find pairs of algebras
related by SU(2) × U(1) symmetry, as well as a group of three algebras related by SO(4) symmetry.
In Section 2, two types of SU(2) × SU(2) algebras are described. In Section 3, we investigate the
units of measure for the conserved quantity due to invariance under temporal rotations. In Section 4,
we consider the implications of breaking time reversal symmetry. In Section 5, the symmetry between
algebras is explored.
Symmetry 2020, 12, 817; doi:10.3390/sym12050817
www.mdpi.com/journal/symmetry
Symmetry 2020, 12, 817
2 of 10
2. SU(2) × SU(2) Subalgebras
One form of SU(2) × SU(2) Lie algebra is related to the proper Lorentz group O(3,1). This Lie
group can be associated with transformations in a four-dimensional space containing three space
dimensions and one time dimension [4]. It has six generators [2],
J1 , J2 , J3 , K1 , K2 , K3
(1)
where the J’s are spatial rotation generators and the K’s are boosts. The commutation relations for this
algebra are,
[Jj , Jk ] = i jkm Jm
[Kj , Kk ] = −i jkm Jm
[Jj , Kk ] = i jkm Km
(2)
where is the Levi-Civita symbol, i is the imaginary unit and the indexes j, k, m can assume any value
from 1 to 3. Using a complexification and a change of basis the Lie algebra becomes a direct product of
two SU(2) algebras [5],
1
1
1
1
1
1
(J + iK1 ), (J2 + iK2 ), (J3 + iK3 ), (J1 − iK1 ), (J2 − iK2 ), (J3 − iK3 )
2 1
2
2
2
2
2
(3)
with commutation relations
[ 12 (Jj + iKj ), 12 (Jk + iKk )] = i jkm 21 (Jm + iKm )
[ 12 (Jj - iKj ), 12 (Jk − iKk )] = i jkm 12 (Jm − iKm )
[ 21 (Jj + iKj ), 12 (Jk − iKk )] = 0
(4)
where the indexes j, k, m = 1, 2, 3. This SU(2) × SU(2) algebra is associated with the description of
spin angular momentum in quantum mechanics [2,5]. Please note that in the text that follows, an
SU(2) × SU(2) algebra will often be written in a format like
1
1
1
{ (J1 ± iK1 ), (J2 ± iK2 ), (J3 ± iK3 )}
2
2
2
(5)
where the curly brackets are delimiters for a list of generators.
This article investigates SU(2) × SU(2) algebras in the context of the Lorentz group O(3,3). This Lie
group can be associated with transformations in a six-dimensional space containing three space
dimensions and three time dimensions [3,4]. Another label for this group is the special orthogonal
Lie group SO(3,3), which has fifteen generators [3,6,7]. The group has three space rotation generators,
here labelled Ji (i = 1, 2, 3), it has three time rotation generators, labelled Ti (i = 1, 2, 3), and it has nine
boost generators, labelled Kij , where the i index denotes the time dimension (i = 1, 2, 3) and the j index
denotes the space dimension (j = 1, 2, 3) (see Appendix A for a matrix representation of the generators).
The commutation relations in this notation are,
[Tj , Tk ] = i jkm Tm
[Kjn , Kkn ] = −i jkm Tm
[Tj , Kkn ] = i jkm Kmn
[Jj , Jk ] = i jkm Jm
[Tj , Jk ] = 0
[Knj , Knk ] = −i jkm Jm
[Jj , Knk ] = i jkm Knm
(6)
where the indexes j, k, m, n = 1, 2, 3
The complexification of the Lie algebra of SO(3,3) used in this article is one in which all the boost
generators are multiplied by the imaginary unit, while the rotation generators are left unchanged.
This is the same complexification commonly used on the Lie algebra of the Lorentz group O(3,1) [5].
This results in the following commutation relations,
[Tj , Tk ] = i jkm Tm
[iKjn , iKkn ] = i jkm Tm
[Tj , iKkn ] = i jkm iKmn
where the indexes j, k, m, n = 1,2,3.
[Jj , Jk ] = i jkm Jm
[Tj , Jk ] = 0
[iKnj , iKnk ] = i jkm Jm
[Jj , iKnk ] = i jkm iKnm
(7)
Symmetry 2020, 12, 817
3 of 10
Complexified SO(3,3) has three complexified SO(3,1) subspaces which give rise to three
SU(2) × SU(2) subalgebras containing spatial rotation generators:
e1 = { 12 (J1 ± iK11 ), 12 (J2 ± iK12 ), 21 (J3 ± iK13 )}
e2 = { 12 (J1 ± iK21 ), 12 (J2 ± iK22 ), 12 (J3 ± iK23 )}
e3 = { 12 (J1 ± iK31 ), 12 (J2 ± iK32 ), 21 (J3 ± iK33 )}.
(8)
These have the standard form [2], and we are encouraged to think of them as a family, as they
differ only by the value of the time index in the boost generators.
Complexified SO(3,3) also has three complexified SO(1,3) subspaces which give rise to a family of
SU(2) × SU(2) subalgebras containing temporal rotation generators:
m1 = { 12 (T1 ± iK11 ), 21 (T2 ± iK21 ), 12 (T3 ± iK31 )}
m2 = { 12 (T1 ± iK12 ), 12 (T2 ± iK22 ), 12 (T3 ± iK32 )}
m3 = { 12 (T1 ± iK13 ), 12 (T2 ± iK23 ), 12 (T3 ± iK33 )}.
(9)
These algebras differ only by the value of the space index in the boost generators.
3. Invariance under Temporal Rotations
We would like to determine the units of measurement for the conserved quantity due to invariance
under temporal rotations. The field theory treatment of Noether’s theorem that follows is adopted
from Schwichtenberg [5] and applied to O(3,3) space. We use the Einstein summation convention in
this section.
For O(3,3) space, a 6-vector is defined as having the form,
xµ = (x1 , x2 , x3 , x4 , x5 , x6 )
(10)
where the first three components are space dimensions and the last three components are time
dimensions. In the following investigation we will restrict ourselves to the action, S4 , with respect to
the time variable x4 . We define,
Z
Z
S4 =
dx4 L4 L4 =
d5 xL4 L4 = L4 Ψ xµ , ∂µ Ψ xµ , xµ
(11)
where Ψ xµ is a scalar field, L4 is the Lagrangian, and the Lagrangian density, L4 , is a density over an
element (δx1 , δx2 , δx3 , δx5 , δx6 ). The equations of motion for this Lagrangian density are then given by
the Euler-Lagrange equations:
∂L4
∂L4
−
∂µ
= 0.
(12)
∂Ψ
∂∂ Ψ
µ
3.1. Infinitesimal Space-Time Translations for a Scalar Field
For an infinitesimal space-time translation we have,
xµ → x0µ = xµ + δxµ = xµ + aµ
(13)
where aµ is an arbitrary infinitesimal change. If the transformation does not change the Lagrangian
density we get,
∂L
∂Ψ
ν
− δµ Laµ = 0
δL = −∂ν
(14)
∂(∂ν Ψ) ∂xµ
where δ is the Kronecker delta. If aµ is arbitrary then we must have,
Symmetry 2020, 12, 817
4 of 10
∂ν Tµν
= 0 where
Tµν
∂L
=
∂ ( ∂ν Ψ )
∂Ψ
ν
− δµ L
∂xµ
(15)
which gives us one continuity equation for each component µ. The elements Tµν are said to define
components of the energy-momentum tensor.
For L4 , there are six continuity equations given by
∂1 T11 + ∂2 T12 + ∂3 T13 + ∂4 T14 + ∂5 T15 + ∂6 T16 = 0
∂1 T21 + ∂2 T22 + ∂3 T23 + ∂4 T24 + ∂5 T25 + ∂6 T26 = 0
∂1 T31 + ∂2 T32 + ∂3 T33 + ∂4 T34 + ∂5 T35 + ∂6 T36 = 0
∂1 T41 + ∂2 T42 + ∂3 T43 + ∂4 T44 + ∂5 T45 + ∂6 T46 = 0
(16)
∂1 T51 + ∂2 T52 + ∂3 T53 + ∂4 T54 + ∂5 T55 + ∂6 T56 = 0
∂1 T61 + ∂2 T62 + ∂3 T63 + ∂4 T64 + ∂5 T65 + ∂6 T66 = 0
Taking into consideration the fourth equation, we can rearrange it and integrate both sides over
an infinite volume,
∂1 T41 + ∂2 T42 + ∂3 T43 + ∂4 T44 + ∂5 T45 + ∂6 T46 = 0
−∂4
R
−∂4 T44 = ∂1 T41 + ∂2 T42 + ∂3 T43 + ∂5 T45 + ∂6 T46
R
5 xT 4 =
5 x ∂ T1 + ∂ T2 + ∂ T3 + ∂ T5 + ∂ T6
d
d
2
3
5
6
1
4
4
4
4
4
4
V
V
R
R
−∂4 V d5 xT44 = V d5 x∇T
R
H
−∂4 V d5 xT44 = δV d4 xT
(17)
where ∇T = ∂1 T41 + ∂2 T42 + ∂3 T43 + ∂5 T45 + ∂6 T46 , δV is the boundary of volume V and we have used
the divergence theorem in the last step. The surface integral on the right hand side of this equation
vanishes because the field vanishes at infinity and we are left with,
Z
∂4
V
d5 xT44 = 0
(18)
R
which implies that d5 xT44 is conserved.
Using a similar method with the other equations gives us six conserved quantities. We know
already that the conserved quantities for invariance under time and space translations in O(3,1) are
energy and momentum, respectively. We make the following assignments for the conserved quantities,
Z
and
Z
d5 xT44
E1 =
Z
P1 =
Z
d5 xT54
E2 =
E3 =
Z
5
d
xT14
P2 =
d5 xT64
(19)
d5 xT34
(20)
Z
5
d
xT24
P3 =
where E1 , E2 , E3 are energies and P1 , P2 , P3 are momentums.
3.2. Infinitesimal Space-Time Rotations for a Scalar Field
For an infinitesimal space-time rotation we have,
xµ → x0µ = xµ + δxµ = xµ + Mσµ xσ
(21)
where the Mσµ are generators of rotations. Setting the change in the Lagrangian density to zero results in,
Symmetry 2020, 12, 817
5 of 10
∂L
δL = −∂ν
∂ ( ∂ν Ψ )
→ ∂ν
(Tµν xσ
∂Ψ
ν
− δµ LMµσ xσ = 0
∂xµ
− Tσν xµ )M
µσ
(22)
=0
where there is one continuity equation for each rotation generator Mµσ .The values of µ and σ for the
spatial rotation generators, Ji , are obtained from the relation,
1
M
2 ijk jk
where is again the Levi-Civita symbol. This gives:
Ji =
J1 =
1
M23
2
J2 =
1
M31
2
(23)
J3 =
1
M12 .
2
For L4 , there are three equations:
∂1 T21 x3 − T31 x2 + ∂2 T22 x3 − T32 x2 + ∂3 T23 x3 − T33 x2
+∂4 T24 x3 − T34 x2 + ∂5 T25 x3 − T35 x2 + ∂6 T26 x3 − T36 x2 = 0
∂1 T31 x1 − T11 x3 + ∂2 T32 x1 − T12 x3 + ∂3 T33 x1 − T13 x3
+∂4 T34 x1 − T14 x3 + ∂5 T35 x1 − T15 x3 + ∂6 T36 x1 − T16 x3 = 0
∂1 T11 x2 − T21 x1 + ∂2 T12 x2 − T22 x1 + ∂3 T13 x2 − T23 x1
+∂4 T14 x2 − T24 x1 + ∂5 T15 x2 − T25 x1 + ∂6 T16 x2 − T26 x1 = 0.
(24)
(25)
We can again use the divergence theorem to obtain the three continuity equations corresponding
to conserved quantities:
R
∂4 d5 x T24 x3 − T34 x2 = 0
R
∂4 d5 x T34 x1 − T14 x3 = 0
(26)
R
∂4 d5 x T14 x2 − T24 x1 = 0.
The terms in each integrand are a product of a momentum density (associated with one of P1 , P2 , P3 )
and a space variable (one of x1 , x2 , x3 ). We conclude that these have units of angular momentum,
as required.
To determine the conserved quantities related to the temporal rotation generators, Ťi , we can get
the values of µ and σ using the relation,
Ťi =
1
M
2 ijk jk
(27)
This gives:
1
1
M56 Ť5 = M64
2
2
The resulting three continuity equations are,
Ť4 =
Ť6 =
1
M45 .
2
∂1 T51 x6 − T61 x5 + ∂2 T52 x6 − T62 x5 + ∂3 T53 x6 − T63 x5
+∂4 T54 x6 − T64 x5 + ∂5 T55 x6 − T65 x5 + ∂6 T56 x6 − T66 x5 = 0
∂1 T61 x4 − T41 x6 + ∂2 T62 x4 − T42 x6 + ∂3 T63 x4 − T43 x6
+∂4 T64 x4 − T44 x6 + ∂5 T65 x4 − T45 x6 + ∂6 T66 x4 − T46 x6 = 0
∂1 T41 x5 − T51 x4 + ∂2 T42 x5 − T52 x4 + ∂3 T43 x5 − T53 x4
+∂4 T44 x5 − T54 x4 + ∂5 T45 x5 − T55 x4 + ∂6 T46 x5 − T56 x4 = 0
(28)
(29)
Symmetry 2020, 12, 817
6 of 10
which simplify to the equations,
d5 x T54 x6 − T64 x5 = 0
R
∂4 d5 x T64 x4 − T44 x6 = 0
R
∂4 d5 x T44 x5 − T54 x4 = 0
∂4
R
(30)
Here, the terms in each integrand are a product of an energy density (associated with one of
E1 , E2 , E3 ) and a time variable (one of x4 , x5 , x6 ). If we consider the first equation then the units of
measure for the first term are,
[d5 x]MKS = m5
[T54 ]MKS = kg m−3 s−2
[x6 ]MKS = s
(31)
giving
h
(d5 x (T54 ) x6 ]MKS = kg m2 s−1 .
(32)
We conclude that these have the same units of measure as the Planck constant.
We note that the units of measure for the conserved quantity due to invariance under spatial
rotations are also the same units of measure as the Planck constant and that the conserved quantity,
for some non-scalar fields, has been associated with spin angular momentum [5].
4. Breaking Time Reversal Symmetry
The spatial SU(2) × SU(2) algebras in complexified SO(3,3) have the basic form
left chirality:
right chirality:
{ 12 (J1 + iKa1 ), 21 (J2 + iKa2 ), 12 (J3 + iKa3 )}
{ 12 (J1 − iKa1 ), 21 (J2 − iKa2 ), 12 (J3 − iKa3 )}
(33)
where a = 1, 2, 3 and the two chiralities are related by a spatial parity transformation [2]. The temporal
SU(2) × SU(2) algebras have the basic form
first chirality:
{ 12 (T1 + iK1b ), 12 (T2 + iK2b ), 12 (T3 + iK3b )}
second chirality: { 12 (T1 − iK1b ), 21 (T2 − iK2b ), 21 (T3 − iK3b )}
(34)
where b = 1, 2, 3 and the two chiralities are related by a time reversal transformation.
The two chiral parts of a spatial SU(2) × SU(2) algebra are related by a spatial parity transformation
and so appear to be unaffected by breaking time reversal symmetry. The two chiral parts of a temporal
SU(2) × SU(2) algebra are related by a time reversal transformation. This suggests that breaking time
reversal symmetry affects the chiral properties of a temporal SU(2) × SU(2) algebra.
5. Symmetry between Algebras
The special orthogonal Lie group SO(4) can be associated with the group of rotations in a
four-dimensional Euclidean space [4]. The group has six generators, here labelled aj ,bj (j = 1, 2, 3),
and commutation relations:
[aj , ak ] = i jkm am
(35)
[bj , bk ] = i jkm am
[aj , bk ] = i jkm bm
where the indexes j, k, m = 1, 2, 3. The Lie group SO(3), associated with the group of rotations in three
dimensions, has three generators, here labelled wj (j = 1, 2, 3), and commutation relations,
[wj , wk ] = i jkm wm
(36)
Symmetry 2020, 12, 817
7 of 10
where the indexes j, k, m = 1, 2, 3. The direct product SO(3) × SO(2) has four generators, here labelled
wj (j = 0, 1, 2, 3), and commutation relations,
[wj , wk ] = i jkm wm
[w0 , wk ] = 0
(37)
where the indexes j, k, m = 1, 2, 3. We also note that SU(2) and SO(3) have the same Lie algebra, and
that U(1) and SO(2) are isomorphic [5].
5.1. SO(3) × SO(2) symmetry
The e1 spatial SU(2) × SU(2) algebra might be represented in tabular form as,
1
(a + b1 )
2 1
1
2 (J1 + iK11 )
1
(a + b2 )
2 2
1
2 (J2 + iK12 )
1
(a
2 3
1
2 (J3
+ b3 )
+ iK13 )
1
(a
2 1
1
2 (J1
− b1 )
− iK11 )
1
(a − b2 ) 12 (a3 − b3 )
2 2
1
1
2 (J2 − iK12 ) 2 (J3 − iK13 )}
(38)
where the a’s and b’s are the generic SO(4) labels given in (35). With a change of basis this becomes:
a1
J1
a2
J2
a3
J3
b1
iK11
b2
iK12
b3
iK13
(39)
This SO(4) contains four SO(3) subalgebras. There is a spatial SO(3) algebra:
w1
J1
w2
J2
w3
J3
(40)
Here, the w’s are the generic SO(3) labels given in (36). There are also three other SO(3) algebras:
w1
J2
J3
J1
w2
iK13
iK11
iK12
w3
iK11
iK12
iK13.
(41)
Additionally, the SO(4) commutes with a rotation generator, T1 , which will give us three SO(3) ×
SO(2) algebras,
w1
w2
w3
w0
J2
iK13
iK11
T1
(42)
J3
iK11
iK12
T1
J1
iK12
iK13
T1
where the w’s are the generic SO(3) × SO(2) labels given in (37). Changing the basis to 12 (w1 ± w2 ) and
1
2 (w0 ± w3 ) yields
1
1
(w1 ± w2 )
(w0 ± w3 )
2
2
1
2 (J2
1
2 (J3
1
2 (J1
± iK13 )
± iK11 )
± iK12 )
1
2 (T1
1
2 (T1
1
2 (T1
± iK11 )
± iK12 )
(43)
± iK13 )
If the columns are considered to be six component algebras then in horizontal form we have
1
2 (w1
1
2 (w0
± w2 ) = { 21 (J2 ± iK13 ), 21 (J3 ± iK11 ), 12 (J1 ± iK12 )}
± w3 ) = { 21 (T1 ± iK11 ), 21 (T1 ± iK12 ), 12 (T1 ± iK13 )}.
(44)
Symmetry 2020, 12, 817
8 of 10
Rotating 21 (w1 ± w2 ) within the vector space of the SO(4) then gives
1
1
1
1
(w ± w2 )0 = { (J1 ± iK11 ), (J2 ± iK12 ), (J3 ± K13 )}.
2 1
2
2
2
(45)
We conclude that 12 (w1 ± w2 )0 and 12 (w0 ± w3 ) are related by SO(3) × SO(2) symmetry plus
a rotation.
Inspection shows that the 12 (w1 ± w2 )0 algebra is the same as e1 algebra. This suggests that the
e-family is related to another family of algebras by SO(3) × SO(2) symmetry plus a rotation. This is
the n-family:
n1 = { 12 (T1 ± iK11 ), 21 (T1 ± iK12 ), 12 (T1 ± iK13 )}
n2 = { 21 (T2 ± iK21 ), 21 (T2 ± iK22 ), 12 (T2 ± iK23 )}
n3 =
{ 21 (T3
± iK31 ),
1
2 (T3
± iK32 ),
1
2 (T3
(46)
± iK33 )}.
These algebras are associated with three spatial dimensions, as indicated by the boost generators.
The n-family members are not SU(2) × SU(2) algebras.
5.2. SO(4) Symmetry
The members of the n-family are related by SO(4) symmetry. This can be illustrated by constructing
an array of generators:
m1
m2
m3
1
2 (T1
1
2 (T1
1
2 (T1
n1
± iK11 )
± iK12 )
± iK13 )
1
2 (T2
1
2 (T2
1
2 (T2
n2
± iK21 )
± iK22 )
± iK23 )
1
2 (T3
1
2 (T3
1
2 (T3
n3
± iK31 )
± iK32 )
(47)
± iK33 ).
Here, the rows are the m-family algebras which have SO(4) = SO(3) × SO(3) symmetry, and the
columns are the n-family. We also note that the n1 algebra shares two of its components with each
of m1 , m2 , and m3 . This suggests that an n-family algebra might be described as a mixture of
m-family components.
6. Conclusions
This article has considered some of the mathematical properties and relationships associated with
SU(2) × SU(2) subalgebras in an O(3,3) space. In particular, we find the following:
1.
2.
3.
4.
5.
6.
The e-family members are the standard type of SU(2) × SU(2) algebra, associated with three space
dimensions and one time dimension.
The e1 algebra is related to the n1 algebra by SU(2) × U(1) symmetry, plus a rotation.
We can describe the n1 algebra as being a mixture of components from the three m-family algebras.
The m-family members are a second type of SU(2) × SU(2) algebra, associated with one space
dimension and three time dimensions.
Breaking of time reversal symmetry affects the chiral properties of the m-family algebras.
The units of measure of the conserved quantity due to invariance under temporal rotations are
the same as those of the Planck constant.
Funding: This research received no external funding.
Acknowledgments: I would like to thank Torsten Schoeneberg for his input. I would also like to thank the
reviewers for their suggestions.
Conflicts of Interest: The author declares no conflict of interest.
Symmetry 2020, 12, 817
9 of 10
Appendix A. SO(3,3) Generators (Referenced in Section 2)
Time rotation generators:
T1 =
0
0
0
0
0
0
0
0
i
0
0
0
0
−i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
T2 =
0 0 i 0 0 0
0 0 0 0 0 0
−i 0 0 0 0 0
T3 =
0 0 0 0 0 0
0 0 0 0 0 0
0 0 0 0 0 0
0
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
−i
0
0
0
0
0
0
i
0
0
0
0
Space rotation generators:
J1 =
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
−i
0
J2 =
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
−i
0
0
0
0
0
0
0
0
0
i
0
0
J3 =
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
−i
0
0
0
0
0
0
0
0
Boost generators:
K11 =
0
0
0
i
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
K12 =
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
K13 =
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
K21 =
0 0 0
0 0 0
0 0 0
0 i 0
0 0 0
0 0 0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
K22 =
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
K23 =
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
K31 =
0 0 0 0
0 0 0 0
0 0 0 i
0 0 i 0
0 0 0 0
0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
0
K32 =
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
i
0
0
0
0
0
0
0
0
0
K33 =
0
0
0
0
0
0
0
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0 i
0 0 0 0
0 0 0 0
i 0 0 0
0
0
0
0
0
0
References
1.
2.
3.
4.
5.
Cartan, É. The Theory of Spinors; Hermann: Paris, France, 1966.
Tung, W. Group Theory in Physics; World Scientific Publishing: Singapore, 1985.
Kim, Y.S.; Noz, M.E. Dirac Matrices and Feynman’s Rest of the Universe. Symmetry 2012, 4, 626–643.
[CrossRef]
Hall, B.C. Lie Groups, Lie Algebras, and Representations: An Elementary Introduction, 2nd ed.; Springer: Berlin,
Germany, 2015.
Schwichtenberg, J. Physics from Symmetry, 2nd ed.; Springer: Berlin, Germany, 2017.
Symmetry 2020, 12, 817
6.
7.
10 of 10
Dirac, P.A.M. A Remarkable Representation of the 3 + 2 de Sitter Group. J. Math. Phys. 1963, 4, 901–909.
[CrossRef]
Lee, D.G. The Dirac gamma matrices as “relics” of a hidden symmetry?: As fundamental representation of
the algebra Sp(4,r). J. Math. Phys. 1995, 36, 524–530. [CrossRef]
© 2020 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 (http://creativecommons.org/licenses/by/4.0/).
|
|
https://openalex.org/W2577481235
|
https://europepmc.org/articles/pmc5193191?pdf=render
|
English
| null |
Psychotropics in different causes of itch: systematic review with controlled studies
|
Anais brasileiros de dermatologia/Anais Brasileiros de Dermatologia
| 2,016
|
cc-by
| 7,043
|
Review Review 791 DOI: http://dx.doi.org/10.1590/abd1806-4841.20164878 Abstract: Among the wide range of symptoms neglected or resistant to conventional treatments in clinical practice, itch is
emerging gradually as a theme to be studied. Itch complaints and the negative effects in the quality of life are observed in
several medical fields. Although the partially obscure pathophysiology, some researchers decided to check and test the use
of psychotropic drugs in resistant itch to conventional topical treatments and antihistamines. The objective of this study was
to evaluate scientific evidence in psychotropic use in the treatment of itch of various causes. This is a systematic review of
scientific literature. The following databases were used: PubMed, Web of Science, Scopus and Scielo. Randomized controlled
trials that should focus on treatment with psychotropic drugs of pruritus of various causes were the inclusion criteria. All
articles were analyzed by the authors, and the consensus was reached in cases of disagreement. Fifteen articles were included
after analysis and selection in databases, with the majority of clinical trials focusing on psychopharmacological treatment of
itch on account of chronic kidney disease. Clinical trials with psychotropic drugs mostly indicated significant improvement in
the itching. In most trials of chronic kidney disease as basal disease for itch, greater effectiveness was observed with the use
of psychotropic drugs compared with placebo or other antipruritic. However, the small amount of controlled trials conducted
precludes the generalization that psychiatric drugs are effective for itch of various causes. Keywords: Pruritus; Psychotropic drugs; Controlled clinical trial Psychotropics in different causes of itch: systematic review with
controlled studies* Lízie Emanuelle Eulalio Brasileiro1
Emerson Arcoverde Nunes2 Dayanna Patrícia de Carvalho Barreto1 DOI: http://dx.doi.org/10.1590/abd1806-4841.20164878 1
Universidade Federal do Rio Grande do Norte (UFRN) – Natal (RN), Brazil. ©2016 by Anais Brasileiros de Dermatologia (
)
2
Universidade de São Paulo (USP) – São Paulo (SP), Brazil. d and accepted for publication on 02.10.2015
de Psiquiatria do Hospital Universitário Onofre Lopes - Universidade Federal do Rio Grande do Norte (HUOL-UFRN) – Natal Received on 25.06.2015
Approved by the Advisory Board and accepted for publication on 02.10.2015
*
Study conducted at Serviço de Psiquiatria do Hospital Universitário Onofre Lopes - Universidade Federal do Rio Grande do Norte (HUOL-UFRN) – Natal
(RN), Brazil.
Financial Support: None.
Conflict of Interest: None. Universidade Federal do Rio Grande do Norte (UFRN) – Natal (RN), Brazil.
2
Universidade de São Paulo (USP) – São Paulo (SP), Brazil. Received on 25.06.2015 INTRODUCTION There are no itch specialized receptors at the end of the peripheral
nerves, but the specificity of the neurons that transmit the itch is in
connection with the spinal cord tracts.9 Itch was defined for a long time as an unpleasant cutane-
ous sensation that provokes the desire to scratch.1 The chronicity of
itch involves multidimensional phenomenon that includes physio-
logical (as the central neurons and nerve pathways), cognitive and
emotional aspects.1,2 Itch can be classified into four dimensional
categories, according to the neurophysiologic considerations: pruri-
toceptive, with peripheral origin; neuropathic; neurogenic; and psy-
chogenic, with central origin.3-5 The diagnosis of psychogenic itch
has the need to discard skin causes or underlying systemic disease,
representing a diagnostic challenge.6,7 As a consequence of chronic itch, a person may have skin
lesions caused by excessive scratching, pinching (ulcers, secondary
infections, bleeding, permanent discoloration or scarring), just as it
can cause sleep disturbance, depression, overall stress or anxiety,
among other psychiatric disorders.9,10,13 The treatment of itch often include antihistamines, antide-
pressants, opioid antagonists and 5HT3 receptors, since endogenous
pruritogenic mediators (serotonin, histamine, neuropeptides, pros-
taglandins, endogenous opioids) have been implicated in triggering
pruritic stimulus.15,8,14 Often, chronic itch is refractory to conven-
tional therapies, which leads to the search for medications initial-
ly underused as psychotropics, reason why clinical trials are being
conducted in this area. When the itch is related to chronic diseases or diseases that
cause major limitation for life, such as chronic kidney disease and
liver or skin diseases, it can cause considerable influence on the be-
havior and, just like pain, it can cause loss of quality of life. 8-10 The itch sensation originates in free nerve endings in the
skin and is transmitted by C fibers to the dorsal horn of the spinal
cord and then to the cerebral cortex via the spinothalamic tract.11,12 It is worth mentioning that pharmacological therapy that An Bras Dermatol. 2016;91(6):791-8. Brasileiro LEE, Barreto DPC, Nunes EA 792 has been used in clinical practice is based on small studies, and that
there are few controlled clinical trials. in this review would be presented grouped by the cause of itch to
allow some inferences on the observed heterogeneity of etiologies. Where it was not feasible a more homogeneous grouping, we per-
formed the methodological, results and other possible outcomes
descriptions of each study. Itch secondary to chronic kidney disease or dialysis This review was conducted following PRISMA (Preferred
Reporting Items for Systematic Reviews and Meta-Analysis) guideline. Table 1 shows the six clinical trials included in the study on
itch due to chronic kidney disease. In the six studies of patients with chronic kidney disease
(CKD) at advanced stage, and therefore performing hemodialysis
(HD), there was the participation of a total of 179 individuals with a
mean age of 56 years (± 11.8). The preparation of the systematic review was initiated in
December 2014, initially through retrospective consultation of elec-
tronic databases PubMed, Web Of Science, Scopus and Scielo, using
the following keywords: Pruritus, itch, treatment, psychotropic drugs,
organized as follows: “pruritus” OR “itch” AND treatment AND psy-
chotropic drugs, and their descriptors in Portuguese for searching
in Scielo. In most of the six studies, the characteristics of patients was
similar as well as the criteria that led to the selection of the partici-
pating subjects. As inclusion criteria, the subjects should be at least
3 to 6 months in hemodialysis, with a frequency of 2 to 3 sessions
per week, with stabilized patterns (normal calcium, phosphatase,
parathyroid hormone, aluminum, serum magnesium and hemoglo-
bin values). They should also present medium to severe intensity
itch, with duration varying from 6 to 8 weeks, not respondent to
traditional pre-treatments (antihistamines and emollients). Exclu-
sion criteria followed the same line, therefore subjects with liver,
dermatologic and other metabolic disorders were excluded from
the study. Solak et al. stablished broader criteria, thus excluding in-
dividuals with uncontrolled mental illness.15 The antipruritic agents
used should be suspended at least 1 week before the start of the
intervention. Included articles should be Controlled Trials, in humans, in
English or Portuguese. Articles focusing on drugs other than psy-
chotropic drugs or on diseases that didn’t have itch as a studied
variable were excluded from the analysis. The first step consisted of reading the titles and abstracts,
and selecting articles that contained the keywords previously ex-
plained or derived words (MeSH terms). Based on selected articles,
the second step consisted of thorough and independent analysis of
each article by the authors, assessing the consistency with the pro-
posed theme and methodological rigor. The authors met before the
collection to ensure uniform understanding of the eligibility criteria. In cases of disagreement, the authors tried to reach a consensus. An Bras Dermatol. 2016;91(6):791-8. INTRODUCTION This paper is a systematic review, which aims to observe the
published studies that demonstrate the effectiveness of psychotro-
pic drugs in different primary causes of itch. Itch secondary to chronic kidney disease or dialysis Finally, in order to find useful items that were not contained
in the initial search, the reference lists of articles originally included
were used. Itch, just like pain, is a subjective and difficult to quantify
symptom, given its subjectivity. Thus, among the six clinical tri-
als, five used the Visual Analogue Scale (VAS) for measuring the
symptomatic itching before, during and after surgery. Therefore,
the results assumed are based on the changes observed over this
scale. Only in the trial conducted by Pour-Reza-Gholi et al. a dif-
ferent method was used, through the categorization of subjective RESULTS Based on the terms used for the search and specifiers, 134
articles were found in PubMed; 12 articles in Web of Science; 97 in
Scopus; and 0 in Scielo database. All items found were in English. Among these, 15 articles fulfilling the inclusion criteria were includ-
ed in this systematic review. From PubMed database, 114 articles
were excluded because they were not related to the keywords and
06 for not being controlled clinical trials; 01 article was included
from a reference list (Figure 1). In the other databases, all items were
excluded because none was a controlled trial.i Figure 1: Flowchart. Process of selection of articles
Identification
Article
selection
Eligibility
Eligibility
Articles found after the
search:
• PubMed: 134
• Scopus: 97
• Web of Science: 12
• Scielo: 0
Included from reference
lists: N= 01
Articles according to inclu-
sion criteria: N = 14
Articles excluded:229
- PubMed: N = 120
Other theme (114)
Uncontrolled study (06)
- Scopus: N = 97
- Web of Science: N= 12
First step (titles and
abstracts): N = 14
Included: N = 14
Total: N = 15 Articles according to inclu-
sion criteria: N = 14
Articles excluded:229
- PubMed: N = 120
Other theme (114)
Uncontrolled study (06)
- Scopus: N = 97
- Web of Science: N= 12 We observed significant heterogeneity in clinical trials as
the primary types that led to itch as well as to the psychotropic and
methodologies used. Identification The articles included in this review presented as the origin
of itch: chronic kidney disease or dialysis (6); itch after burn (2); mus-
tard gas lesions and itch for other dermatological causes (2); chronic
liver disease or cholestasis (2); chronic idiopathic urticaria (2); and
one for not dermatologic causes (with a predominance of cancer). It should also be evidenced that there has been no controlled study
addressing the psychogenic or somatoform as an origin of itch. The psychotropic drugs used in these clinical trials for the
various causes of itch were: pregabalin, gabapentin, doxepin, par-
oxetine and sertraline. It was decided that clinical trials comprised Figure 1: Flowchart. RESULTS Process of selection of articles Psychotropics in different causes of itch: systematic review with controlled studies 793 improvement: complete improvement, relative improvement and
no effect, respectively.16 significant.17 Furthermore, three of the six studies presented very sim-
ilar methodologies, but one of the differences observed in these stud-
ies was the dosage of gabapentin 300 mg 3 times a week, 400 mg 2
times a week and 100 mg 3 times a week.18-20 These three trials demon-
strate the effectiveness of gabapentin over the placebo group.18-20 We found that, of the six trials on CKD as underlying dis-
ease for itch, five used gabapentin 100-300 mg following dialysis
sessions as medication to be compared and/or tested. Doxepin was
used in one study on the grounds that, until then, it had not been
tested in patients with CKD in controlled trials.16 The rationale for
the use of such tricyclic antidepressant would be because it presents
strong anti-histaminergic action, acting in one of the neurotransmit-
ters of itch mechanism.16 As for secondary endpoints, all six trials demonstrated
greater adverse events in the groups using psychotropics, especial-
ly dizziness, drowsiness and fatigue, more intense after the first
dose and with gradual tolerance. However, some subjects could not
continue in the trial due to the intensity and persistence of adverse
events. This occurred in the study of Marquez et al., in which four
subjects discontinued the study due to gabapentin (fatigue and
drowsiness) and one abandoned it when used desloratadine due to
“nervousness”. 17 In Razeghi et al., there was a loss caused by ad-
verse events of gabapentin. 20 Among these six studies, we observed that in five of them
there was a significant improvement of itch with use of psychotropic,
although Solak et al. study compared gabapentin 300 mg, 3 times a
week, with pregabalin 75 mg daily, two anticonvulsants.15 Interest-
ingly, in one of the trials, the outcome was different. Marquez et al. compared a group using desloratadine 5 mg/day (3 weeks) with
other group using gabapentin 300 mg after hemodialysis sessions (3
weeks), with cross over and a week washout. The study showed that
desloratadine presented significant improvement in itch, while gab-
apentin, although it also improved the symptom, was not statistically Legend: WO- washout; VAS: Visual Analogue Scale Itch with dermatological cause Two articles included in this review addressed different
skin impairment (in sequence, lesion resulting from sulfuric gas and
atopic eczema), so it was not possible to unify methods or actions Table 1: Itch secondary to chronic kidney disease (CKD)
Study
Solak Y et
al., 2012
Marquez
D et al.,
2012
Gunal
AI et al.,
2004
Razeghi
E et al.,
2009
Naini
AE et al.,
2007
Pour-
Reza-
GholiF. et
al., 2007
Country
England
Argentina
Turkey
Iran
Iran
Iran
Design
Prospective,
randomized,
crossover, com-
parative
prospective,
nonrandomized,
Prospective, ran-
domized, place-
bo-controlled,
double-blind
Prospective, pla-
cebo-controlled,
double-blind
Longitudinal,
randomized pla-
cebo-controlled
Prospective, ran-
domized, place-
bo-controlled,
double-blind,
crossover
N of
subjects
50
22
25
34
34
24
Drugs
Gabapentin
300 mg 3x/
week
Pregabalin
75mg/day
Gabapentin
300 mg 3x/
week of deslo-
ratadine 5mg
3x/week
Gabapentin
300mg or
placebo
Gabapentin
100mg or
placebo
3x/weeks
Gabapentin
400mg
Placebo
Doxepin 10mg
2x/day
Placebo
Via
Oral
Oral
Oral
Oral
Oral
Oral
Duration
14 weeks
(6 weeks for
each drug; 2
weeks WO)
7 weeks
(3 weeks
each drug; 1
week WO)
9 weeks
(4 weeks
each drug; 1
week WO)
9 weeks
4 weeks
3 weeks
(1 week each
group; 1
week WO)
Instrument
VAS
VAS
VAS
VAS
VAS
Clinical im-
provement
Type of itch
Peripheral
neuropathy
in patients
in dialysis
dialysis
dialysis
dialysis
dialysis
dialysis
Results
Significantly
decrease in
symptoms, with
no difference
between the two
drugs
Only deslorata-
dine had statisti-
cal significance
Decreased score
showed highly
significance
(0.0001) after
gabapentin
Effectiveness of
gabapentin in low
doses
Gabapentin was
effective
Doxepin with sig-
nificant improve-
ment Table 1: Itch secondary to chronic kidney disease (CKD) Legend: WO- washout; VAS: Visual Analogue Scale An Bras Dermatol. 2016;91(6):791-8. Itch for chronic idiopathic urticaria Itch for chronic idiopathic urticaria Also in dermatologic causes, in the 1980’s two studies were
performed with doxepin oral for the treatment of chronic idiopathic
urticaria, with similar methodology, not replicated until the time of
writing this article (Table 2). In 1985, Greene et al. published a study of 14 months du-
ration in which 50 patients were evaluated in a randomized dou-
ble-blind controlled trial of diphenhydramine 75 mg/day.23 The
study included patients refractory to previous treatments and ex-
cluded patients younger than 18 years old, pregnant and nursing
women. The sample was divided into three groups of types of urti-
caria as follows: simple urticaria, leukocytoclastic urticaria and lym-
phoblastic urticaria; and it was also subdivided by severity. It was found a significant improvement in almost all af-
fected parts of the body in both groups, with no significant differ-
ence between groups, according to VAS and DLQI (Dermatology
Life Quality Index). Doxepin cream 5% resulted in fewer adverse
events compared with betamethasone topical. Topical presentation
of doxepin compared with oral presentation presented advantages
because it did not cause adverse events typical of tricyclic antide-
pressants. 21 This trial showed clinical and statistical superiority of dox-
epin at a dosage of 30 mg/day, with complete remission of symp-
toms in 43% of patients compared with 5% remission when in use
of diphenhydramine (p<0.001). The study also showed 74% of
partial improvement in symptoms with the use of antidepressant,
compared with 10% improvement with the use of antihistamine
(p<0.001), being, therefore, a safe alternative for the treatment of
patients with simple itch that do not respond to common antihis-
tamines. Despite these positive results, the only statistical test used
was the McNemar test, which is used to compare dichotomous
variables, which was not the case of this study that quantified the
symptoms in four categories. Thus, the inadequate statistical analy-
sis limited the conclusions presented by the authors.24 Groene et al. conducted a quite diverse clinical trial in terms
of methodology compared with the studies above. He performed
the induction of itch by acetylcholine in order to test doxepin cream
5% on itch improvement in patients with atopic eczema. The study
used the front side of the forearms of 11 subjects, randomizing them,
with the aim of comparing doxepin cream 5%, applied to a forearm,
with placebo, applied to the other forearm, 4 times a day during 3
days. An Bras Dermatol. 2016;91(6):791-8. Itch with dermatological cause Brasileiro LEE, Barreto DPC, Nunes EA 794 Table 2: Itch secondary to skin diseases Table 2: Itch secondary to skin diseases Study
Greene et
al., 1985
Goldso-
bel et al.,
1986
Panahi
Y et al.,
2011
Groene
D et al.,
2001
Country
USA
USA
Iran
Germany
Design
Longitudinal,
controlled,
double-blind,
randomized
Longitudi-
nal, place-
bo-controlled,
double-blind,
randomized
Prospective,
randomized,
single-blind
Longitudinal,
placebo-control,
randomized,
crossover
N of
subjects
50
16
75
11
Drugs
Doxepin 10mg
or diphenhy-
dramine 25mg
3x/day
Doxepin 25mg
Placebo
3x/day
Doxepin
cream 5% or
betamethasone
cream
Doxepin
cream 5%;
Wolff basis
cream (pla-
cebo)
Via
Oral
Oral
Topic
Topic
Duration
31 days
8 weeks
6 weeks
4 days
Instrument
McNemar
test
Clinical
evaluation
VAS
Itch score
DLQI
SDS
(self-rating
depression
scale)
Type of itch
Chronic
idiopathic
urticaria
Chronic
idiopathic
urticaria
Lesion due
to mustard
gas
Atopic
eczema
Results
Itch decreased
with doxepin
according to the
instrument used
Itch decrease with
doxepin
Significant de-
crease of pruritus
with both topical
drugs
No significant
antipruritic action
of doxepin com-
pared with basis
cream
Legend: WO- washout; VAS: Visual Analogue Scale Legend: WO- washout; VAS: Visual Analogue Scale Legend: WO- washout; VAS: Visual Analogue Scale though the dimension of the papule was lower in the forearm that
received doxepin, suggesting that acetylcholine caused less reac-
tion. 22 to be taken, although the tested medication was the same, doxepin
cream 5% (Table 2). Panahi’s clinical trial aimed to check whether doxepin could
be an effective alternative to relieve the itching caused by mustard
gas. The study included 75 men who were exposed to mustard gas
decades ago and who presented itch. Subjects were excluded if they
presented itch secondary to systemic or cutaneous disease, not trig-
gered by mustard gas. Patients were divided into two groups: in
one group (40 men), doxepin 5% was applied in lesions two times a
day; in the other group (35 males), betamethasone 0.1% was applied
for 6 weeks. 21 Itch for chronic idiopathic urticaria In the following day (Day 4), acetylcholine or sodium hydro-
chloride applications were made in the pretreated regions. It was
observed that doxepin didn’t have a greater antipruritic effect when
compared with placebo, and the author referred it should be due
to the moisturizing effect of the base solution used as a placebo, al- In the following year, Goldsobel et al. also published a dou-
ble-blind controlled study, this time with placebo, with duration Psychotropics in different causes of itch: systematic review with controlled studies 795 of four weeks.25 This study included a sample population of 16 pa-
tients, 14 women and 2 men, but not all had undergone previous
treatment (1 patient). This trial also showed clinically and statistical-
ly significant results of doxepin, with improvement in all evaluated
categories, when compared with placebo: 51% reduction in number
of lesions (2.02 versus 0.98 placebo; p<0.001), 64% reduction in dis-
comfort with the symptoms (1:38 versus 0.49 placebo; p<0.001), and
75% reduction in edema (0.85 versus 0.21 placebo; p<0.001). sis) and presence of itch (Table 3). The factors necessary to enter
in both studies were: evidence of cholestatic liver disease and itch
over three months. The exclusion criteria in common were: patients
could not have dermatologic disease associated with itch or make
use of antipruritic agents. These, if used, should be suspended un-
til two weeks before starting the study; also, patients could not be
taking antidepressant, opioid, corticosteroids, phenothiazines and
antiviral for hepatitis or they should be suspended five days before
the start of the trial.26,27 Bergasa et al. proposed further exclusion cri-
teria: history of hepatic encephalopathy, ascites, visceral bleeding,
malignancy, cannot prevent pregnancy or being pregnant, creati-
nine greater than 1.7 mg/dL, hemoglobin less than 10 g/dL, have
received a liver transplant or having human immunodeficiency vi-
rus.27 Despite this improvement, methodological flaws limited
incisively the conclusions from these observations. For example,
the symptoms were grouped into categories, resulting in ordinal
variables, which were compared using paired t-tests, which are ap-
plied to mean of interval variables, beacuse it does not make sense
to calculate mean of ordinal of categorical variables. Thus, the data
presented as statistically significant actually were not calculated
properly. 24 Both studies were randomized, placebo-controlled and
double-blind. However, both compared different medications with
placebo, in view of possible different pathophysiological mecha-
nisms of pruritus. Mayo et al. Itch for chronic idiopathic urticaria used sertraline, a serotonin receptor
inhibitor, understanding that the serotonergic pathway is important
in the perception of itch.26 Bergasa et al., differently, used gabapen-
tin, having the hypothesis that the itch is part of nociceptive stimu- Itch for chronic liver disease (CLD) or cholestasis study was composed of two phases: the first had
the function of dose titration of sertraline in 38 subjects, as well as
laboratory monitoring of sertraline and its metabolite; the second,
after excluding 26 subjects, had the function of demonstrating the
effectiveness of sertraline. In this second phase, VAS and evalua-
tion of lesions secondary to itch were used during follow-up for 13
weeks, and a higher effectiveness was verified, with significance in
the group using sertraline 75 mg to 100 mg daily compared with
placebo.26 In mild to moderate itch, the almost complete remission
was achieved in the combined and pregabalin group, with a signif-
icant difference compared with the antihistamine and the placebo
group. Regarding the severe itch, there was also a significant im-
provement in the combined and pregabalin groups when VAS was
evaluated on the 28th day. There were no reported adverse events
in the placebo and pregabalin groups, however, the antihistamine
and combined groups, 11 and 9 patients complained of drowsiness,
respectively.28 It was also observed that doses higher than 100 mg/day
does not necessarily caused benefits related to itch, but increased
adverse events like insomnia, fatigue and increased bowel habits. In the clinical trial conducted by Bergasa et al., it was found
unexpected and initially paradoxical result. It was concluded that
there was no significant improvement after therapeutic trial of four
weeks in the group receiving gabapentin in variable dose of 300 mg
to 2400 mg per day, compared with placebo, in aspects related to the
assessment of itch (little decrease in VAS) and frequency in scratching
(HSA- Hourly Scratching Activity). In apparent contradiction, it was
observed that the HSA of patients in the placebo group decreased and
approached zero, which allowed the conclusion that there was a sub-
jective improvement of itch and a change in behavior. 27 The second trial included in this review was conducted by
the same author, with the objective to test gabapentin in itch second-
ary to severe burns as well as to test if the association of gabapentin
to an antihistamine would result in significant superiority of the
junction of the two neurophysiological mechanisms.29 Therefore, 60
patients were divided into three groups with dosage quantitatively
related to VAS: A- Cetirizine 10 to 20 mg/day; B - Gabapentin 300
to 900 mg/day; C- Cetirizine and gabapentin. Itch for chronic liver disease (CLD) or cholestasis Two clinical trials were conducted in 2006, randomized,
placebo-controlled, totaling 28 subjects with chronic liver disease
(the leading cause of both studies was the primary biliary cirrho- Table 3: Itch secondary to chronic liver disease; malignancies; after-burns
Study
Ahuja
RB et al.,
2012
Ahuja
RB et al.,
2010
Mayo
MJ et al.,
2006
Bergasa
NV et al.,
2006
Zylicz
Z et al.,
2003
Country
India
India
USA
USA
Nether-
lands
Design
Randomized,
double-blind,
placebo-con-
trolled
Longitudinal,
randomized,
double-blind
Randomized,
placebo-con-
trolled, dou-
ble-blind
Randomized,
placebo-con-
trolled, dou-
ble-blind
Prospective, ran-
domized, place-
bo-controlled,
double-blind,
crossover
N of
subjects
80
60
12
16
26
Drugs
G1: cetirizine and
chlorpheniramine;
G2: pregabalin, ceti-
rizine and chlorphe-
niramine;
G3: Vit.B
G4: Pregabalin
G1: Cetirizine 10mg
G2: Gabapentin
300mg
G3: Gabapentin +
cetirizine
Sertraline 75-100mg
per day, and placebo
Gabapentin 100 mg
to 2400mg / day or
placebo
Paroxetine 20 mg or
placebo
Via
Oral
Oral
Oral
Oral
Oral
Duration
9 months
4 months
13 weeks
4 weeks
Variable
according
to the
response
of each
subject
Instrument
VAS
VAS
VAS
VAS
HSA
HDRS
SCID
NAS
(Numerical
Analogue
Scale)
Type of itch
After burns
After burns
CLD
CLD
No derma-
tological
(malignan-
cy)
Results
Decrease in symp-
toms significantly,
with no difference
between the two
drugs
VAS reduced 95%
in the gabapentin
group (52% in the
cetirizine)
Sertraline shown
to be statistically
significant
Gabapentin
demonstrated no
significant im-
provement in itch
Effectiveness of
paroxetine
Legend: SCID: Structured Clinical Interview; DLQI: Dermatology Life Quality Index); HDRS: Hamilton Rating Scale for Depression; CLD: chronic liver disease Table 3: Itch secondary to chronic liver disease; malignancies; after-burns An Bras Dermatol. 2016;91(6):791-8. Brasileiro LEE, Barreto DPC, Nunes EA 796 lation and therefore would improve with this medication.27 lation and therefore would improve with this medication.27 other group which received only pregabalin. In this trial, VAS was
used not only for subsequent itch ratings, but also to determine the
dosage of each drug used in accordance with the categorization in
mild itch (2-5 VAS), moderate itch (6-8) and severe itch (9 and 10). Therefore, cetirizine doses ranged from 10 to 30 mg/day; pregabalin
doses ranged from 75 to 300 mg; and pheniramine doses was main-
tained at 25 mg/da. Mayo et al. An Bras Dermatol. 2016;91(6):791-8. Itch for non-dermatological causes (malignant diseases) A single study was included, whose central theme was the
psychopharmacological treatment of non-dermatological itch due
to, mostly, malignancies and serious systemic diseases in order to
test paroxetine 20 mg/day (Table 3). Itch for chronic liver disease (CLD) or cholestasis The study concluded
that there was significant difference in the decrease of itch assessed
by VAS when comparing group A (52%) with group B (95%). Also,
all patients in group B presented complete improvement in itch on
the 28th day, compared with 3 patients (out of 20) in group A. There
was no significant difference between groups B and C. 29 Also the adverse events observed in the group receiving ga-
bapentin were: increase in serum bilirubin (1 patient), fatigue, dizzi-
ness, worsening of symptoms of carpal tunnel syndrome, vomiting,
and fluctuations in serum creatinine.27 Another outcome observed
in this trial was the presence of depressive symptoms – assessed
by HDRS (Hamilton Depression Rating Scale) – presented mildly
in eight patients, and moderate in three and absent or minimal in
two patients; and by SCID (Structured Clinical Interview Question-
naire) axis 1 – 12 patients were classified with mood changes due
to general medical condition and one with depressive episode with
atypical presentation. DISCUSSION However, the way doxepin was used (in a study as treatment and in
another as pretreatment) and the type of skin disorder diverge.23,25
So it is not possible to infer that doxepin shows good results related
to itch. When the use of doxepin was directed to chronic urticaria,
the two studies evaluated showed efficacy of antidepressant, yet
both suffered from methodological flaws and follow without fur-
ther replication of results.24 Part of this may be explained by the fact
that these are two publications with about 30 years and can be used
as an example of the lack of more objective tools for evaluation of
itch, already present in the most recent publications. It is important to add that psychosomatic factors modulate
the perception of itch in a large amount of skin diseases.35 It is estimat-
ed that, in dermatological clinics, the prevalence of psychogenic itch
complaints can be considered high, about 2% of patients treated. 36 Most of patients enrolled in studies relating to itch conse-
quent to CKD had diabetes mellitus as underlying disease of renal
dysfunction. Symptoms of pain and itch resulting from peripheral
neuropathy were perceived as chronic complaints and were prov-
en through the assessment by electromyography.15 Solak conducted
this test carefully correlating the cause of itch to medication tested
for itch - gabapentin and pregabalin.15 It would be a great addition
to the knowledge that is being established in this branch of medicine
studies comparing gabapentin, pregabalin and placebo. Furthermore, the diagnosis of psychogenic itch is performed
excluding other causes. However, there may occur simultaneously
the other type of itch.6,7 Given this difficult differential diagnosis, we understand
the objections to conduct controlled clinical trials for psychogenic
pruritus. However, these studies are needed, as itch requires an in-
terface between specialties as well as more evidence is needed on
the effectiveness of psychotropics. It is understood that the pathophysiological mechanism of
itch in renal and hepatic causes is the same (neuropathic pruritus). Therefore, the use of gabapentin and pregabalin would have a good
indication, although we can highlight the lack of uniformity related
to difference of dose used in these trials (100 to 300 mg after hemo-
dialysis; or daily in cases of hepatic impairment). We can conclude that most of the clinical trials performed so
far show statistically significant efficacy of the psychotropic in the
treatment of itch for various causes. DISCUSSION We observed in the studies compiled in this review that, re-
currently, the authors stressed the importance that the itch can have
in the lives of patients, regardless of the cause of the itch. They ad-
dress cases that the traditional treatment with antipruritic agents
does not show effectiveness in clinical practice, thus justifying the
use of psychotropic drugs in their studies. Not failing to appreciate
the fact that different methodologies were used in these 15 articles
reviewed, we noticed that 12 of them have concluded that the use of
psychotropic drugs (gabapentin in 7; pregabalin in 2 - one of them
compared gabapentin and pregabalin -; doxepin in 5; paroxetine in
1; and sertraline in 1) represented a significant improvement in itch. To treat itch with skin disease origin (pruritoceptive), cap-
saicin cream, doxepin and aspirin can be used.9 Nevertheless, this
review did not show enough clinical trials able to reinforce this be-
havior in clinical practice in a guided way. Even with these results, we cannot fail to emphasize the
low amount of controlled clinical trials using psychotropic drugs
for itch of various causes, given the importance of this theme. Fur-
thermore, we observe the absence of clinical trials including or fo-
cusing on mental disorders that may cause or be comorbid to itch
of several causes. Possibly one of the factors that justify this lack is
the high prevalence of mental disorders in the general population. That said, it was observed that only one of the clinical trials used
as an exclusion criterion “uncontrolled” mental illness, which may
have influenced so as to cause biased results in other studies.15 In
another study, the authors were careful to investigate the depression
according to HDRS and SCID, and concluded that the underlying
disease and itch could be one of the causes of the depressive symp-
tomatology.27 It was observed that there is a direct and statistically
significant correlation between the severity of itch and the severity
of depressive symptoms.31 Even in the treatment of itch in skin diseases, the psychotro-
pic drug most commonly used in clinical practice is doxepin, a tri-
cyclic antidepressant with a strong antihistamine action.23 Doxepin
was used in two trials whose basal cause of itch was dermatologic. Itch after burning The inclusion criteria used were: adult patients, who expe-
rience intense itching during at least one month, not associated with
skin disease, able to assess the intensity, as well as having more than
a month of life expectancy. Patients with known hypersensitivity to
paroxetine, such as pregnant women or women who were breast-
feeding, with bipolar disorder, uncontrolled epilepsy, use of MAOIs
(monoamine oxidase inhibitors), chronic nausea, dependence on
antihistamines and creatinine clearance estimated at less than 30 ml
per minute were excluded from the study. Two articles related to burning as a primary cause of itch
were included, both with methodological design of randomized,
double-blind, and only the first described below held control with
placebo group (Table 3). Inclusion criteria were nearly identical between the two
clinical trials as well as the exclusion criteria. To enter these studies,
patients should be aged between 18-60 years (in the second study,
between 12-70 years), present percentage of burned body area high-
er than 5%, including predominance of second degree burns and
lesions in healing process or already healed up to 3 months. As ex-
clusion criteria, the same comorbid conditions were marked: dia-
betes mellitus, skin disease, kidney disease, pregnancy, lactation or
surface of grafted area greater than 2%. From a total of 26 patients included in this trial, 17 had solid
tumors, 4 had hematologic malignancy and 5 did not have cancer
nor had idiopathic origin for the itch. Two primary endpoints were evaluated in this clinical trial:
rating by VAS of the mean of the itch, assessed 7 days after random-
ization and a crossover; and individual overall response to treat-
ment (minimum of 50% improvement in the 3 days prior compared
to baseline). The study found greater effectiveness and significance
of paroxetine compared with placebo (p=0.027), especially in the
mean of the last 3 days of evaluation with the NAS (Numerical An-
alog Scale). Itch after burning A statistical significance was also observed (p=0.067) In the first study, Ahuja conducted a clinical trial with 80
subjects who were subdivided into four groups, divided equally,
arranged in the subsequent order: cetirizine and pheniramine ma-
leate (antihistamine group); pregabalin, cetirizine and pheniramine
maleate (combined group); placebo (vitamin B complex); and an- Psychotropics in different causes of itch: systematic review with controlled studies 797 depolarization changes in calcium and potassium channels and may
be related to improvement of itch.32,33 when a greater proportion of patients reported having preference
for the week of paroxetine. Adverse events, such as drowsiness and
nausea, were responsible for study discontinuation for 2 patients.30 In one of the results in that the hypotheses of the author
has been refuted (that psychotropic would have greater statistical
effectiveness in itch), it was discussed why the itch showed better
response and behavior change after use of placebo.27 In this study, in
a contradictory manner, even with high doses of gabapentin (max-
imum of 2400 mg/day), there was no significant improvement in
itch. The author argues that the itch caused by the cholestatic dis-
ease is mediated by tone of opioid neuromodulators and should
therefore be treated with opioid antagonists, demonstrated by con-
trolled clinical trials. 27,34 An Bras Dermatol. 2016;91(6):791-8. REFERENCES 11. Mazeh D, Melamed Y, Cholostoy A, Aharonovitzch V, Weizman A, Yosipovitch G. Itching in the psychiatric ward. Acta Derm Venereol. 2008;88:128-31. 12. Savin JA. How should we define itching? J Am Acad Dermatol. 1998;39:268-9. 13. Krishnan A, Koo J. Psyche, opioids, and itch: therapeutic consequences. Dermatol
Ther. 2005;18:314-22. 13. Krishnan A, Koo J. Psyche, opioids, and itch: therapeutic consequences. Dermatol
Ther. 2005;18:314-22. 14. Paus R, Schmelz M, Bíró T, Steinhoff M. Frontiers in pruritus research: scratching
the brain for more effective itch therapy. J Clin Invest. 2006;116:1174-86. 14. Paus R, Schmelz M, Bíró T, Steinhoff M. Frontiers in pruritus research: scratching
the brain for more effective itch therapy. J Clin Invest. 2006;116:1174-86. 15. Solak Y, Biyik Z, Atalay H, Gaipov A, Guney F, Turk S, et al. Pregabalin versus
gabapentin in the treatment of neuropathic pruritus in maintenance haemodialysis
patients: a prospective, crossover study. Nephrology (Carlton). 2012;17:710-7. p
p
p
y
p
gy (
)
16. Pour-Reza-Gholi F, Nasrollahi A, Firouzan A, Nasli Esfahani E, Farrokhi F. Low-dose
doxepin for treatment of pruritus in patients on hemodialysis. Iran J Kidney Dis. 2007;1:34-7. 17. Marquez D, Ramonda C, Lauxmann JE, Romero CA, Vukelic VL, Martinatto C, et
al. Uremic pruritus in hemodialysis patients: treatment with desloratidine versus
gabapentin. J Bras Nefrol. 2012;34:148-52. 18. Gunal AI, Ozalp G, Yoldas TK, Gunal SY, Kirciman E, Celiker H. Gabapentin therapy
for pruritus in haemodialysis patients: a randomized, placebo-controlled, double-
blind trial. Nephrol Dial Transplant. 2004;19:3137-9. p
p
19. Naini AE, Harandi AA, Khanbabapour S, Shahidi S, Seirafiyan S, Mohseni M. Gabapentin: a promising drug for the treatment of uremic pruritus. Saudi J Kidney
Dis Transpl. 2007;18:378-81. p
;
20. Razeghi E, Eskandari D, Ganji MR, Meysamie AP, Togha M, Khashayar P. Gabapentin
and uremic pruritus in hemodialysis patients. Ren Fail. 2009;31:85-90. 21. Panahi Y, Davoudi SM, Beiraghdar F, Amiri M. Doxepin cream vs betamethasone
cream for treatment of chronic skin lesions due to sulfur mustard. Skinmed. 2011;9:152-8. 22. Groene D, Martus P, Heyer G. Doxepin affects acetylcholine induced cutaneous
reactions in atopic eczema. Exp Dermatol. 2001;10:110-7. 23. Greene SL, Reed CE, Schroeter AL. Double-blind crossover study comparing
doxepin with diphenhydramine for the treatment of chronic urticaria. J Am Acad
Dermatol. 1985;12:669-75. 24. Marino MJ. The use and misuse of statistical methodologies in pharmacology
research. Biochem Pharmacol. 2014;87:78-92. 25. Goldsobel AB, Rohr AS, Siegel SC, Spector SL, Katz RM, Rachelefsky GS, et al. REFERENCES 1. Greaves MW. Pathogenesis and Treatment of Pruritus. Curr Allergy Asthma Rep. 2010;10:236-42. g
31. Gupta MA, Gupta AK, Schork NJ, Ellis CN. Depression modulates pruritus
perception: a study of pruritus in psoriasis, atopic dermatitis, and chronic
idiopathic urticaria. Psychosom Med. 1994;56:36-40. 2. Greaves MW, Wall PD. Pathophysiology of itching. Lancet. 1996;348:938-40. 3. Greaves MW, Khalifa N. Itch: more than skin deep. Int Arch Allergy Immunol. 2004;135:166-72. 32. Aperis G, Paliouras C, Zervos A, Arvanitis A, Alivanis P. The use of pregabalin
in the treatment of uraemic pruritus in haemodialysis patients. J Ren Care. 2010;36:180-5 4. Pogatzki-Zahn E, Marziniak M, Schneider G, Luger TA, Ständer S. Chronic pruritus:
targets, mechanisms and future therapies. Drug News Perspect. 2008;21:541-51. 4. Pogatzki Zahn E, Marziniak M, Schneider G, Luger TA, Ständer S. Chronic pruritus:
targets, mechanisms and future therapies. Drug News Perspect. 2008;21:541-51. ets, mechanisms and future therapies. Drug News Perspect. 2008;21:541-5 5. Twycross R, Greaves MW, Handwerker H, Jones EA, Libretto SE, Szepietowski JC, 5. Twycross R, Greaves MW, Handwerker H, Jones EA, Libretto SE, Szepietowski JC,
et al. Itch: scratching more than the surface. QJM. 2003;96:7-26. 5. Twycross R, Greaves MW, Handwerker H, Jones EA, Libretto SE, Szepietowski JC,
et al. Itch: scratching more than the surface. QJM. 2003;96:7-26. 33. Tzellos TG, Papazisis G, Toulis KA, Sardeli Ch, Kouvelas D. A2delta ligands
gabapentin and pregabalin: future implications in daily clinical practice. Hippokratia. 2010;14:71-5. 6. Shaw RJ, Dayal S, Good J, Bruckner AL, Joshi SV. Psychiatric medications for the
treatment of pruritus. Psychosom Med. 2007;69:970-8. 34. Wolfhagen FH, Sternieri E, Hop WC, Vitale G, Bertolotti M, Van Buuren HR. Oral
naltrexone treatment for cholestatic pruritus: A double-blind, placebo-controlled
study. Gastroenterology. 1997;113:1264-9. 7. Yosipovitch G, Samuel LS. Neuropathic and psychogenic itch. Dermatol Ther. 2008;21:32-41. 8. To TH, Clark K, Lam L, Shelby-James T, Currow DC. The role of ondansetron in
the management of cholestatic or uremic pruritus--a systematic review. J Pain
Symptom Manage. 2012;44:725-30. y
gy
35. Gupta MA, Gupta AK. Psychodermatology: an update. J Am Acad Dermatol. 1996;34:1030-46. 36. Arnold LM, Auchenbach MB, McElroy SL. Psychogenic excoriation. Clinical
features, proposed diagnostic criteria, epidemiology and approaches to treatment. CNS Drugs. 2001;15:351-9. y p
g
9. Yosipovitch G, Greaves MW, Schmelz M. Itch. Lancet. 2003;361:690-4. 10. Kini SP, DeLong LK, Veledar E, McKenzie-Brown AM, Schaufele M, Chen SC. The
impact of pruritus on quality of life: the skin equivalent of pain. Arch Dermatol. 2011;147:1153-6. Mailing address:
Lízie Emanuelle Eulalio Brasileiro
Hospital Universitário Onofre Lopes
Av. Nilo Peçanha, 620 - Petrópolis
59012-300 - Natal - RN
Brazil
Email: lizie.eeb@gmail.com DISCUSSION Controlled studies stood out,
whose basic cause of the itch is chronic kidney disease. In these, a
greater effectiveness was observed in most psychiatric drugs used
compared with placebo. However, due to the small amount of con-
trolled studies, especially in the other causes of itch, it is not possible
to generalize that psychotropic drugs are effective in the treatment
of itch of various causes. It was also shown that, depending on the
underlying cause of itch, different medications may be used.q Gabapentin and pregabalin are part of the class of anticon-
vulsant, derivatives of the inhibitory neurotransmitter GABA (gam-
ma amino butyric acid), but may have indications for peripheral
neuropathy and peripheral neuralgia. Through the mechanism of
action, they act so the post-synaptic excitability attenuates through Brasileiro LEE, Barreto DPC, Nunes EA 798 30. Zylicz Z, Krajnik M, Sorge AA, Costantini M. Paroxetine in the treatment of severe
non-dermatological pruritus: a randomized, controlled trial. J Pain Symptom
Manage. 2003;26:1105-12. Mailing address:
Lízie Emanuelle Eulalio Brasileiro
Hospital Universitário Onofre Lopes
Av. Nilo Peçanha, 620 - Petrópolis
59012-300 - Natal - RN
B
il How to cite this article: Brasileiro LEE, Barreto DPC, Nunes EA. Psychotropics in different causes of itch: systematic
review with controlled studies. An Bras Dermatol. 2016;91(6):791-8. REFERENCES Efficacy of doxepin in the treatment of chronic idiopathic urticaria. J Allergy Clin
Immunol. 1986;78:867-73. 26. Mayo MJ, Handem I, Saldana S, Jacobe H, Getachew Y, Rush AJ. Sertraline as a
first-line treatment for cholestatic pruritus. Hepatology. 2007;45:666-74. 27. Bergasa NV, McGee M, Ginsburg IH, Engler D. Gabapentin in patients with the
pruritus of cholestasis: a double-blind, randomized, placebo-controlled trial. Hepatology. 2006;44:1317-23. 28. Ahuja RB, Gupta GK. A four arm, double blind, randomized and placebo controlled
study of pregabalin in the management of post-burn pruritus. Burns. 2013;39:24-9. 29. Ahuja RB, Gupta R, Gupta G, Shrivastava P. A comparative analysis of cetirizine,
gabapentin and their combination in the relief of post-burn pruritus. Burns. 2011;37:203-7. 29. Ahuja RB, Gupta R, Gupta G, Shrivastava P. A comparative analysis of cetirizine,
gabapentin and their combination in the relief of post-burn pruritus. Burns. 2011;37:203-7. 29. Ahuja RB, Gupta R, Gupta G, Shrivastava P. A comparative analysis of cetirizine,
gabapentin and their combination in the relief of post-burn pruritus. Burns. 2011;37:203-7. How to cite this article: Brasileiro LEE, Barreto DPC, Nunes EA. Psychotropics in different causes of itch: systematic
review with controlled studies. An Bras Dermatol. 2016;91(6):791-8. An Bras Dermatol. 2016;91(6):791-8.
|
https://openalex.org/W4312266543
|
https://journal.ump.edu.my/jmmst/article/download/8571/2541
|
English
| null |
Study of Underfill Flow in Microchip Packaging Using Ansys
|
Journal of Modern Manufacturing Systems and Technology
| 2,022
|
cc-by
| 4,167
|
INTRODUCTION In this century, all sectors of technology are competing each other to be the finest and enhance their flaws in industry. They emphasize the capability of machines or materials used during the manufacturing process of a product to increase
the productivity and efficiency. Fluids are widely used in industry since they are one of the aspects that contribute to
machines performance. Metalworking fluids, which are mineral-based oils, are widely used as feedstock in the
manufacturing industry for a variety of lubricating and cooling applications. However, the excessive consumption of it
creating environmental and health problems. Crude Tamanu Oil (CTO) is one of manufacturing effort to improve 'greener'
metalworking applications, including the world's agricultural and socioeconomic sectors [1]. Newtonian and non-Newtonian fluids are two different types of liquids that have different properties. Newtonians
include air, water, steam, all gases, and most fluids made up of simple molecules and it follows Newton’s law of viscosity
which is the physical characteristic that describes flow resistance. The viscosity of Newtonian systems is independent of
shear rate [2], while non-Newtonian fluid, such as resin solution and molten polymer does not necessarily follow a linear
relationship with the shear strain rate. Non-Newtonian fluid behaviors can be characterized in one of four ways, dilatant,
pseudoplastic, rheopectic and thixotropic fluid. p
p
p
p
Die fitting, solderless connectors, component refurbishment, display connectivity, and heat dissipation are just a few
of the areas where electrically conductive adhesives (ECAs) are used. Due to their eco-friendliness, ECAs attract the
attention of scientific researchers as a green and sustainable lead-free connecting material that outperforms traditional
solders [3]. This adhesive is used to stick the electrical components such as microchip onto the flat base. The injection
fluid between the microchip surface and flat base is the same approach as the liquid injected between two plates and the
parameters such as fluid viscosity and flowrate are being observed to ensure the conducting fillers or ECAs spread and
solidifies uniformly. Lubrication in vehicles such as diesel emphasizes the rate of viscosity to ensure the efficiency of the
engine. The viscosity of the lubricant impacts the friction, which affects heat and it influences the pace of oil consumption
at the same time [4]. Manufacturing equipment generally requires proper lubrication. However, overly viscous lubricants
can choke and block pipes. Too thin lubricants provide insufficient protection for moving components [5]. INTRODUCTION The viscosity
of a product is measured in relation to its quality and efficiency. The study of fluids in motion lead to better understand
the functional structure and behavior so that both elements can be manipulated. For example, the type of fluid that
frequently used in electrical industries as a bonding technology is Electrically Conductive Adhesive (ECAs). Different
viscosity of ECAs gives different solderable quality on the ECAs interconnection formation [7]. JOURNAL OF MODERN MANUFACTURING SYSTEMS AND TECHNOLOGY (JMMST)
e-ISSN: 2636-9575 VOL. 6, ISSUE 2, 76 – 82
DOI: https://doi.org/10.15282/jmmst.v6i2.8571 Study of Underfill Flow in Microchip Packaging Using Ansys N.F.R. Hussin1 and N. Rosli1* 1Faculty of Manufacturing and Mechatronic Engineering Technology, Universiti Malaysia Pahang, 26600 Pahang, Malaysia. ARTICLE HISTORY
Received: 27th July 2022
Revised: 1st September 2022
Accepted: 26th September 2022 ARTICLE HISTORY
Received: 27th July 2022
Revised: 1st September 2022
Accepted: 26th September 2022 ABSTRACT – The project was related to microchip application where adhesive fluid is used to
stick the microchip onto the electrical flat base. In this project, 5mm of width and length of microchip
with 3 different types of solder ball diameter sizes are used in this project. The objective of the
project is to study the flow pattern and velocity of fluid during injection based on the decided
parameters, which are initial velocity, fluid viscosity, and the diameter of the solder ball. Each
parameter that has been set produces a different outcome in terms of flow pattern and velocity of
fluid. To maximise the performance of fluid flow in the aspect of uniformity of the fluid flow to fill the
gap around the solder ball, the flow pattern and the velocity are being observed and recorded
throughout the simulation process. KEYWORDS
Underfill flow
Microchip packaging
Non-Newtonian flow ARTICLE HISTORY
Received: 27th July 2022
Revised: 1st September 2022
Accepted: 26th September 2022 N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) Ansys is a flexible CFD program that may be used for a variety of applications. Ansys used to generate, and import
meshes, set boundary conditions, parameterize cases, run simulations, and post-process results. Ansys software is a
complete numerical tool that can handle compressible and incompressible fluid flows which is suitable for observing the
flow pattern of Newtonian fluid that is incompressible without having a change in viscosity which means they need
pressure or high temperature to change their viscosities. But for Newtonian fluid such as water, the viscosity will not
change even the pressure and temperature applied. METHODOLOGY A simulation study of fluid behaviors that will be carry out by observing the flow pattern and measuring the fluid
filling time based on the parameters such as initial velocity, fluid viscosity and the diameter of solder ball. This project
involves the study of underfill flow in microchip using Ansys workbench 2021 which is to observe fluid behaviors by
observing the flow pattern and velocity of liquid injected. The dimension of microchip is 5mm length and width with
pitch every solder ball is 1mm. Parameters include to conduct the simulation are the initial velocity, fluid viscosity and
the diameter of solder ball. The flow pattern and viscosity were observed by all the dependence stated. Experiment will
be conducted by CFD approach using Ansys software, fluid fluent. *CORRESPONDING AUTHOR | N. Rosli | nurrinarosli@ump.edu.my
© The Authors 2022. Published by Penerbit UMP. This is an open access article under the C journal.ump.edu.my/jmmst ◄ Effects of solder ball diameter There are 3 different diameters of solder ball, which are 0.2 mm, 0.3 mm, and 0.4 mm with a pitch of 1 mm, and the
observation can be made in relation to the flow pattern around the solder ball. The pitch is bigger than the previous
simulation because the recent simulation only focuses on the diameter of the solder ball that might influence the flow
pattern of the fluid and only replicates the microchip with a solder ball. Cases 1, 2 and 3 in Table 1 show the flow pattern at 0.03s with constant viscosity and initial velocity with different
solder ball diameters. The fluid flow pattern that passes through the gap between the solder balls can be seen reacting
with the solder ball diameter when the fluid hits obstacles or the solder ball during injection. The fluid surrounds the
solder ball with different continuity from 0.006s up to 0.03s. The front flow pattern at diameter 0.2 mm with initial
velocity 0.1 m/s and viscosity, 0.165 kg/ms conclude the same result as previous research which is lower velocity produce
curvy line at the edges to the middle at the first 2mm distance and the filling time increases with an increase of the solder
diameter. In case 1, the fluid flow permeability is less than in cases 2 and 3 because the smaller solder ball diameter makes the
fluid flow more uniform and there is no frequent collision with the solder ball wall, making the fluid pass through neatly
between the solder balls. In case 2, the fluid flow pattern around the solder balls is different with case 1 as it is not
uniformly filled with fluid. There is a space that is not passed by the fluid, causing there to be empty space around the
solder ball. This phenomenon shows that increasing the diameter of the solder ball in the underfill process causes non-
uniform flow as the fluid collides with the wider area of the solder ball's wall and the fluid bounces back and forth, making
the velocity in the area increase, causing the fluid to spread unevenly around the solder ball. The fluid spread wider than
the solder ball diameter, causing the empty space to occur during the fluid flow passing through the solder ball. Effects of initial velocity High initial velocity values affect the continuity of the flow pattern that passes through the space between the solder balls. The higher the initial velocity, the more tapered the fluid flow pattern becomes, and this can be observed at the beginning
of the injection. By visual observation based on Figure 1, the fluid flow pattern, or to be more precise, the front flow
pattern, may be seen clearly when the fluid breaks a barrier in front, which is a solder ball with high velocity, and the
volume of fluid filled at the edges exhibits a more tapered flow pattern than in the centre, and this also shows that the
volume of fluid filled unevenly in the middle of the plate. The parameter considered in this case is only dependent on the
initial velocity. Figure 1. Flow pattern at initial velocity 0.5m/s with viscosity 0.165 kg/ms at 0.006s. Figure 1. Flow pattern at initial velocity 0.5m/s with viscosity 0.165 kg/ms at 0.006s. Figure 2 shows that the front flow pattern is more uniform as the initial velocity is lower, which is 0.1 m/s, and the
fluid spreads evenly even if it breaks the barrier or the solder ball. The volume of fluid was filled at the centre evenly but
narrowed at the edges. The tapered flow pattern cannot be seen at the beginning of the injection or at the first 0.001 mm. Past research used a constant initial velocity for both cases, having a uniform flow at the beginning of injection but a
different filling time depending on the gap between the 2 plates or the thickness of the solder ball, while Figures 1 and 2
show a different initial velocity but the thickness is neglected as it is in a 2D simulation. The comparison that can be made
is by visual observation, specifically at the front flow, whereby increasing the initial velocity can increase the probability
of the fluid to taper at the edges and spaces around the solder ball.is by visual observation specifically at the front flow
weather by increasing the initial velocity can higher the probability of the fluid to tapered at the edges and while break
through solder ball. Figure 2. Flow pattern at initial velocity 0.1m/s with viscosity 0.165 kg/ms at 0.012s. Figure 2. Flow pattern at initial velocity 0.1m/s with viscosity 0.165 kg/ms at 0.012s. 77 journal.ump.edu.my/jmmst ◄ N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) Effects of viscosity The last parameter used in the recent simulation involves fluid viscosity. Previous research used 3 viscosities of fluid:
0.165, 0.34, and 0.7 kg/m s and this simulation also used the same parameter but used different solder ball diameters,
different from previous research where it used the factor of the thickness of the solder ball using the viscosities stated. However, a comparison can still be made by looking at the dependence of the flow pattern on the viscosity of the fluid,
whether it has a large impact or not on the fluid flow pattern. Table 2. Comparison of fluid flow pattern for viscosity 0.165,0.34,0.7 kg/ms. Experimental conditions
Flow pattern
Case 1
Viscosity = 0.165
Initial velocity = 0.1
Filling time = 0.03
Solder ball diameter = 0.4
Case 2
Viscosity = 0.34
Initial velocity = 0.1
Filling time = 0.03
Solder ball diameter = 0.4
Case 3
Viscosity = 0.7
Initial velocity = 0.1
Filling time = 0.03
Solder ball diameter = 0.4
al observation regarding the viscosity used for cases 1, 2 and 3, the fluid flow pattern as can be seen in
niform at low viscosity at 0.165 kg/ms and unevenly at 0.7 kg/ms. However, the viscosity factor does
nfluence the flow pattern because only a small change occurs in the flow pattern when high viscosity is
y space around the solder ball can still be seen and the tapered pattern can also be detected using different
at 0.7 kg/ms, the flow pattern is more tapered than at 0.34 kg/ms and 0.165 kg/ms. Table 2. Comparison of fluid flow pattern for viscosity 0.165,0.34,0.7 kg/ms. Through visual observation regarding the viscosity used for cases 1, 2 and 3, the fluid flow pattern as can be seen in
Table 2 is more uniform at low viscosity at 0.165 kg/ms and unevenly at 0.7 kg/ms. However, the viscosity factor does
not significantly influence the flow pattern because only a small change occurs in the flow pattern when high viscosity is
used but the empty space around the solder ball can still be seen and the tapered pattern can also be detected using different
viscosities, which at 0.7 kg/ms, the flow pattern is more tapered than at 0.34 kg/ms and 0.165 kg/ms. journal.ump.edu.my/jmmst ◄ Effects of solder ball diameter For the
last case, which is case 3 with the largest solder ball diameter, the flow pattern at 0.03s shows that there is empty space
at the end of the flow where the fluid does not fill the space around the solder ball evenly. The same goes for case 1 at
0.03s, but the consistency of the flow is better than in case 3, and the volume of fluid filled in case 3 is higher than in
cases 1 and 2, as both cases still have unfilled space at 0.03s, while case 3 almost fills the plate surface area at 0.03s. journal ump edu my/jmm
Table 1. Fluid flow pattern for diameter 0.2mm,0.3mm and 0.4mm. Experimental conditions
Flow pattern
Case 1
Diameter = 0.2mm
Initial velocity = 0.5 m/s
Viscosity = 0.165
Filling time = 0.03s
Case 2
Diameter = 0.3mm
Initial velocity = 0.5 m/s
Viscosity = 0.165
Filling time = 0.03s
Case 3
Diameter = 0.4mm
Initial velocity = 0.5 m/s
Viscosity = 0.165
Filling time = 0.03s Table 1. Fluid flow pattern for diameter 0.2mm,0.3mm and 0.4mm. 78 journal.ump.edu.my/jmmst ◄ N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) Fluid in Figure 3 with viscosity 0.7 kg/ms having a lower velocity at initial injection and increase at distance 0.003mm
while for viscosity 0.34 kg/ms and 0.165 kg/ms having a decreasing pattern at distance 0.003 m. This occur when fluid
passes through the solder ball and the velocity changes depend on how hard the collision happened towards the solder
ball’s wall that influence the fluid to move back and forth to push the fluid forward. C.Y. Khor previous research was
regarding the pressure drop and the gap height plotted graph which the pressure drop increased with decreased gap height. Thus, the gap height changes significantly affected the filling time and pressure drop in the underfill process. For this
project, the plotted graph is referring to velocity, viscosity, and distance x with different diameter solder ball. Solder ball
distract the fluid flow from going forward and by increasing the solder ball diameter, the velocity will be decreasing at
the initial distance of injection but rose at the end of plate, 5 mm as shown in Figure 3 and 4. Solder ball with diameter 0.4 mm show a different pattern of plotted graph shown in Figure 5 as the velocity is
increasing at the initial of injection but going down at the end of the plate, 5 mm because the fluid passes through larger
obstacle and collision occur more frequently causing the velocity at start higher than diameter 0.2 mm and 0.3 mm. The
velocity for viscosity 0.7 kg/ms is almost constant and consistent at viscosity 0.165 kg/ms from 0 mm to 5 mm and been
shown in Figure 6 because the initial velocity is higher, and the solder ball diameter is also small than others causing the
laminar flow to occur directly from inlet to outlet. However, for viscosity 0.34 kg/ms, the velocity rose at distance 4mm
because the inconsistency of fluid flow passes through solder ball and make a tapered flow pattern causing some area
uncovered with fluid. For Figures 7 and 8 have a same condition as Figures 4 and 5 that show a same pattern for plotted
graph but only with a different initial velocity that cause the velocity is higher than them. Figure 5. Velocity vs distance x for diameter 0.4mm with initial velocity 0.1m/s. Effects of viscosity Through visual observation regarding the viscosity used for cases 1, 2 and 3, the fluid flow pattern as can be seen in
Table 2 is more uniform at low viscosity at 0.165 kg/ms and unevenly at 0.7 kg/ms. However, the viscosity factor does
not significantly influence the flow pattern because only a small change occurs in the flow pattern when high viscosity is
used but the empty space around the solder ball can still be seen and the tapered pattern can also be detected using different
viscosities, which at 0.7 kg/ms, the flow pattern is more tapered than at 0.34 kg/ms and 0.165 kg/ms. Figure 3. Velocity vs distance x for diameter 0.2mm with initial velocity 0.1m/s. 0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.000
0.002
0.004
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 3. Velocity vs distance x for diameter 0.2mm with initial velocity 0.1m/s. Figure 4. Velocity vs distance x for diameter 0.3mm with initial velocity 0.1m/s. 0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 4. Velocity vs distance x for diameter 0.3mm with initial velocity 0.1m/s. 79 N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) 0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0.160
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 5. Velocity vs distance x for diameter 0.4mm with initial velocity 0.1m/s. 0.000
0.020
0.040
0.060
0.080
0.100
0.120
0.140
0.160
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 5. Velocity vs distance x for diameter 0.4mm with initial velocity 0.1m/s. Figure 6. Velocity vs distance x for diameter 0.2mm with initial velocity 0.5 m/s. 0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 6. Velocity vs distance x for diameter 0.2mm with initial velocity 0.5 m/s. 80 journal.ump.edu.my/jmmst ◄ N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022)
Figure 7. Velocity vs distance x for diameter 0.3mm with initial velocity 0.5 m/s. 0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022)
Figure 7. Velocity vs distance x for diameter 0.3mm with initial velocity 0.5 m/s. Figure 8. Velocity vs distance x for diameter 0.4mm with initial velocity 0.5 m/s. 0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Fi
7 V l
i
di
f
di
0 3
i h i i i l
l
i
0 5
/
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 7. Velocity vs distance x for diameter 0.3mm with initial velocity 0.5 m/s. Figure 8. Velocity vs distance x for diameter 0.4mm with initial velocity 0.5 m/s. 0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 7. Velocity vs distance x for diameter 0.3mm with initial velocity 0.5 m/s. Figure 7. Velocity vs distance x for diameter 0.3mm with initial velocity 0.5 m/s. N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) Figure 8. Velocity vs distance x for diameter 0.4mm with initial velocity 0.5 m/s. 0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
0.000
0.001
0.002
0.003
0.004
0.005
0.006
Velocity, m/s
Distance X, m
0.7
0.34
0.165 Figure 8. Velocity vs distance x for diameter 0.4mm with initial velocity 0.5 m/s. CONCLUSION In conclusion, all objectives have been achieved with respect to flow pattern and filling time. The simulation that has
been carried out shows that the initial velocity affects the uniformity of the fluid flow pattern. The higher the initial
velocity, the less uniform the fluid flow pattern between the two plates. Initial velocity, which is 0.1 m/s, produces a better
and even flow than 0.5 m/s. The diameter of the solder ball also proves that the larger the size of the solder ball, the bigger
the empty space around the solder ball when fluid passes through and most probably leads to tapered flow, causing the
surface of the plate to not be covered with fluid evenly and the probability of the microchip losing grip is higher. Other
than that, with respect to fluid viscosity, which can be seen through visual observation where the response of the flow
pattern to the viscosity is not very significant, as only slight differences can be seen in 0.7 kg/ms, 0.34 kg/ms, and 0.165
kg/ms for all cases. Filling time takes longer at 0.1 m/s than at 0.5 m/s, but in terms of uniformity of fluid flow, the lower the initial
velocity, the higher the time taken to fill the plate, and the better the front flow and flow pattern from 0 mm to 5 mm. Solder ball diameter influence the velocity in fluid flow. The larger the diameter of solder ball, the collision of fluid with
solder ball make the velocity decrease while when the fluid spread to fill the gap around the solder ball, the velocity
become increase. The authors would like to thank International Islamic University Malaysia and Universiti Malaysia Pahang for funding
this work under the IIUM-UMP Sustainable Research Collaboration Grant (RDU223221). ACKNOWLEDGEMENT The authors would like to thank International Islamic University Malaysia and Universiti Malaysia Pahang for funding
this work under the IIUM-UMP Sustainable Research Collaboration Grant (RDU223221). 81 journal.ump.edu.my/jmmst ◄ N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) N.F.R. Hussin and N. Rosli │ Journal of Modern Manufacturing Systems and Technology │ Vol. 6, Issue 2 (2022) journal.ump.edu.my/jmmst ◄ REFERENCES [1]
A. S. Abdul Sani, S. Baharom, N. A. Mamat, A. S. Mohd Rozlan, and N. Talib, “Comparative evaluation
il
f
l
ki
fl id ” M
i l T d
P
di
S
2021 d i 10 1016/J MATPR [1]
A. S. Abdul Sani, S. Baharom, N. A. Mamat, A. S. Mohd Rozlan, and N. Talib, “Comparative evaluation
oil performance as metalworking fluids,” Materials Today: Proceedings, Sep. 2021, doi: 10.1016/J.MATPR [1]
A. S. Abdul Sani, S. Baharom, N. A. Mamat, A. S. Mohd Rozlan, and N. Talib, Comparative evaluation of crude Tamanu
oil performance as metalworking fluids,” Materials Today: Proceedings, Sep. 2021, doi: 10.1016/J.MATPR.2021.08.232. [2]
E. Katoueizadeh, M. Rasouli, and S. M. Zebarjad, “The rheological behavior of the non-Newtonian thixotropic colloidal silica
gels from sodium silicate,” Materials Chemistry and Physics, vol. 272, p. 124994, Nov. 2021, doi:
10.1016/J.MATCHEMPHYS.2021.124994. p
g
,
y
g ,
p
,
[2]
E. Katoueizadeh, M. Rasouli, and S. M. Zebarjad, “The rheological behavior of the non-Newtonian thixotropic colloidal silica
gels from sodium silicate,” Materials Chemistry and Physics, vol. 272, p. 124994, Nov. 2021, doi:
10.1016/J.MATCHEMPHYS.2021.124994. [3]
R. Aradhana, S. Mohanty, and S. K. Nayak, “A review on epoxy-based electrically conductive adhesiv
Journal of Adhesion and Adhesives, vol. 99, p. 102596, Jun. 2020, doi: 10.1016/J.IJADHADH.2020.10259 [4]
M. J. Souza De Carvalho, P. Rudolf Seidl, C. R. Pereira Belchior, and J. Ricardo Sodr, “Lubricant viscosity and viscosity
improver additive effects on diesel fuel economy,” Tribology International, vol. 43, no. 12, pp. 2298–2302, Dec. 2010, doi:
10.1016/J.TRIBOINT.2010.07.014. [5]
A. Maltby, “Internal lubricants yield multiple benefits in injection moulding,” Plastics, Additives and Compounding, vol. 7,
no. 6, pp. 28–31, Nov. 2005, doi: 10.1016/S1464-391X (05)70487-6. [6]
S. Entezari, A. Shakiba, and H. Niazmand, “Numerical investigation of the effects of cannula geometry on hydraulic blood
flow to prevent the risk of thrombosis,” Computers in Biology and Medicine, vol. 134, p. 104484, Jul. 2021, doi:
10.1016/J.COMPBIOMED.2021.104484. [7]
B. S. Yim et al., “Characteristics of solderable electrically conductive adhesives (ECAs) for electronic packaging,”
Microelectronics Reliability, vol. 52, no. 6, pp. 1165–1173, Jun. 2012, doi: 10.1016/J.MICROREL.2011.12.004. 82 82
|
https://openalex.org/W2030739006
|
https://liu.diva-portal.org/smash/get/diva2:1238367/FULLTEXT01
|
English
| null |
Tension-dependent removal of pericentromeric shugoshin is an indicator of sister chromosome biorientation
|
Genes & development
| 2,014
|
cc-by
| 19,480
|
2014 Nerusheva et al.
This article, published in Genes & Develop-
ment, is available under a Creative Commons License (Attribution 4.0
International), as described at http://creativecommons.org/licenses/by/
4.0. Cold Spring Harbor Laboratory Pre
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Pre
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Tension-dependent removal of
pericentromeric shugoshin is an indicator
of sister chromosome biorientation
Olga O. Nerusheva, Stefan Galander, Josefin Fernius, David Kelly, and Adele L. Marston1
The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR,
United Kingdom During mitosis and meiosis, sister chromatid cohesion resists the pulling forces of microtubules, enabling the
generation of tension at kinetochores upon chromosome biorientation. How tension is read to signal the
bioriented state remains unclear. Shugoshins form a pericentromeric platform that integrates multiple functions
to ensure proper chromosome biorientation. Here we show that budding yeast shugoshin Sgo1 dissociates from the
pericentromere reversibly in response to tension. The antagonistic activities of the kinetochore-associated Bub1
kinase and the Sgo1-bound phosphatase protein phosphatase 2A (PP2A)-Rts1 underlie a tension-dependent
circuitry that enables Sgo1 removal upon sister kinetochore biorientation. Sgo1 dissociation from the pericentromere
triggers dissociation of condensin and Aurora B from the centromere, thereby stabilizing the bioriented state. Conversely, forcing sister kinetochores to be under tension during meiosis I leads to premature Sgo1 removal and
precocious loss of pericentromeric cohesion. Overall, we show that the pivotal role of shugoshin is to build
a platform at the pericentromere that attracts activities that respond to the absence of tension between sister
kinetochores. Disassembly of this platform in response to intersister kinetochore tension signals the bioriented
state. Therefore, tension sensing by shugoshin is a central mechanism by which the bioriented state is read. [Keywords: mitosis; meiosis; shugoshin; biorientation; tension; kinetochore] Supplemental material is available for this article. Supplemental material is available for this article. Received February 19, 2014; revised version accepted May 20, 2014. Received February 19, 2014; revised version accepted May 20, 2014. For accurate dissemination of the genome, chromosomes
are first duplicated during S phase of the cell cycle to
generate identical sister chromatids that are held together
by cohesion. During mitosis, to ensure that each daughter
cell receives one copy of each chromosome after cohesion
is lost, sister kinetochores must attach to microtubules
from opposite poles. Cohesion resists the pulling force of
microtubules, resulting in the generation of tension at
sister kinetochores. Sister kinetochore tension is critical
in enabling biorientation to be sensed, thereby allowing
chromosome segregation to proceed. Although central to
the segregation process, the underlying mechanism by
which this state of tension is read is not known. on the cohesin complex and phosphorylation of histone
2A on residue S121 by the kinetochore-associated kinase
Bub1 (Kawashima et al. 2010; Liu et al. 2013a). Shugosh-
ins are emerging as important pericentromeric ‘‘adaptor’’
proteins that integrate multiple functions that contribute
to accurate chromosome segregation (Gutie´rrez-Caballero
et al. 2012; Rattani et al. 2013; Verzijlbergen et al. 2014). 1Corresponding author
E-mail adele.marston@ed.ac.uk
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.240291.
114. Freely available online through the Genes & Development Open
Access option. Nerusheva et al. cosegregation of sister chromatids. Once homologs are
aligned on the meiotic spindle, cohesion is lost from
chromosome arms, resolving chiasmata and triggering
the segregation of homologs. However, cohesion in the
pericentromere must be protected from loss during meio-
sis I to allow the biorientation of sister chromatids during
meiosis II. The protection of pericentromeric cohesin
during meiosis I depends on shugoshin, which recruits
protein phosphatase 2A associated with its B9-type regula-
tory subunit (PP2A-B9) to the pericentromere (Kitajima
et al. 2006; Riedel et al. 2006). PP2A-B9 reverses phosphor-
ylation of the meiosis-specific Rec8 subunit of cohesin in
the pericentromere, making it refractory to cleavage by the
protease separase (Brar et al. 2006; Ishiguro et al. 2010;
Katis et al. 2010; Attner et al. 2013). Shugoshin similarly
spatially regulates cohesin loss during mammalian mito-
sis, where the bulk of cohesin dissociates from chromo-
some arms during prophase due to the activity of the
destabilizing protein Wapl (Waizenegger et al. 2000; Hauf
et al. 2005; Kueng et al. 2006; Tang et al. 2006; Shintomi
and Hirano 2009). In this case, the shugoshin–PP2A-B9
complex dephosphorylates the Wapl-counteracting pro-
tein sororin, thereby maintaining its pericentromeric
localization (Nishiyama et al. 2010; Liu et al. 2013b). close proximity. In contrast, tension moves the outer kinet-
ochore away from the inner centromere. This spatial separa-
tion is thought to allow outer kinetochore substrates to
evade the reach of Aurora B phosphorylation, thereby
stabilizing kinetochore–microtubule attachments (Keating
et al. 2009; Liu et al. 2009; Welburn et al. 2010). However,
this model has recently been challenged by the finding that
the centromere localization of the CPC does not need to be
tightly regulated for tension sensing by Aurora B in budding
yeast, suggesting that other mechanisms may contribute
(Campbell and Desai 2013). (
p
)
Interestingly, in both mitosis and meiosis, shugoshin
plays its critical roles at pericentromeres only when sister
kinetochores are not under tension. This suggests that
shugoshins may govern the tension-sensing process. In-
deed, shugoshin undergoes a tension-dependent relocation
from the inner centromere to the kinetochores in sper-
matocytes, oocytes, and human somatic cells (Gomez
et al. 2007; Lee et al. 2008; Liu et al. 2013a). In human
cells, the tension-dependent relocalization of shugoshin to
kinetochores is triggered by dephosphorylation and is
important for accurate segregation (Liu et al. 2013a). However the underlying mechanism of this relocation
and its role is not well understood. Nerusheva et al. Here we use budding
yeast to address the role of spindle tension in the regula-
tion and function of its single shugoshin, Sgo1. We show
that intersister kinetochore tension negatively regulates
Sgo1 association with pericentromeric chromatin. Spatial
separation of the kinetochore-associated Bub1 kinase trig-
gers Sgo1 removal from the pericentromere, facilitated by
Sgo1 association with PP2A. We further show that Sgo1
release from the pericentromere triggers Aurora B removal
upon biorientation, thereby initiating the silencing of the
error correction process. Finally, we demonstrate that the
protection of pericentromeric cohesin in meiosis I by Sgo1
relies on the suppression of sister kinetochore biorienta-
tion. Overall, our findings reveal tension-dependent Sgo1
removal from the pericentromere as a fundamental sign
that a pair of sister kinetochores has bioriented. In addition to protecting pericentromeric cohesin dur-
ing meiosis and mammalian mitosis, shugoshins play
a conserved role in promoting biorientation of sister
chromatids (Indjeian et al. 2005; Huang et al. 2007; Kiburz
et al. 2008). Biorientation is achieved owing to a bias for
sister kinetochores to be captured from opposite poles
together with an error correction mechanism that de-
stabilizes incorrect attachments that lack tension (for
review, see Tanaka 2010). We recently found that shu-
goshins contribute to sister kinetochore biorientation by
both enabling the bias to capture by microtubules from
opposite poles and engaging the error correction machin-
ery (Verzijlbergen et al. 2014). Error correction relies on
the chromosomal passenger complex (CPC), which is
comprised of Aurora B kinase (Ipl1 in budding yeast)
and its centromere targeting factor, survivin (Bir1), to-
gether with INCENP (Sli15) and borealin (Nbl1) (for
review, see Carmena et al. 2012). Maintenance of the
CPC at centromeres requires shugoshin (Kawashima
et al. 2007; Vanoosthuyse et al. 2007; Yamagishi et al. 2010; Rivera et al. 2012; Verzijlbergen et al. 2014). Additionally, shugoshin recruits the chromosome-orga-
nizing complex condensin to the pericentromere to bias
sister kinetochores toward biorientation (Verzijlbergen
et al. 2014). Therefore, overall, Shugoshin acts as an
adaptor
that
attracts
multiple
activities,
including
PP2A-B9, CPC, and condensin, to the pericentromere to
safeguard accurate chromosome segregation. Results Spindle tension between sister kinetochores promotes
Sgo1 removal from the pericentromere during mitosis Tension-dependent removal of
pericentromeric shugoshin is an indicator
of sister chromosome biorientation
Olga O. Nerusheva, Stefan Galander, Josefin Fernius, David Kelly, and Adele L. Marston1
The Wellcome Trust Centre for Cell Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JR,
United Kingdom Shugoshins were first identified as regulators of chromo-
some segregation during meiosis (Katis et al. 2004;
Kitajima et al. 2004; Marston et al. 2004; Rabitsch et al. 2004; Kerrebrock et al. 1992). During meiosis I, a unique
segregation event occurs in which the maternal and
paternal chromosomes (homologs) are separated and that
requires homologs to be linked, usually by chiasmata, the
products of meiotic recombination (for reviews, see
Marston and Amon 2004; Marston 2014). Also during
meiosis I, sister kinetochore biorientation is suppressed,
and sister kinetochores attach to microtubules from the
same pole, known as mono-orientation, to ensure the The conserved shugoshin family of proteins has been
implicated in the sensing of intersister kinetochore ten-
sion (Indjeian et al. 2005; Huang et al. 2007; Kiburz et al. 2008; Kawashima et al. 2007; Vanoosthuyse et al. 2007). Shugoshins are localized to the region surrounding the
centromere (the pericentromere) in a manner dependent Corresponding author
E-mail adele.marston@ed.ac.uk
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.240291. 114. Freely available online through the Genes & Development Open
Access option. DEVELOPMENT 28:1291–1309 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/14; www.genesdev.org 1291 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension Wild-type (AM6390) and stu2-277 (AM9093) cells carrying pMET3-CDC20 and SGO1-6HA as well as a no tag control (AM2508) were
treated as in G except that cells were shifted to 37°C after release from G1. Cells were harvested for Sgo1-6HA ChIP-qPCR after 1.5 h
(wild type) or 2.25 h (stu2-277) to obtain similar numbers of cells arrested in metaphase. In H and I, the average of three independent
repeats is shown, with error bars representing standard error. Figure 1. Sgo1 is removed from the peri-
centromere in metaphase in the presence
of microtubules. (A,B) Sgo1 dispersal into
the nucleus in metaphase is dependent on
microtubules. Cells carrying SGO1-6HA
and pMET3-CDC20 (strain AM6390) were
arrested in G1 with a factor. The culture
was split, a factor was washed out, and
both cultures were released into medium
containing methionine to repress CDC20
and induce arrest in metaphase. Either
DMSO (A; tension) or nocodazole (B; no
tension) was added. Samples were extracted
at the indicated intervals after release
from G1 for Sgo1-6HA and tubulin immu-
nofluorescence, and Sgo1 localization (no,
dot/stripe, nuclear) and spindle morphol-
ogy were scored. Schematic diagrams in-
dicate chromosome configuration in the
presence (A) or absence (B) of tension. (C)
Loss of Sgo1-yeGFP from the pericentro-
mere coincides with the appearance of
a bilobed kinetochore signal. Cells carry-
ing SGO1-yeGFP and MTW1-tdTomato
(strain AM9233) were imaged on a micro-
fluidics device at 15-min intervals after re-
lease from G1 arrest. (D–F) Sgo1-yeGFP loses
its pericentromeric localization as kineto-
chore signals split. Strain AM9233 (pMET3-
CDC20 SGO1-yeGFP MTW1-tdTomato)
was arrested in G1 using a factor and
released in medium containing 8 mM
methionine to deplete Cdc20. Images of
multiple cells were taken every 15 min,
with the first time point taken 0.5 h after
the release from G1. (D) Line scans across
kinetochore foci of single cells were as-
sembled from 100 images to generate a V
plot showing Sgo1-GFP localization as
interkinetochore distance increases. Bar,
2mm. (E) Bar chart showing the fraction of
cells with the indicated Sgo1 localization
at each time point. (F) The distance be-
tween Mtw1-tdTomato signals and the
localization of Sgo1-yeGFP was scored in
200 cells. The bean plot shows the distri- p
bution of interkinetochore distances for which each localization type was scored. The horizontal line represents the mean. (G) Sgo1 is
removed from the pericentromere at metaphase in the presence of microtubules. Sgo1 regulated by tension Sgo1 regulated by tension were not treated with nocodazole, Sgo1 first appeared as
a bright dot within the nucleus, likely representing the
pericentromere (Kiburz et al. 2005). Interestingly, by 100 min after release from G1, the Sgo1-GFP signal had
dissipated throughout the nucleus (Fig. 1A). However,
in nocodazole-treated cells, the dot-like Sgo1-6HA local- pericentromere (Kiburz et al. 2005). Interestingly, by 100
in nocodazole-tre
bution of interkinetochore distances for which each localization type was scored. The hor
removed from the pericentromere at metaphase in the presence of microtubules. Strains
AM2508 (pMET3-CDC20; no tag control) were released from G1 into medium containin
nocodazole (+NOC). After 2 h, cells were harvested, and Sgo1-6HA levels at the indicated
ChIP-qPCR. The average of three experimental repeats (qPCR performed in triplicate in e
bars representing standard error. For the no tag control (AM2508), representative values ar
also Supplemental Figure S2, G and H, for Sgo1-6HA association with sites on chromosome
as5 (AM8217) cells carrying pMET3-CDC20 and SGO1-6HA as well as a no tag control (AM
PP1 (50 mM) was added to inhibit Ipl1 when bud formation was observed after release from
on chromosome IV were measured by ChIP-qPCR in cells harvested 2 h (wild type) or 2. a similar number of cells arrested in metaphase. (I) The stu2-277 mutation prevents Sgo
Wild-type (AM6390) and stu2-277 (AM9093) cells carrying pMET3-CDC20 and SGO1-6HA
treated as in G except that cells were shifted to 37°C after release from G1. Cells were har
(wild type) or 2.25 h (stu2-277) to obtain similar numbers of cells arrested in metaphase. I
t i
h
ith
b
ti
t
d
d Figure 1. Sgo1 is removed from the peri-
centromere in metaphase in the presence
of microtubules. (A,B) Sgo1 dispersal into
the nucleus in metaphase is dependent on
microtubules. Cells carrying SGO1-6HA
and pMET3-CDC20 (strain AM6390) were
arrested in G1 with a factor. The culture
was split, a factor was washed out, and
both cultures were released into medium
containing methionine to repress CDC20
and induce arrest in metaphase. Either
DMSO (A; tension) or nocodazole (B; no
tension) was added. Samples were extracted
at the indicated intervals after release
from G1 for Sgo1-6HA and tubulin immu-
nofluorescence, and Sgo1 localization (no,
dot/stripe, nuclear) and spindle morphol-
ogy were scored. Schematic diagrams in-
dicate chromosome configuration in the
presence (A) or absence (B) of tension. Sgo1 regulated by tension (C)
Loss of Sgo1-yeGFP from the pericentro-
mere coincides with the appearance of
a bilobed kinetochore signal. Cells carry-
ing SGO1-yeGFP and MTW1-tdTomato
(strain AM9233) were imaged on a micro-
fluidics device at 15-min intervals after re-
lease from G1 arrest. (D–F) Sgo1-yeGFP loses
its pericentromeric localization as kineto-
chore signals split. Strain AM9233 (pMET3-
CDC20 SGO1-yeGFP MTW1-tdTomato)
was arrested in G1 using a factor and
released in medium containing 8 mM
methionine to deplete Cdc20. Images of
multiple cells were taken every 15 min,
with the first time point taken 0.5 h after
the release from G1. (D) Line scans across
kinetochore foci of single cells were as-
sembled from 100 images to generate a V
plot showing Sgo1-GFP localization as
interkinetochore distance increases. Bar,
2mm. (E) Bar chart showing the fraction of
cells with the indicated Sgo1 localization
at each time point. (F) The distance be-
tween Mtw1-tdTomato signals and the
localization of Sgo1-yeGFP was scored in
200 cells. The bean plot shows the distri-
bution of interkinetochore distances for which each localization type was scored. The horizontal line represents the mean. (G) Sgo1 is
removed from the pericentromere at metaphase in the presence of microtubules. Strains AM6390 (pMET3-CDC20 SGO1-6HA) and
AM2508 (pMET3-CDC20; no tag control) were released from G1 into medium containing methionine and either DMSO (NOC) or
nocodazole (+NOC). After 2 h, cells were harvested, and Sgo1-6HA levels at the indicated sites on chromosome IV were analyzed by
ChIP-qPCR. The average of three experimental repeats (qPCR performed in triplicate in each case) is shown for AM6390, with error
bars representing standard error. For the no tag control (AM2508), representative values are shown from one of these experiments. See
also Supplemental Figure S2, G and H, for Sgo1-6HA association with sites on chromosomes III and V. (H) Wild-type (AM6390) and ipl1-
as5 (AM8217) cells carrying pMET3-CDC20 and SGO1-6HA as well as a no tag control (AM2508) were treated as in G except that NA-
PP1 (50 mM) was added to inhibit Ipl1 when bud formation was observed after release from G1. Sgo1-6HA levels at the indicated sites
on chromosome IV were measured by ChIP-qPCR in cells harvested 2 h (wild type) or 2.5 h (ipl1-as) after release from G1 to obtain
a similar number of cells arrested in metaphase. (I) The stu2-277 mutation prevents Sgo1 removal in the presence of microtubules. Spindle tension between sister kinetochores promotes
Sgo1 removal from the pericentromere during mitosis Budding yeast have a single shugoshin protein, Sgo1, that
localizes to the pericentromere and functions to both
protect cohesin in meiosis I and promote sister kineto-
chore biorientation in mitosis. To explore the possibility
that intersister kinetochore tension regulates Sgo1 distri-
bution, we monitored Sgo1-6HA localization by immuno-
fluorescence as cells progressed from G1 into a metaphase
arrest in either the presence or absence of microtubules. We
used cells in which the essential APC regulator CDC20 was
placed under the control of the methionine-repressible
promoter pMET3 (pMET3-CDC20) to induce metaphase
arrest by addition of methionine. Cells carrying SGO1-6HA
and pMET3-CDC20 were released from G1 into medium
containing methionine and either nocodazole (to depoly-
merize microtubules) or DMSO (as a control). In cells that Although the ability to discriminate between tension-
generating and tension-less attachments is the key to achiev-
ing chromosome biorientation (Nicklas and Ward 1994), it is
not well understood. One way in which changes in kineto-
chore tension can be sensed is distance-dependent substrate
accessibility (for review, see Lampson and Cheeseman 2011). Indeed, in the absence of tension, the outer kinetochore and
the inner centromere (where the CPC is localized) are in 1292 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
wnloaded from Sgo1 dispersal into the nucleus occurs as sister
kinetochores biorient To more accurately determine the relative timing of the
establishment of intersister kinetochore tension and Sgo1
removal from the pericentromere, we released live cells
with labeled kinetochores (MTW1-tdTomato) and SGO1-
GFP from a G1 arrest and imaged them at 15-min intervals
as they progressed into a metaphase arrest induced by
CDC20 depletion (Fig. 1C; Supplemental Movie S1). This
confirmed that Sgo1 initially appears as a bright pericen-
tromeric dot before dispersing into the nucleus during
metaphase (Fig. 1C; Supplemental Movie S1), and this was
also observed in cells that were not arrested in metaphase
or previously arrested in G1 (Supplemental Fig. S1H,I). Fluorescence intensity measurements confirmed deple-
tion of Sgo1-GFP from the area occupied by the kineto-
chores and spindle during metaphase (Supplemental Fig. S1J,K). Assembled line scans of kinetochore foci separated
by increasing distance suggested that Sgo1 release from the
pericentromere correlated with increased interkineto-
chore distance (Fig. 1D). We measured the longest distance
covered by the Mtw1-tdTomato foci and scored the Sgo1-
GFP signal in at least 200 live cells at 15-min intervals
after release from G1. Figure 1, E and F, shows that release
of Sgo1-GFP into the nucleus occurred as Mtw1-tdTomato
distance increased to ;1.5 mm (120 min after release from
G1). Therefore, Sgo1 removal from the pericentromere
occurs concomitant with the establishment of intersister
kinetochore tension and biorientation. With this in mind, we used ChIP to analyze Sgo1
association with the pericentromere in cells arrested in
metaphase in the presence and absence of microtubules. Cells carrying pMET3-CDC20 and SGO1-6HA as well as
a no tag control were treated with methionine to induce
a metaphase arrest in either the presence or absence of
nocodazole, and Sgo1-6HA levels were analyzed by ChIP-
qPCR on chromosomes III, IV, and V (Fig. 1G; Supplemental
Fig. S2G,H). Spindle length measurements and Pds1 stain-
ing confirmed that the majority of cells remained arrested in
metaphase at the time of harvesting (Supplemental Fig. 2I,J). As our live-cell analysis revealed, Sgo1 associates with the
pericentromere only when sister kinetochores are not under
tension (Fig. 1G; Supplemental Fig. S2G,H). Sgo1 associa-
tion with the pericentromere is not dependent on spindle
checkpoint activation in response to unattached kineto-
chores generated by the nocodazole treatment because
deletion of the spindle checkpoint component MAD2 did
not reduce Sgo1 protein levels or its association with the
pericentromere (Supplemental Fig. S2K,L). Nerusheva et al. Nerusheva et al. Eckert et al. 2007; Ocampo-Hafalla et al. 2007; Kogut
et al. 2009). However, live-cell microscopy experiments
have shown that cohesin remains localized at pericen-
tromeres during metaphase, questioning the significance
of the ChIP experiments (Mc Intyre et al. 2007; Yeh et al. 2008; Rowland et al. 2009). Indeed, we found that cen-
tromeric quantitative PCR (qPCR) values were also re-
duced by the presence of microtubules when the consti-
tutive kinetochore subunits Mtw1 and Ndc10 were
immunoprecipitated (Supplemental Fig. S2D,E). More-
over, the levels of TetR-GFP artificially tethered to tetOs
adjacent to CEN3 were also reduced twofold by the
presence of microtubules as measured by ChIP (Supple-
mental Fig. S2F). It is unlikely that tension causes re-
moval of core kinetochore proteins and tethered TetR-
GFP from the centromere, as no such change was ob-
served by microscopy (e.g., Fig. 1C; OO Nerusheva and
AL Marston, unpubl.). Instead, we suggest that the dif-
ference relates to a reduced ChIP efficiency of pericen-
tromeric sequences separated by tension. Importantly,
only where the pericentromeric ChIP-qPCR signal is
reduced more than twofold by microtubule-dependent
tension can we be confident that this is due to a decrease
in the association of the protein measured. ization persisted, and uniform nuclear staining was not
observed (Fig. 1B). Consistently, treatment of live cells
with increasing doses of microtubule-depolymerizing
drugs was shown to increase Sgo1 levels at the pericen-
tromere (Haase et al. 2012). These findings suggest that
metaphase spindle formation triggers the release of
Sgo1-6HA from the pericentromere into the nucleus. Sgo1 is absent in a-factor-arrested G1 cells, accumulates
upon cell cycle entry, and is degraded during anaphase
(Marston et al. 2004). In cells released from a G1 arrest,
chromatin immunoprecipitation (ChIP) showed that Sgo1
associates with the pericentromere and is later dispersed
into the nucleus prior to its degradation in anaphase,
demonstrating that release from the pericentromere is
not a consequence of the metaphase arrest (Supplemental
Fig. S1A–G). Sgo1 is absent from pericentromeres under tension To test whether the disappearance of the subnuclear
Sgo1-GFP dot upon tension establishment corresponds
to Sgo1 release from the pericentromeric chromatin, we
sought to use ChIP. Based on ChIP assays, the localization
of cohesin and its Scc2 loader in the pericentromere is
thought to be negatively regulated by tension (Eckert
et al. 2007; Ocampo-Hafalla et al. 2007; Kogut et al. 2009). Indeed, the recovery of pericentromeric sequences after
ChIP of the cohesin subunit Scc1 is lower when cells are
arrested in metaphase with microtubules compared with
those without microtubules (Supplemental Fig. S2A–C; Sgo1 dispersal into the nucleus occurs as sister
kinetochores biorient Addition of
nocodazole to cells already arrested in metaphase led to
Sgo1 accumulation at the centromere, indicating that Sgo1
removal under tension is reversible (Supplemental Fig. S2M,N). We conclude that Sgo1 associates with the peri-
centromere only in the absence of microtubules and that
this reduction can be readily observed by ChIP. Sgo1 regulated by tension Strains AM6390 (pMET3-CDC20 SGO1-6HA) and
AM2508 (pMET3-CDC20; no tag control) were released from G1 into medium containing methionine and either DMSO (NOC) or
nocodazole (+NOC). After 2 h, cells were harvested, and Sgo1-6HA levels at the indicated sites on chromosome IV were analyzed by
ChIP-qPCR. The average of three experimental repeats (qPCR performed in triplicate in each case) is shown for AM6390, with error
bars representing standard error. For the no tag control (AM2508), representative values are shown from one of these experiments. See
also Supplemental Figure S2, G and H, for Sgo1-6HA association with sites on chromosomes III and V. (H) Wild-type (AM6390) and ipl1-
as5 (AM8217) cells carrying pMET3-CDC20 and SGO1-6HA as well as a no tag control (AM2508) were treated as in G except that NA-
PP1 (50 mM) was added to inhibit Ipl1 when bud formation was observed after release from G1. Sgo1-6HA levels at the indicated sites
on chromosome IV were measured by ChIP-qPCR in cells harvested 2 h (wild type) or 2.5 h (ipl1-as) after release from G1 to obtain
a similar number of cells arrested in metaphase. (I) The stu2-277 mutation prevents Sgo1 removal in the presence of microtubules. Wild-type (AM6390) and stu2-277 (AM9093) cells carrying pMET3-CDC20 and SGO1-6HA as well as a no tag control (AM2508) were
treated as in G except that cells were shifted to 37°C after release from G1. Cells were harvested for Sgo1-6HA ChIP-qPCR after 1.5 h
(wild type) or 2.25 h (stu2-277) to obtain similar numbers of cells arrested in metaphase. In H and I, the average of three independent
repeats is shown, with error bars representing standard error. GENES & DEVELOPMENT 1293 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Bub1 removal is sufficient for Sgo1 removal Bub1 removal is sufficient for Sgo1 removal To determine whether the continued presence of Bub1 is
essential for the maintenance of Sgo1 in the pericentro-
mere, we used the auxin-inducible degron (aid) system
(Nishimura et al. 2009) to conditionally degrade Bub1 in
metaphase-arrested cells in the presence of nocodazole. Cells were harvested 1 h after release from G1 into
nocodazole, and Sgo1 levels were measured by ChIP-
qPCR. Subsequently, the culture was split: One-half of
the culture was treated with auxin (NAA) to induce Bub1
degradation, while the other half received no NAA (Fig. 2F). Prior to Bub1 degradation, as expected, Sgo1 was
localized throughout the pericentromere, although levels
in the Bub1-aid strain were considerably lower, presum-
ably due to the partial functionality or stability of the
Bub1-aid fusion protein (Fig. 2H). However, addition of
auxin (NAA) led to Bub1 degradation, and Sgo1 was
delocalized from the pericentromere (Fig. 2G,H). We
conclude that continued Bub1 presence is required for
Sgo1 maintenance at the pericentromere. Sgo1 is increased at the pericentromere in the absence
of Rts1 Sgo1 is increased at the pericentromere in the absence
of Rts1 The finding that inactivation of Bub1 kinase leads to Sgo1
removal from the pericentromere predicts the existence of
a phosphatase that reverses Bub1-dependent phosphoryla-
tion. A prime candidate is the PP2A, a tripartite enzyme
comprised of a scaffold (A), regulatory (B), and catalytic (C)
subunit (Shi 2009). In budding yeast, there are two alter-
native regulatory subunits, Rts1 and Cdc55. PP2A-Rts1
associates with Sgo1 during mitosis and meiosis (Riedel
et al. 2006; Xu et al. 2009), whereas PP2A-Cdc55 acts
downstream from Sgo1 in preventing anaphase onset (Clift
et al. 2009; Bizzari and Marston 2011; Yaakov et al. 2012). We examined the levels of Sgo1 at the pericentromere in
cells lacking the PP2A regulatory subunits Rts1 or Cdc55. While pericentromeric levels of Sgo1 were modestly in-
creased in cdc55D cells arrested in metaphase without
microtubules, deletion of RTS1 led to an approximately
fourfold increase in pericentromeric Sgo1, although total
cellular levels remained unchanged (Fig. 3A; Supple-
mental Fig. S3A). However, the majority of Sgo1 was
removed when sister kinetochores were under tension
even in cells lacking RTS1 (Fig. 3A). This suggests that
PP2A-Rts1 plays the predominant role in reducing Sgo1
levels at the pericentromere, with other phosphatases,
including PP2A-Cdc55, also being important. Intersister kinetochore tension is responsible for Sgo1
removal from the pericentromere Our findings suggest that Sgo1 association with the
pericentromere is negatively regulated by microtubules. To further investigate the effect of tension on Sgo1
removal, we employed two methods that reduce kineto-
chore tension. First, we inactivated the Aurora B kinase
(Ipl1) using a version (ipl1-as5) sensitive to the ATP GENES & DEVELOPMENT 1294 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension analog NAPP1, which results in syntelic attachments
(both sister kinetochores attached to microtubules from
the same pole) (Fig. 1H; Pinsky et al. 2006b). Although
reduced compared with the wild type, Sgo1-6HA ChIP-
qPCR values were similar in NAPP1-treated ipl1-as
metaphase-arrested cells in the presence and absence of
microtubules. Although we cannot rule out a direct ef-
fect of Ipl1, which is known to associate with Sgo1
(Verzijlbergen et al. 2014), this finding supports the idea
that intersister kinetochore tension triggers Sgo1 removal
from the pericentromere. As an alternative way to abolish
intersister kinetochore tension while kinetochores are
attached to microtubules, we used a strain in which the
function of the microtubule assembly protein Stu2
(ortholog of XMAP215/Dis1) is impaired. Strains harbor-
ing the stu2-277 allele grown at the restrictive tempera-
ture have reduced microtubule dynamics, resulting in
prevalent monotelic and syntelic attachments (He et al. 2001; Pearson et al. 2003; Marco et al. 2013). In stu2-277
metaphase-arrested cells, similar levels of Sgo1-6HA were
associated with the pericentromere in both the presence
and absence of nocodazole (Fig. 1I). The observation that
Sgo1 is not removed from the pericentromere in the
presence of microtubules either upon Ipl1 inhibition or
in the presence of the stu2-227 allele, both of which
reduce tension, is strong support for the idea that inter-
sister kinetochore tension triggers Sgo1 removal from the
pericentromere. stretching upon tension is sufficient to move Bub1 away
from substrates important for Sgo1 localization. Biorientation of sister kinetochores removes Bub1
from the centromere (F–H) Continued Bub1 presence at kinetochores is required for Sgo1
localization at the pericentromere. (F) Scheme of the experiment is shown. Wild-type (AM6390) and bub1-aid OsTir1 (AM9096) cells
carrying SGO1-6HA and a no tag control (AM2508), all carrying pMET3-CDC20, were released from G1 into methionine and nocodazole-
containing medium. After 1 h, one-third of the culture was harvested for ChIP and Western blotting, the remaining culture was split, and
NAA was added to one half. After 2 h total, the remaining cultures were harvested. (G) Western immunoblot analysis was performed with
anti-aid, anti-HA, and anti-Pgk1 antibodies to confirm that Bub1 is degraded upon NAA treatment, but Sgo1 is not. Pgk1 is shown as
a loading control. (H) ChIP-qPCR analysis of Sgo1 localization at the indicated sites on chromosome IV. The mean of three experimental
repeats is shown, with error bars indicating standard error. Student’s t-test was used to calculate confidence values. (*) P < 0.05. Figure 2. Bub1 is removed from kinetochores later than Sgo1 dissociates from the pericentromere. (A) Bub1 associates with centromeres
in metaphase-arrested cells only in the absence of spindle tension. Cells (strain AM7449) carrying BUB1-6HA and pMET3-CDC20 and
a no tag control (AM2508) were treated as described in Figure 1G. Bub1-6HA levels at the indicated sites were measured by ChIP-qPCR. The average of three experimental repeats is shown, with error bars representing standard error. (B–E) Bub1 is retained at kinetochores
upon separation of kinetochore clusters. Cells carrying BUB1-yeGFP and MTW1-tdTomato (strain AM9229) were imaged on a micro-
fluidics device at 15-min intervals after release from G1 arrest. (B) Cells exhibiting different types of Bub1-GFP localization at the
indicated time points are shown. Bar, 5 mm. (C) Line scans across kinetochore foci of single cells were assembled from 100 images to
generate a V plot showing Bub1-yeGFP localization as interkinetochore distance increases. Bar, 2 mm. (D) Bar chart with the fraction of
cells with the indicated Bub1 localization at each time point is shown. (E) The distance between Mtw1-tdTomato signals and the
localization of Bub1-yeGFP was scored in at least 90 cells for each time point. The bean plot shows the distribution of interkinetochore
distances for which each localization type was scored. Lines within the beans represent individual cells. Beans for small sets of cells (N <
10) are not shown. The horizontal line represents the mean. Biorientation of sister kinetochores removes Bub1
from the centromere Sgo1 association with the pericentromere depends on the
Bub1 kinase (Fernius and Hardwick 2007). Bub1-dependent
phosphorylation of histone H2A on Ser121 (H2A-S121) is
important for Sgo1 recruitment to the pericentromere
(Kawashima et al. 2010). Bub1 is positioned closer to the
inner centromere than Sgo1 (Haase et al. 2012) but is also
released from kinetochores as mitosis proceeds (Gillett
et al. 2004). To test the idea that tension may also regulate
Bub1 localization, we used ChIP-qPCR in cells arrested in
metaphase in both the presence and absence of microtu-
bules. Bub1-6HA was restricted to the core centromere, as
expected, but is localized only in the absence of microtu-
bules (Fig. 2A). This indicates that, like Sgo1, Bub1 is
distant from the chromatin when sister kinetochores are
under tension. We used live cells carrying BUB1-GFP and
MTW1-tdTomato to correlate Bub1 disappearance with
sister kinetochore separation (Fig. 2B–E; Supplemental
Movie S2). Bub1-GFP colocalized with the kinetochore
cluster soon after release from G1. As the Mtw1-tdTomato
signal became bilobed, two Bub1-GFP foci were observed
(Fig. 2B,C). Indeed, Bub1-GFP colocalized with kinetochore
clusters separated to distances of >1.5 mm (Fig. 2D,E),
where Sgo1-GFP was predominantly nuclear (Fig. 1F). This
is consistent with a previous report that Bub1 and Sgo1 are
spatially separated at metaphase (Haase et al. 2012). Together, our observations indicate that either Bub1
removal from kinetochores is not the trigger for Sgo1-
GFP release from the pericentromere or kinetochore To determine whether direct association with PP2A is
important for controlling the pericentromeric levels of
Sgo1, we analyzed the sgo1-3A mutant, which fails to
associate with PP2A (Xu et al. 2009). Similar to wild-type
Sgo1 in cells lacking RTS1, the levels of the mutant Sgo1-
3A protein were not increased overall (Supplemental Fig. S3B), accumulated to high levels on the pericentromere
in metaphase-arrested cells lacking microtubules, and
decreased in the presence of tension (Fig. 3B). The 1295 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
wnloaded from 2. Bub1 is removed from kinetochores later than Sgo1 dissociates from the pericentromere. (A) Bub1 associates with cen
taphase-arrested cells only in the absence of spindle tension. Cells (strain AM7449) carrying BUB1-6HA and pMET3-CD
ag control (AM2508) were treated as described in Figure 1G. Bub1-6HA levels at the indicated sites were measured by Ch
verage of three experimental repeats is shown, with error bars representing standard error. Biorientation of sister kinetochores removes Bub1
from the centromere (B–E) Bub1 is retained at kin
separation of kinetochore clusters. Cells carrying BUB1-yeGFP and MTW1-tdTomato (strain AM9229) were imaged on
s device at 15-min intervals after release from G1 arrest. (B) Cells exhibiting different types of Bub1-GFP localizati
ted time points are shown. Bar, 5 mm. (C) Line scans across kinetochore foci of single cells were assembled from 100
te a V plot showing Bub1-yeGFP localization as interkinetochore distance increases. Bar, 2 mm. (D) Bar chart with the f
with the indicated Bub1 localization at each time point is shown. (E) The distance between Mtw1-tdTomato signal
ation of Bub1-yeGFP was scored in at least 90 cells for each time point. The bean plot shows the distribution of interkin
ces for which each localization type was scored. Lines within the beans represent individual cells. Beans for small sets of
e not shown. The horizontal line represents the mean. (F–H) Continued Bub1 presence at kinetochores is required Figure 2. Bub1 is removed from kinetochores later than Sgo1 dissociates from the pericentromere. (A) Bub1 associates with centromeres
in metaphase-arrested cells only in the absence of spindle tension. Cells (strain AM7449) carrying BUB1-6HA and pMET3-CDC20 and
a no tag control (AM2508) were treated as described in Figure 1G. Bub1-6HA levels at the indicated sites were measured by ChIP-qPCR. The average of three experimental repeats is shown, with error bars representing standard error. (B–E) Bub1 is retained at kinetochores
upon separation of kinetochore clusters. Cells carrying BUB1-yeGFP and MTW1-tdTomato (strain AM9229) were imaged on a micro-
fluidics device at 15-min intervals after release from G1 arrest. (B) Cells exhibiting different types of Bub1-GFP localization at the
indicated time points are shown. Bar, 5 mm. (C) Line scans across kinetochore foci of single cells were assembled from 100 images to
generate a V plot showing Bub1-yeGFP localization as interkinetochore distance increases. Bar, 2 mm. (D) Bar chart with the fraction of
cells with the indicated Bub1 localization at each time point is shown. (E) The distance between Mtw1-tdTomato signals and the
localization of Bub1-yeGFP was scored in at least 90 cells for each time point. The bean plot shows the distribution of interkinetochore
distances for which each localization type was scored. Lines within the beans represent individual cells. Beans for small sets of cells (N <
10) are not shown. The horizontal line represents the mean. Biorientation of sister kinetochores removes Bub1
from the centromere Wild-type (AM9233) or rts1D (AM9735) cells producing SGO1-yeGFP and SGO1-3A-yeGFP (AM9873) cells, all carrying
pMET3-CDC20 and MTW1-tdTomato, were released from a G1 arrest on a microfluidics plate, and images were grabbed every 15 min. (C) Sgo1 localization was scored in at least 150 cells from each time point. (D) The number of frames in which pericentromeric Sgo1
signal was observed was scored for 100 cells per strain. (E) Bilobed Mtw1-tdTomato signal was scored in at least 150 cells as a marker of
cell cycle progression. Figure 3. Association with PP2ARts1 is required for timely Sgo1 removal from the pericentromere. (A) Pericentromeric Sgo1 levels are
regulated by Rts1 and Cdc55. Wild-type (AM6390), rts1D (AM8859), and cdc55D (AM8957) cells carrying SGO1-6HA and pMET3-
CDC20 and a no tag pMET3-CDC20 control (AM2508) were arrested in metaphase in the presence or absence of microtubules as
described in Figure 1G, and anti-HA ChIP was performed followed by qPCR with primer sets at the indicated locations on chromosome
IV. The average of four experimental repeats is shown, with error bars representing standard error. Student’s t-test was used to calculate
confidence values. (*) P < 0.05. (B) Interaction with PP2A is required to control Sgo1 levels on the centromere. Wild-type and rts1D cells
carrying SGO1-6HA (AM6390 and AM8859) or SGO1-3A-6HA (AM10143 and AM11902) and pMET3-CDC20 together with a no tag
control (AM2508) were grown and processed for ChIP-qPCR as described in A. The average of three experimental replicates are shown,
with error bars representing standard error. (C,D) Sgo1 removal from the pericentromere is delayed in the absence of associated
PP2ARts1. Wild-type (AM9233) or rts1D (AM9735) cells producing SGO1-yeGFP and SGO1-3A-yeGFP (AM9873) cells, all carrying
pMET3-CDC20 and MTW1-tdTomato, were released from a G1 arrest on a microfluidics plate, and images were grabbed every 15 min. (C) Sgo1 localization was scored in at least 150 cells from each time point. (D) The number of frames in which pericentromeric Sgo1
signal was observed was scored for 100 cells per strain. (E) Bilobed Mtw1-tdTomato signal was scored in at least 150 cells as a marker of
cell cycle progression. tdTomato (Fig. 3C–E). Deletion of RTS1 led to an ;15-
min delay in overall cell cycle progression, as judged by
the splitting of Mtw1 foci (Fig. 3E). However, release of
Sgo1 from the pericentromere was delayed by a further
15 min in rts1D cells (Fig. 3C). Biorientation of sister kinetochores removes Bub1
from the centromere (F–H) Continued Bub1 presence at kinetochores is required for Sgo1
localization at the pericentromere. (F) Scheme of the experiment is shown. Wild-type (AM6390) and bub1-aid OsTir1 (AM9096) cells
carrying SGO1-6HA and a no tag control (AM2508), all carrying pMET3-CDC20, were released from G1 into methionine and nocodazole-
containing medium. After 1 h, one-third of the culture was harvested for ChIP and Western blotting, the remaining culture was split, and
NAA was added to one half. After 2 h total, the remaining cultures were harvested. (G) Western immunoblot analysis was performed with
anti-aid, anti-HA, and anti-Pgk1 antibodies to confirm that Bub1 is degraded upon NAA treatment, but Sgo1 is not. Pgk1 is shown as
a loading control. (H) ChIP-qPCR analysis of Sgo1 localization at the indicated sites on chromosome IV. The mean of three experimental
repeats is shown, with error bars indicating standard error. Student’s t-test was used to calculate confidence values. (*) P < 0.05. Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
wnloaded from Sgo1 regulated by tension Figure 3. Association with PP2ARts1 is required for timely Sgo1 removal from the pericentromere. (A) Pericentromeric Sgo1 levels are
regulated by Rts1 and Cdc55. Wild-type (AM6390), rts1D (AM8859), and cdc55D (AM8957) cells carrying SGO1-6HA and pMET3-
CDC20 and a no tag pMET3-CDC20 control (AM2508) were arrested in metaphase in the presence or absence of microtubules as
described in Figure 1G, and anti-HA ChIP was performed followed by qPCR with primer sets at the indicated locations on chromosome
IV. The average of four experimental repeats is shown, with error bars representing standard error. Student’s t-test was used to calculate
confidence values. (*) P < 0.05. (B) Interaction with PP2A is required to control Sgo1 levels on the centromere. Wild-type and rts1D cells
carrying SGO1-6HA (AM6390 and AM8859) or SGO1-3A-6HA (AM10143 and AM11902) and pMET3-CDC20 together with a no tag
control (AM2508) were grown and processed for ChIP-qPCR as described in A. The average of three experimental replicates are shown,
with error bars representing standard error. (C,D) Sgo1 removal from the pericentromere is delayed in the absence of associated
PP2ARts1. Biorientation of sister kinetochores removes Bub1
from the centromere The Sgo1-3A protein
showed a similar delay in release from the pericentromere
(Fig. 3C), although overall cell cycle progression was not
perturbed in this mutant (Fig. 3E). The 15-min delay in
Sgo1 relocalization in rts1D and sgo1-3A cells was con-
firmed by scoring the number of time points in which
pericentromeric Sgo1 was observed (Fig. 3D). We con-
clude that association with PP2A-Rts1 is required for the
timely dissociation of Sgo1 from the pericentromere. pericentromeric levels of Sgo1-3A were not further in-
creased by deletion of RTS1, indicating that PP2A-Rts1
controls Sgo1 levels at the pericentromere through a di-
rect association (Fig. 3B). Furthermore, deletion of RTS1
did not increase the levels of centromeric Bub1 (Supple-
mental Fig. S3C). We conclude that association with
PP2A-Rts1 negatively regulates the pericentromeric lo-
calization of Sgo1. PP2A-Rts1 promotes timely release of Sgo1
from the pericentromere If the interaction with PP2A-Rts1 is important for Sgo1
removal in the context of the cell cycle, we expected that
Sgo1 dispersal into the nucleus would be delayed in rts1D
or sgo1-3A cells. Wild-type and rts1D cells carrying
SGO1-yeGFP and MTW1-tdTomato were released from
a G1 arrest and imaged at 15-min intervals. We simulta-
neously analyzed a strain in which Sgo1-3A was tagged
with GFP (SGO1-3A-yeGFP) and that also carried MTW1- Bub1 targets other than H2A-S121-P are important
for Sgo1 removal under tension Bub1 targets other than H2A-S121-P are important
for Sgo1 removal under tension Our findings suggest that the antagonistic activities of
a kinetochore-localized kinase (Bub1) and a Sgo1-bound GENES & DEVELOPMENT 1297 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Nerusheva et al. version by mutating S121 to alanine. The H2A-S121D
(phospho-mimic) or H2A-S121A (phospho-null) alleles
were introduced into cells carrying pMET3-CDC20 and
SGO1-6HA as the sole source of H2A, and the pericen-
tromeric levels of Sgo1-6HA in metaphase-arrested cells
were measured by ChIP-qPCR in the presence and absence
of nocodazole. Figure 4B shows that the H2A-S121A
mutation abolished Sgo1 localization at the pericentro-
mere, as expected, confirming that phosphorylation at this
residue is important for Sgo1 recruitment (Kawashima
et al. 2010). Interestingly, cells carrying the H2A-S121D
mutation behaved similarly to wild-type cells: Sgo1 was
localized to the pericentromere only in the absence of
spindle tension (Fig. 4B). Neither mutant affected total
cellular levels of Sgo1 (Supplemental Fig. S4A). Therefore, phosphatase (PP2A-Rts1) control Sgo1 localization in the
pericentromere (Fig. 4A). Bub1 is known to phosphorylate
histone H2A at residue S121, and this is important for
Sgo1 association with the pericentromere (Fernius and
Hardwick 2007; Kawashima et al. 2010; Haase et al. 2012). Since maintenance of Sgo1 at the pericentromere
also requires Bub1 (Fig. 2H), we reasoned that dephos-
phorylation of H2A-S121 might be responsible for Sgo1
dispersal into the nucleus when sister kinetochores are
under tension. Unfortunately, we were unable to monitor
the phosphorylation status of H2A-S121 directly, as
several attempts to raise antibodies to this site were not
successful. As an alternative approach, we replaced S121
of H2A with aspartic acid to mimic the phosphorylated
state. For comparison, we generated a phospho-null Figure 4. Bub1 substrates other than H2A-S121 are important for Sgo1 localization. (A) Hypothetical model for the regulation of Sgo1
localization by spindle tension. In the absence of tension, kinetochore-associated Bub1 phosphorylates chromatin-associated
substrates, including H2A-S121, to create a binding site for Sgo1 in the pericentromere. Sgo1-bound PP2ARts1 antagonizes these
phosphorylations to release Sgo1 so that Sgo1 cycles on and off the pericentromere. In the presence of tension, Bub1 is moved away
from the pericentromeric chromatin, and the pericentromeric binding site for Sgo1 is not maintained. (B) Dephosphorylation of H2A-
S121 is not required for release of Sgo1 from the pericentromere. Bub1 targets other than H2A-S121-P are important
for Sgo1 removal under tension Wild type (AM10120), H2A-S121A (AM10128), and H2A-S121D
(AM10137) carrying SGO1-6HA and pMET3-CDC20 as well as a no tag control (AM2508) were arrested in metaphase with or without
microtubules. The localization of Sgo1 was analyzed by ChIP-qPCR as described in Figure 1G. The mean of three experimental repeats
is shown, with error bars representing standard error. (C) Bub1 is required for Sgo1 localization to the pericentromere in H2A-S121D
cells. Wild-type (AM6390), bub1D (AM11962), H2A-S121D (AM10137), and bub1D H2A-S121D (AM11683) cells carrying SGO1-6HA
and pMET3-CDC20 as well as a no tag control (AM2508) were arrested in metaphase with or without microtubules, and the
localization of Sgo1 was analyzed by ChIP-qPCR as described in Figure 1G. The mean of three experimental replicates is shown, with
error bars representing standard error. (D) PP2ARts1 affects Sgo1 levels independently of the phosphorylation status of H2A-S121. Wild-
type (AM10123), rts1D (AM11977), H2A-S121D (AM10140), and rts1D H2A-S121D (AM11979) cells carrying SGO1-6HA as well as a no
tag control (AM1176) were arrested in metaphase in the presence of nocodazole, and the localization of Sgo1 was analyzed by ChIP-
qPCR at the indicated sites. Mean values of experimental replicates (n = 10 for AM1176, AM10123, AM11977; n = 7 for AM10140; n = 6
for AM11979) are shown, with error bars indicating standard error. The unpaired Student’s t-test was used to calculate significance. (**)
P < 0.001; (*) P < 0.05. Figure 4. Bub1 substrates other than H2A-S121 are important for Sgo1 localization. (A) Hypothetical model for the regulation of Sgo1
localization by spindle tension. In the absence of tension, kinetochore-associated Bub1 phosphorylates chromatin-associated
substrates, including H2A-S121, to create a binding site for Sgo1 in the pericentromere. Sgo1-bound PP2ARts1 antagonizes these
phosphorylations to release Sgo1 so that Sgo1 cycles on and off the pericentromere. In the presence of tension, Bub1 is moved away
from the pericentromeric chromatin, and the pericentromeric binding site for Sgo1 is not maintained. (B) Dephosphorylation of H2A-
S121 is not required for release of Sgo1 from the pericentromere. Wild type (AM10120), H2A-S121A (AM10128), and H2A-S121D
(AM10137) carrying SGO1-6HA and pMET3-CDC20 as well as a no tag control (AM2508) were arrested in metaphase with or without
microtubules. The localization of Sgo1 was analyzed by ChIP-qPCR as described in Figure 1G. The mean of three experimental repeats
is shown, with error bars representing standard error. (C) Bub1 is required for Sgo1 localization to the pericentromere in H2A-S121D
cells. Bub1 targets other than H2A-S121-P are important
for Sgo1 removal under tension Wild-type (AM6390), bub1D (AM11962), H2A-S121D (AM10137), and bub1D H2A-S121D (AM11683) cells carrying SGO1-6HA
and pMET3-CDC20 as well as a no tag control (AM2508) were arrested in metaphase with or without microtubules, and the
localization of Sgo1 was analyzed by ChIP-qPCR as described in Figure 1G. The mean of three experimental replicates is shown, with
error bars representing standard error. (D) PP2ARts1 affects Sgo1 levels independently of the phosphorylation status of H2A-S121. Wild-
type (AM10123), rts1D (AM11977), H2A-S121D (AM10140), and rts1D H2A-S121D (AM11979) cells carrying SGO1-6HA as well as a no
tag control (AM1176) were arrested in metaphase in the presence of nocodazole, and the localization of Sgo1 was analyzed by ChIP-
qPCR at the indicated sites. Mean values of experimental replicates (n = 10 for AM1176, AM10123, AM11977; n = 7 for AM10140; n = 6
for AM11979) are shown, with error bars indicating standard error. The unpaired Student’s t-test was used to calculate significance. (**)
P < 0.001; (*) P < 0.05. 1298 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Pre
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension the regulated dephosphorylation of H2A-S121 cannot be
essential for Sgo1 removal from the pericentromere. unclear (Tanaka et al. 2002; Buvelot et al. 2003; Pereira and
Schiebel 2003; Woodruff et al. 2010; Nakajima et al. 2011;
Zimniak et al. 2012). We found that soon after release
from G1, Ipl1-GFP coalesced from its interphase loca-
lization on microtubules into a bright dot that colocalized
with the kinetochores. As Mtw1-tdTomato foci split,
Ipl1-GFP lost its kinetochore localization and was briefly
released into the nucleus before associating with the
metaphase spindle (Fig. 4D–F; Supplemental Movie S3). Importantly, the average distance occupied by kineto-
chores decorated by Ipl1-GFP (0.925 mm) (Fig. 5G) corre-
lates with the average distance at which Sgo1-GFP is
localized at the pericentromere (1.047 mm) (Fig. 1F), while
the other types of localization occur at longer interkine-
tochore distances. Therefore, like Sgo1, Ipl1 shows ten-
sion-dependent removal from kinetochores. g
p
Next, we considered the possibility that H2A-S121
phosphorylation is not the only way that Bub1 promotes
Sgo1 localization to the pericentromere. Bub1 targets other than H2A-S121-P are important
for Sgo1 removal under tension We deleted
BUB1 in cells where H2A-S121D is the only source of
H2A and measured Sgo1 levels at the pericentromere in
metaphase-arrested cells in both the presence and ab-
sence of spindle tension. Figure 4C shows that although
H2A-S121D can support normal Sgo1 localization, this is
dependent on Bub1. Again, cellular levels of Sgo1 were
not affected (Supplemental Fig. S4B). Therefore, in addi-
tion to H2A-S121 phosphorylation, Bub1 plays other
critical, as yet unknown, roles in promoting Sgo1 associ-
ation with the pericentromere. As a final test of the importance of regulating phos-
phorylation at residue S121 on H2A in controlling the
different localization states of Sgo1, we examined the
combined effect of H2A mutations and deletion of RTS1. If dephosphorylation of H2A contributes to Sgo1 removal,
we would anticipate higher levels of Sgo1 at the pericen-
tromere in the H2A-S121D mutant cells, but this is not
the case (Fig. 4B). Moreover, deletion of RTS1 led to an
elevation of pericentromeric Sgo1 in H2A-S121D cells
similar to that in wild-type cells, although total levels
were not affected (Fig. 4D; Supplemental Fig. S4C). Therefore, like Bub1, PP2A-Rts1 exerts its effects on
Sgo1 localization at the pericentromere in ways other
than regulating H2A-S121 phosphorylation. p
Next, we asked whether Sgo1 removal from the peri-
centromere is sufficient to relocate the CPC, condensin,
and PP2A from this region. We generated an auxin-
inducible degron version of Sgo1 to enable artificial
removal of Sgo1 from the pericentromere in cells arrested
in mitosis. Wild-type or sgo1-aid cells carrying tagged
PP2A (RTS1-3PK), condensin (BRN1-6HA), or CPC (BIR1-
6HA, IPL1-6HA) components were arrested in metaphase
by treatment with nocodazole, and the levels of the
tagged proteins at CEN4 were measured by ChIP-qPCR. Subsequently, we treated half the culture with NAA (to
induce Sgo1 degradation), while the other half received no
treatment (Fig. 5H–J; Supplemental Fig. S5B). After a fur-
ther 1 h, the levels of the proteins at CEN4 were again
measured by ChIP-qPCR. In all cases, NAA treatment
induced degradation of Sgo1 in metaphase and led to
almost complete removal of the effector proteins from the
pericentromere, while in untreated cells, Sgo1 was main-
tained, and the localization of its effector proteins per-
sisted (Fig. 5H–J; Supplemental Fig. S5B). We conclude
that Sgo1 removal from the pericentromere in metaphase
is sufficient for the release of condensin, CPC, and PP2A-
Rts1 from this region (Fig. 5K). Sgo1 removal from the pericentromere disengages the
biorientation machinery During mitosis, Sgo1 engages Ipl1 and condensin to
promote chromosome biorientation (Verzijlbergen et al. 2014). Importantly, once biorientation is established, the
error correction machinery must be deactivated, presum-
ably in a chromosome-autonomous manner. We reasoned
that Sgo1 removal from the pericentromere could con-
tribute to this chromosome-autonomous response to
tension by triggering dissociation of its effectors from
the pericentromere. Indeed, we found that the pericen-
tromeric association of the PP2A regulatory subunit Rts1,
the condensin component Brn1, and the CPC subunit
Bir1 (which depends on Sgo1 for its maintenance at the
centromere) (Supplemental Fig. S5A) were all negatively
regulated by tension. Centromeric ChIP-qPCR values
were reduced more than fourfold in the presence, com-
pared with the absence, of tension for all three proteins
(Fig. 5A–C). This suggests that disassembly of the peri-
centromeric platform of Sgo1 leads to the dispersal of its
effector proteins from this region. Nerusheva et al. (H–J) Sgo1 is
ild-type and sgo1-aid strains carrying RTS1-
treatment with nocodazole for 2 h, and one-
h NAA to induce Sgo1-aid degradation, and
codazole. Anti-aid, anti-Pgk1, and anti-PK (H)
s a loading control. Also shown are the mean
with error bars representing standard error. chematic diagram summarizing disassembly tors are removed from the centromere in
response to intersister kinetochore ten-
sion. The
association
of
PP2ARts1
(A;
Rts1), condensin (B; Brn1), and CPC (C;
Bir1) subunits with the pericentromere is
reduced in the presence of spindle tension. Strains carrying pMET3-CDC20 and pro-
ducing the indicated tagged proteins were
arrested in metaphase with or without
microtubules as described in Figure 1G,
and the levels of the indicated proteins
were examined by ChIP-qPCR using anti-
PK (A) or anti-HA (B,C) antibodies and
primer sets at the locations shown. Strains
used were AM2508 (no tag), AM9639
(RTS1-3PK),
AM8955
(BRN1-6HA),
and
AM6941 (BIR1-6HA). Mean values are
given, and error bars represent standard
error, except where n = 2 (no tag in A),
where they represent range. In A, the
number of experimental repeats was four
(AM9639; RTS1-3PK) or two (AM2508, no
tag). In B, data are shown from three
experimental
repeats
for
both
no
tag
(AM2508) and BRN1-6HA (AM8955). In
C, data are from three experimental repli-
cates (AM6941; BIR1-6HA) or one experi-
ment (AM2508; no tag). The unpaired
Student’s t-test was used to calculate sig-
nificance. (*) P < 0.05. (D–G) Ipl1 relocalizes
from kinetochores during metaphase. Cells
carrying IPL1-yeGFP and MTW1-tdTomato
(strain AM9231) were imaged on a micro-
fluidics device at 15-min intervals after
release from G1 arrest. (D) Examples of
Ipl1-GFP localization observed are shown. Time is given relative to release from G1. Bar, 5 mm. See also Supplemental Movie
S3. (E) Line scans across kinetochore foci of
single cells were assembled from 100 im-
ages to generate a V plot showing Ipl1-GFP
localization as interkinetochore distance
increases. Bar, 2 mm. (F) Bar chart with the fraction of cells with the indicated Ipl1 localization at each time point is shown. (G)
The distance between Mtw1-tdTomato signals and the localization of Ipl1-yeGFP was scored in at least 77 cells for each time point. The
bean plot shows the distribution of interkinetochore distances for which each localization type was scored. Lines within the beans
represent individual cells. Beans for small sets of cells (N < 6) are not shown. Nerusheva et al. The horizontal line represents the mean. (H–J) Sgo1 is
required for the maintenance of PP2ARts1, condensin, and the CPC at the centromere. Wild-type and sgo1-aid strains carrying RTS1-
3PK (H), BRN1-6HA (I), or IPL1-6HA (J) and a no tag control were arrested in metaphase by treatment with nocodazole for 2 h, and one-
third of the culture was harvested. The remaining culture was split, half was treated with NAA to induce Sgo1-aid degradation, and
both treated and untreated cultures were harvested after a further 1 h in the presence of nocodazole. Anti-aid, anti-Pgk1, and anti-PK (H)
or anti-HA (I,J) immunoblots are shown to confirm Sgo1-aid degradation. Pgk1 is shown as a loading control. Also shown are the mean
results of qPCR after anti-PK (H) or anti-HA ChIP (I,J) from four experimental replicates, with error bars representing standard error. The two-tailed paired Student’s t-test was used to calculate significance. (*) P < 0.05. (K) Schematic diagram summarizing disassembly
of the pericentromeric signaling platform. increases. Bar, 2 mm. (F) Bar chart with the fraction of cells with the indicated Ipl1 localization at each time point is shown. (G)
The distance between Mtw1-tdTomato signals and the localization of Ipl1-yeGFP was scored in at least 77 cells for each time point. The
bean plot shows the distribution of interkinetochore distances for which each localization type was scored. Lines within the beans
represent individual cells. Beans for small sets of cells (N < 6) are not shown. The horizontal line represents the mean. (H–J) Sgo1 is
required for the maintenance of PP2ARts1, condensin, and the CPC at the centromere. Wild-type and sgo1-aid strains carrying RTS1-
3PK (H), BRN1-6HA (I), or IPL1-6HA (J) and a no tag control were arrested in metaphase by treatment with nocodazole for 2 h, and one-
third of the culture was harvested. The remaining culture was split, half was treated with NAA to induce Sgo1-aid degradation, and
both treated and untreated cultures were harvested after a further 1 h in the presence of nocodazole. Anti-aid, anti-Pgk1, and anti-PK (H)
or anti-HA (I,J) immunoblots are shown to confirm Sgo1-aid degradation. Pgk1 is shown as a loading control. Also shown are the mean
results of qPCR after anti-PK (H) or anti-HA ChIP (I,J) from four experimental replicates, with error bars representing standard error. The two-tailed paired Student’s t-test was used to calculate significance. (*) P < 0.05. Nerusheva et al. Cells
carrying IPL1-yeGFP and MTW1-tdTomato
(strain AM9231) were imaged on a micro-
fluidics device at 15-min intervals after
release from G1 arrest. (D) Examples of
Ipl1-GFP localization observed are shown. Time is given relative to release from G1. Bar, 5 mm. See also Supplemental Movie
S3. (E) Line scans across kinetochore foci of
single cells were assembled from 100 im-
V
l
h
i
I l1 GFP Figure 5. Sgo1 removal from the pericen-
tromere leads to disassembly of the signal-
ing platform that responds to a lack of
tension at kinetochores. (A–C) Sgo1 effec-
tors are removed from the centromere in
response to intersister kinetochore ten-
sion. The
association
of
PP2ARts1
(A;
Rts1), condensin (B; Brn1), and CPC (C;
Bir1) subunits with the pericentromere is
reduced in the presence of spindle tension. Strains carrying pMET3-CDC20 and pro-
ducing the indicated tagged proteins were
arrested in metaphase with or without
microtubules as described in Figure 1G,
and the levels of the indicated proteins
were examined by ChIP-qPCR using anti-
PK (A) or anti-HA (B,C) antibodies and
primer sets at the locations shown. Strains
used were AM2508 (no tag), AM9639
(RTS1-3PK),
AM8955
(BRN1-6HA),
and
AM6941 (BIR1-6HA). Mean values are
given, and error bars represent standard
error, except where n = 2 (no tag in A),
where they represent range. In A, the
number of experimental repeats was four
(AM9639; RTS1-3PK) or two (AM2508, no
tag). In B, data are shown from three
experimental
repeats
for
both
no
tag
(AM2508) and BRN1-6HA (AM8955). In
C, data are from three experimental repli-
cates (AM6941; BIR1-6HA) or one experi-
ment (AM2508; no tag). The unpaired
Student’s t-test was used to calculate sig-
nificance. (*) P < 0.05. (D–G) Ipl1 relocalizes
from kinetochores during metaphase. Cells
carrying IPL1-yeGFP and MTW1-tdTomato
(strain AM9231) were imaged on a micro-
fluidics device at 15-min intervals after
release from G1 arrest. (D) Examples of
Ipl1-GFP localization observed are shown. Time is given relative to release from G1. Bar, 5 mm. See also Supplemental Movie
S3. (E) Line scans across kinetochore foci of
single cells were assembled from 100 im-
ages to generate a V plot showing Ipl1-GFP
localization as interkinetochore distance
calization at each time point is shown. (G)
ed in at least 77 cells for each time point. The
on type was scored. Lines within the beans
ontal line represents the mean. Tethered Sgo1 is sufficient to maintain Aurora B
at the centromere in the presence of microtubules If removal of the CPC from the pericentromere upon
biorientation is triggered by tension-dependent dissocia-
tion of Sgo1, we reasoned that Sgo1 tethered to the
pericentromere would prevent CPC removal even when
sister kinetochores should be under tension. We inte-
grated tetO arrays adjacent to the centromere of chromo-
some IV (Fig. 6A) or chromosome V (Fig. 6B) at sites that
are known to separate when sister kinetochores are under
tension (He et al. 2000; Tanaka et al. 2000) and expressed
Sgo1-TetR-GFP in cells also carrying IPL1-6HA. These
cells were arrested in metaphase by depletion of CDC20
(sister kinetochores are under tension) with or without
nocodazole and in either the presence (+DOX; no Sgo1-
TetR-GFP tethering) or absence (DOX; Sgo1-TetR-GFP
bound to tetOs) of doxycycline. We first confirmed that
tethered Sgo1-TetR-GFP remained bound to tetO repeats
as they separate under tension. In metaphase-arrested To analyze the tension dependence of Aurora B/Ipl1
localization in more detail, we imaged live cells pro-
ducing Ipl1-GFP as they progressed from G1 into a meta-
phase arrest induced by depletion of CDC20 (Fig. 5D–G). Ipl1 relocalization onto the spindle during anaphase is
well documented; however, kinetochore, nuclear, and
spindle localizations have all been observed in metaphase,
and the relative timing of these localizations has been GENES & DEVELOPMENT 1299 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Nerusheva et al. Nerusheva et al. increases. Bar, 2 mm. (F) Bar chart with the fraction of cells with the indicated Ipl1 loc
The distance between Mtw1-tdTomato signals and the localization of Ipl1-yeGFP was score
bean plot shows the distribution of interkinetochore distances for which each localizati
represent individual cells. Beans for small sets of cells (N < 6) are not shown. The horizo
required for the maintenance of PP2ARts1, condensin, and the CPC at the centromere. W
3PK (H), BRN1-6HA (I), or IPL1-6HA (J) and a no tag control were arrested in metaphase by
third of the culture was harvested. The remaining culture was split, half was treated wit
both treated and untreated cultures were harvested after a further 1 h in the presence of noc
or anti-HA (I,J) immunoblots are shown to confirm Sgo1-aid degradation. Pgk1 is shown as
results of qPCR after anti-PK (H) or anti-HA ChIP (I,J) from four experimental replicates,
The two-tailed paired Student’s t-test was used to calculate significance. (*) P < 0.05. (K) Sc Figure 5. Sgo1 removal from the pericen-
tromere leads to disassembly of the signal-
ing platform that responds to a lack of
tension at kinetochores. (A–C) Sgo1 effec-
tors are removed from the centromere in
response to intersister kinetochore ten-
sion. The
association
of
PP2ARts1
(A;
Rts1), condensin (B; Brn1), and CPC (C;
Bir1) subunits with the pericentromere is
reduced in the presence of spindle tension. Strains carrying pMET3-CDC20 and pro-
ducing the indicated tagged proteins were
arrested in metaphase with or without
microtubules as described in Figure 1G,
and the levels of the indicated proteins
were examined by ChIP-qPCR using anti-
PK (A) or anti-HA (B,C) antibodies and
primer sets at the locations shown. Strains
used were AM2508 (no tag), AM9639
(RTS1-3PK),
AM8955
(BRN1-6HA),
and
AM6941 (BIR1-6HA). Mean values are
given, and error bars represent standard
error, except where n = 2 (no tag in A),
where they represent range. In A, the
number of experimental repeats was four
(AM9639; RTS1-3PK) or two (AM2508, no
tag). In B, data are shown from three
experimental
repeats
for
both
no
tag
(AM2508) and BRN1-6HA (AM8955). In
C, data are from three experimental repli-
cates (AM6941; BIR1-6HA) or one experi-
ment (AM2508; no tag). The unpaired
Student’s t-test was used to calculate sig-
nificance. (*) P < 0.05. (D–G) Ipl1 relocalizes
from kinetochores during metaphase. Nerusheva et al. (K) Schematic diagram summarizing disassembly
of the pericentromeric signaling platform. graph). Conversely, in cells where tetO repeats were close
to CEN5, Sgo1-TetR-GFP remained associated with a site
near to CEN5 in the presence of tension (Fig. 6B, top right
graph) but not a site near to CEN4 (Fig. 6B, top left graph). Importantly, Ipl1-6HA localization was significantly in- cells with tetO repeats adjacent to CEN4, similar levels of
Sgo1-TetR-GFP associated with a site close to CEN4 in
the presence and absence of nocodazole (Fig. 6A, top left
graph); however, Sgo1-TetR-GFP close to CEN5 was
removed in the presence of tension (Fig. 6A, top right GENES & DEVELOPMENT 1300 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
wnloaded from Sgo1 regulated by tension Figure 6. Sgo1 removal from the pericentromere upon biorientation is required for Aurora B (Ipl1) dissociation. (A,B) Tethered Sgo1 is
sufficient to retain Ipl1 at the centromere in the presence of spindle tension. Strains carrying SGO1-tetR-GFP, IPL1-6HA, and pMET3-
CDC20 and with tetO repeats integrated ;2.4 kb to the left of CEN4 (AM12151; A) or ;80 bp to the left of CEN5 (AM12148; B) were
released from a G1 arrest into medium containing methionine to induce a metaphase arrest either with or without nocodazole and in
both the presence (+DOX) and absence (DOX) of doxycycline. Anti-GFP (top graphs) and anti-HA (bottom graphs) ChIP was
performed, and samples were analyzed by qPCR with primers specific to the indicated sites. A no tag strain (AM2508) was also
analyzed, and data are reproduced in A and B. The mean values from four experimental replicates are shown, with error bars
representing standard error. The two-tailed paired Student’s t-test was used to calculate significance. (*) P < 0.05. Figure 6. Sgo1 removal from the pericentromere upon biorientation is required for Aurora B (Ipl1) dissociation. (A,B) Tethered Sgo1 is
sufficient to retain Ipl1 at the centromere in the presence of spindle tension. Strains carrying SGO1-tetR-GFP, IPL1-6HA, and pMET3-
CDC20 and with tetO repeats integrated ;2.4 kb to the left of CEN4 (AM12151; A) or ;80 bp to the left of CEN5 (AM12148; B) were
released from a G1 arrest into medium containing methionine to induce a metaphase arrest either with or without nocodazole and in
both the presence (+DOX) and absence (DOX) of doxycycline. Nerusheva et al. Anti-GFP (top graphs) and anti-HA (bottom graphs) ChIP was
performed, and samples were analyzed by qPCR with primers specific to the indicated sites. A no tag strain (AM2508) was also
analyzed, and data are reproduced in A and B. The mean values from four experimental replicates are shown, with error bars
representing standard error. The two-tailed paired Student’s t-test was used to calculate significance. (*) P < 0.05. et al. 2008). However, chromosomes have the opportu-
nity to attach to the spindle in a variety of orientations in
monopolin mutants due to the presence of chiasmata that
provide resistance to spindle forces (Fig. 7A), so it is likely
that not all sister kinetochores are bioriented. As a mea-
sure of sister kinetochore biorientation in cells lacking
monopolin, we examined the separation of TetR-GFP foci
bound to CEN5-proximal tetO repeats in cells arrested in
metaphase I by depletion of CDC20 (by placement under
the control of the mitosis-specific promoter pCLB2) (Lee
and Amon 2003). In wild-type cells, since sister kinetochore
biorientation is suppressed, a single GFP focus is observed
(Supplemental Fig. S7A). In cells lacking the monopolin
component Mam1, separated CEN5-GFP foci were observed
in ;30% of cells (Supplemental Fig. S7A). While this
indicates that mono-orientation is defective in mam1D
cells, the fraction of cells with separated CEN5-GFP foci
is much lower than expected if sister kinetochores on
chromosome V were bioriented in all cells. We reduced
the number of ways that kinetochores could stably attach
to microtubules in metaphase I by deleting SPO11, the
endonuclease required for the initiation of meiotic
recombination, thereby abolishing chiasmata (Fig. 7A;
Keeney et al. 1997; Shonn et al. 2000). In spo11D mam1D
cells, the percentage of cells with separated CEN5-GFP
was increased to ;60%, indicating that eliminating creased adjacent to Sgo1-TetR-GFP tethered to either
CEN4 or CEN5 but not at the site close to the centromere
lacking the tether (Fig. 6A,B, bottom panels). (Note that
integration of tetOs at either CEN4 or CEN5 prevented
recruitment of normal levels of Ipl1 to adjacent sites in
the absence of Sgo1-TetR-GFP tethering for reasons that
are currently unclear [Fig. 6A,B, +DOX condition].)
Therefore, the dissociation of Ipl1 in metaphase requires
Sgo1 release from the pericentromere. Overall, our results
support a model in which tension-triggered Sgo1 removal
leads to disassembly of the pericentromeric platform that
governs error correction (Supplemental Fig. S6). Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I During meiosis I, sister kinetochores must be mono-
oriented (attached to microtubules from the same pole),
and therefore the biorientation of sister kinetochores is
suppressed. Inactivation of the monopolin complex,
which is required for kinetochore mono-orientation, does
not abolish the protection of pericentromeric cohesion
during meiosis I, which has led to the idea that a lack of
tension between sister kinetochores during meiosis I is
not important for the maintenance of Sgo1 (Toth et al. 2000; Rabitsch et al. 2003; Petronczki et al. 2006; Matos GENES & DEVELOPMENT 1301 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Nerusheva et al. Nerusheva et al. Figure 7. Sister kinetochore tension leads
to partial deprotection of cohesin in meiosis
I. (A) Schematic diagram showing possible
kinetochore orientations at meiosis I for the
indicated genotypes. (B) Sgo1 is released
from the pericentromere upon kinetochore
biorientation during meiosis I. Wild-type
(AM15137),
spo11D
(AM15139),
mam1D
(AM15138), and spo11D mam1D (AM15140)
cells carrying SGO1-yeGFP MTW1-tdTomato
and pCLB2-CDC20 were induced to sporu-
late, transferred to a microfluidics device
after 4 h, and imaged every 15 min. The area
occupied by Sgo1-yeGFP was scored in 50
cells in the first frame after Mtw1-tdTomato
kinetochore foci split and categorized as
pericentromere (foci covering <2 mm2) or
dispersed nuclear localization (no distinct
foci, but signal of at least three times the
intensity of the background signal over >2
mm2). Example images are shown. (C–E)
Reduced Rec8 at centromeres during ana-
phase I in mam1D and spo11D mam1D cells. Wild-type (AM13716), spo11D (AM13718),
mam1D (AM13717), and spo11D mam1D
(AM13719) cells carrying REC8-GFP, MTW1-
dtTomato, and PDS1-tdTomato were resus-
pended in sporulation medium for 2 h be-
fore loading onto a microfluidics plate and
imaged at 15-min intervals. (C) Example
sequences are shown, with time shown re-
lative to the first frame in which Pds1 de-
gradation has occurred (t = 0, anaphase I). Arrowheads indicate centromeric Rec8. (D)
The percentage of cells in which Rec8-GFP
colocalized with Mtw1-tdTomato kineto-
chore foci in the first or second time frame
after Pds1 degradation (t = 15 or 30) is given
after scoring the behavior of 50 cells. Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I Wild-type
(AM15137),
spo11D
(AM15139),
mam1D
(AM15138), and spo11D mam1D (AM15140)
cells carrying SGO1-yeGFP MTW1-tdTomato
and pCLB2-CDC20 were induced to sporu-
late, transferred to a microfluidics device
after 4 h, and imaged every 15 min. The area
occupied by Sgo1-yeGFP was scored in 50
cells in the first frame after Mtw1-tdTomato
kinetochore foci split and categorized as
pericentromere (foci covering <2 mm2) or
dispersed nuclear localization (no distinct
foci, but signal of at least three times the
intensity of the background signal over >2
mm2). Example images are shown. (C–E)
Reduced Rec8 at centromeres during ana-
phase I in mam1D and spo11D mam1D cells. Wild-type (AM13716), spo11D (AM13718),
mam1D (AM13717), and spo11D mam1D
(AM13719) cells carrying REC8-GFP, MTW1-
dtTomato, and PDS1-tdTomato were resus-
pended in sporulation medium for 2 h be-
fore loading onto a microfluidics plate and
imaged at 15-min intervals. (C) Example
sequences are shown, with time shown re-
lative to the first frame in which Pds1 de-
gradation has occurred (t = 0, anaphase I). Arrowheads indicate centromeric Rec8. (D)
The percentage of cells in which Rec8-GFP
colocalized with Mtw1-tdTomato kineto-
chore foci in the first or second time frame
after Pds1 degradation (t = 15 or 30) is given
after scoring the behavior of 50 cells. (E) The
average intensity of Rec8-GFP signal was
measured in the area occupied by and be-
tween the Mtw1-tdTomato signal for each
cell. The average ratio of Rec8-GFP/Mtw1-
tdTomato intensity is given for 50 cells. As
a measure of background fluorescence, we
analyzed kinetochore clusters of wild-type
ndard error. The unpaired Student’s t-test was
I in a fraction of mam1D and spo11D mam1D
po11D (AM13979), mam1D (AM13978), and
ing tetR-GFP and carrying CNM67-3mCherry
o a microfluidics plate and imaging at 15-min
dation (t = 0). Arrowheads indicate CENV-GFP
egradation but before SPB reduplication for 50 y
yp
cells in anaphase II, where all Rec8 would be expected to be lost. Error bars represent standard error. The unpaired Student’s t-test was
used to calculate significance. (**) P < 0.001. (F,G) Sister chromatids segregate at meiosis I in a fraction of mam1D and spo11D mam1D
cells, indicating precocious loss of pericentromeric cohesion. Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I (E) The
average intensity of Rec8-GFP signal was
measured in the area occupied by and be-
tween the Mtw1-tdTomato signal for each
cell. The average ratio of Rec8-GFP/Mtw1-
tdTomato intensity is given for 50 cells. As
a measure of background fluorescence, we
analyzed kinetochore clusters of wild-type
cells in anaphase II, where all Rec8 would be expected to be lost. Error bars represent standard error. The unpaired Student’s t-test was
used to calculate significance. (**) P < 0.001. (F,G) Sister chromatids segregate at meiosis I in a fraction of mam1D and spo11D mam1D
cells, indicating precocious loss of pericentromeric cohesion. Wild type (AM13431), spo11D (AM13979), mam1D (AM13978), and
spo11D mam1D (AM13980) with tetO repeats integrated at CEN5 of one homolog expressing tetR-GFP and carrying CNM67-3mCherry
and PDS1-tdTomato were resuspended in sporulation medium for 2 h before loading onto a microfluidics plate and imaging at 15-min
intervals. (F) Representative sequences are shown. Times are given relative to Pds1 degradation (t = 0). Arrowheads indicate CENV-GFP
foci. (G) The greatest distance between sister CENV-GFP foci was measured after Pds1 degradation but before SPB reduplication for 50
cells. cells in anaphase II, where all Rec8 would be expected to be lost. Error bars represent sta
used to calculate significance. (**) P < 0.001. (F,G) Sister chromatids segregate at meiosis
cells, indicating precocious loss of pericentromeric cohesion. Wild type (AM13431), s
spo11D mam1D (AM13980) with tetO repeats integrated at CEN5 of one homolog express
and PDS1-tdTomato were resuspended in sporulation medium for 2 h before loading ont
l
( )
h
l
d
d
d cells in anaphase II, where all Rec8 would be expected to be lost. Error bars represent sta
used to calculate significance. (**) P < 0.001. (F,G) Sister chromatids segregate at meiosis
cells, indicating precocious loss of pericentromeric cohesion. Wild type (AM13431), s
spo11D mam1D (AM13980) with tetO repeats integrated at CEN5 of one homolog express
and PDS1-tdTomato were resuspended in sporulation medium for 2 h before loading ont
intervals. (F) Representative sequences are shown. Times are given relative to Pds1 degra
foci. (G) The greatest distance between sister CENV-GFP foci was measured after Pds1 d
ll Figure 7. Sister kinetochore tension leads
to partial deprotection of cohesin in meiosis
I. (A) Schematic diagram showing possible
kinetochore orientations at meiosis I for the
indicated genotypes. (B) Sgo1 is released
from the pericentromere upon kinetochore
biorientation during meiosis I. Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I Similar to our
observations with mam1D cells, we found that deletion of
SPO11 both increases sister kinetochore biorientation
and decreases Sgo1 levels at centromeres in pCUP1-CLB3
cells (Supplemental Fig. S7C,D). The idea that Sgo1 levels
at the pericentromere are sensitive to all types of tension
at kinetochores was confirmed by treatment of wild-type
and spo11D metaphase I-arrested cells with the microtu-
bule-destabilizing drug benomyl, which resulted in thin
metaphase I spindles and increased levels of Sgo1 at the
pericentromere (Supplemental Fig. S7E). Together, these
findings indicate that the pericentromeric levels of Sgo1
are responsive to spindle tension also during meiosis I and
that Sgo1 levels at the pericentromere are lowest when
sister kinetochores are bioriented. remaining at kinetochore clusters directly after Pds1
degradation and expressed this as a ratio of the Mtw1-
tdTomato signal (Fig. 7E). These measurements con-
firmed a significant overall reduction in Rec8 levels at
centromeres during anaphase I in mam1D cells and
a further reduction in spo11D mam1D cells. This is
consistent with the idea that biorientation of sister
chromosomes during metaphase I impairs the mainte-
nance of pericentromeric Rec8 during anaphase I. To determine whether the reduced pericentromeric
Rec8 in mam1D and spo11D mam1D mutants results in
the segregation of sister chromosomes to opposite poles
in meiosis I, we filmed cells carrying CEN5-GFP foci on
one homolog together with Pds1-tdTomato and the
spindle pole body marker Cnm67-3mCherry (Fig. 7F). We scored the percentage of cells in which CEN5-GFP
segregated away from each other (CEN5-GFP foci sepa-
rated to >2 mm) directly following Pds1 degradation in
meiosis I. Separation of sister CEN5-GFP foci to a distance
of <2 mm suggests that sister kinetochores are bioriented,
but pericentromeric cohesion is retained. As reported
previously for mam1D mutants (Toth et al. 2000), in
a large fraction (58%) of cells, CEN5-GFP separated only
a short distance (<2 mm), indicating sister kinetochore
biorientation without loss of cohesion, and this pheno-
type was also apparent in 40% of spo11D mam1D cells
(Fig. 7G). This indicates that biorientation of individual
kinetochores may not in itself be sufficient for sister
centromeres to segregate to opposite poles. Remarkably,
however, 18% of mam1D cells and 52% of spo11D
mam1D cells segregated sister CEN5-GFP foci to opposite
poles immediately following Pds1 degradation in meiosis
I, indicating a failure to protect pericentromeric cohesion
(Fig. 7G). Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I These results indicate that suppression of sister
kinetochore biorientation during meiosis I is required to
ensure the proper protection of pericentromeric cohesion,
likely through maintaining the localization of Sgo1. Suppression of sister kinetochore biorientation ensures
the retention of pericentromeric Sgo1 during meiosis I Wild type (AM13431), spo11D (AM13979), mam1D (AM13978), and
spo11D mam1D (AM13980) with tetO repeats integrated at CEN5 of one homolog expressing tetR-GFP and carrying CNM67-3mCherry
and PDS1-tdTomato were resuspended in sporulation medium for 2 h before loading onto a microfluidics plate and imaging at 15-min
intervals. (F) Representative sequences are shown. Times are given relative to Pds1 degradation (t = 0). Arrowheads indicate CENV-GFP
foci. (G) The greatest distance between sister CENV-GFP foci was measured after Pds1 degradation but before SPB reduplication for 50
cells. chiasmata facilitates sister kinetochore biorientation in
mam1D cells (Supplemental Fig. S7A). measured in live cells directly after kinetochore clusters
became bilobed (Fig. 7B). Although Sgo1-GFP formed
pericentromeric foci in all wild-type and spo11D cells,
only diffuse nuclear fluorescence was observed in 42% of
mam1D cells and 88% of spo11D mam1D cells (Fig. 7B). Therefore, Sgo1 localization is responsive to kinetochore
orientation in meiosis I too. We confirmed these obser-
vations by ChIP-qPCR: Centromeric Sgo1 levels were
lowest in spo11D mam1D cells in which sister kineto- The
increased
sister
kinetochore
biorientation
of
mam1D spo11D cells gave us the opportunity to test
how tension across sister kinetochores influences Sgo1
association with the pericentromere during meiosis I. Wild-type, spo11D, mam1D, and spo11D mam1D cells
were arrested in metaphase I by depletion of CDC20
(pCLB2-CDC20), and the area occupied by Sgo1-GFP was 1302 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension chore biorientation is most frequent (Supplemental Fig. S7B). Interestingly, centromeric Sgo1 levels were highest
in spo11D cells in which both intersister tension and
interhomolog tension are abolished (Fig. 7A; Supplemen-
tal Fig. S7B). Overall centromeric Sgo1 levels in mam1D
cells were comparable with wild-type cells (Supplemental
Fig. S7B), perhaps representing the average of a population
of cells that includes attachments that lack tension as
well as those that generate intersister tension (Fig. 7A). Expression of the mitotic cyclin CLB3 in meiosis I causes
an albeit milder defect in sister kinetochore mono-orien-
tation than mam1D without affecting overall centro-
meric levels of Sgo1 (Miller et al. 2012). Sister kinetochore biorientation in meiosis I leads
to partial deprotection of cohesin The reduced pericentromeric Sgo1 in metaphase I-
arrested spo11D mam1D cells implied that cohesin may
not be efficiently protected in these cells. Consistent
with this idea, spo11D mam1D cells undergo a single
meiotic division in which sister chromatids separate to
opposite poles (Matos et al. 2008). We examined the
localization of the meiotic cohesin subunit Rec8 on
spread meiotic chromosomes from cells progressing syn-
chronously through meiosis after release from a prophase
I arrest (Carlile and Amon 2008). Compared with wild-
type, spo11D, or mam1D cells, the fraction of cells with
Rec8 only in the vicinity of centromeres (as identified by
costaining the kinetochore subunit Ndc10) was reduced
in spo11D mam1D cells (Supplemental Fig. S7F–I). We
confirmed these observations in live single cells progress-
ing through meiosis by examining Rec8-GFP localization
immediately after Pds1-tdTomato (securin) degradation
in cells that also carried Mtw1-tdTomato (to label kinet-
ochores) (Fig. 7C,D). Interestingly, although 100% of
wild-type and spo11D cells retained Rec8 at kinetochores,
Rec8 was undetectable at kinetochores in 24% of mam1D
and 38% of spo11D mam1D cells directly after separase
activation in meiosis I (Fig. 7D). Since our findings above
suggest that chromosomes can attach to the spindle in
a variety of orientations in mam1D cells and, to a lesser
extent, spo11D mam1D cells, it is likely that not all
chromosomes within each cell behave in a uniform
manner. Therefore, we used fluorescence intensity mea-
surements to quantify the average Rec8-GFP signal Nerusheva et al. tension may play a role in ensuring the step-wise loss of
cohesin in meiosis through controlling shugoshin local-
ization. However, it is unlikely that tension between
sister kinetochores is sufficient for the deprotection of
cohesion, and other mechanisms must contribute. In
a considerable fraction of cells lacking both monopolin
and
chiasmata,
sister
kinetochore
biorientation
is
achieved, yet sister chromatids fail to segregate to oppo-
site poles following securin degradation during meiosis I,
indicating that pericentromeric cohesion persists (Fig. 7;
Toth et al. 2000; Matos et al. 2008). In contrast, in-
activation of SGO1 in monopolin mutant cells allows
nuclear division without a delay, and spo11D mam1D
cells lacking Sgo1 segregate sister chromatids to opposite
poles during meiosis I (Katis et al. 2004; Petronczki et al. 2006; Kiburz et al. 2008). This suggests that even when
sister kinetochores are under tension, a low level of Sgo1
persists at some pericentromeres and that this is suffi-
cient for cohesin protection. Alternatively, these obser-
vations raise the possibility that once cohesin protection
is in place, events downstream from Sgo1 removal are
required to reverse it. While we cannot currently distin-
guish between these models, these observations demon-
strate that sister kinetochore biorientation is unlikely
to be sufficient for the deprotection of cohesion, and
additional mechanisms must contribute. Indeed, in
mouse oocytes, the PP2A inhibitor I2PP2A/Set1b colo-
calizes with Rec8 only in meiosis II, and its depletion
prevents sister chromatid segregation during meiosis II
(Chambon et al. 2013). Therefore, although suppression of
sister chromatid biorientation facilitates the mainte-
nance of pericentromeric cohesion during meiosis I, its
deprotection during meiosis II is likely to require addi-
tional factors. centromere only when sister kinetochores are not un-
der tension. Moreover, we provided evidence that the
crux of the response to sister kinetochore biorientation
is the tension-dependent removal of shugoshin from the
pericentromere. Shugoshin fits all of the criteria for the fundamental
tension sensor. First, shugoshin associates with the
pericentromere only when sister kinetochores are not under
tension. Second, shugoshin can reversibly associate with the
pericentromere during prometaphase and metaphase where
kinetochore-microtubule interactions are perturbed. Third,
the pericentromeric localization of the tension-sensing
machinery depends on shugoshin. Fourth, the tension-
dependent localization of shugoshin to the pericentromere
is chromosome-autonomous. Fifth, shugoshin is irrevers-
ibly destroyed when the commitment to chromosome
segregation is made in anaphase. Nerusheva et al. We propose that shugoshin removal from the pericen-
tromere in mitotic metaphase signals sister kinetochore
biorientation and initiates the transition to anaphase. Dispersal of shugoshin abolishes the platform for Aurora
B at the pericentromere, thereby disengaging the error
correction machinery and reinforcing kinetochore–
microtubule attachments. This will in turn suppress
SAC signaling from unattached kinetochores, ultimately
allowing loss of cohesion and chromosome segregation. However, Sgo1 dispersal cannot be the only mechanism
by which Ipl1 is inactivated in response to tension. Truncation of the CPC component Sli15 allows Ipl1
clustering on microtubules and overrides the require-
ment for its Sgo1-dependent centromeric targeting, yet
chromosomes biorient normally (Campbell and Desai
2013), suggesting that additional factors are able to
counteract Ipl1 activity upon tension establishment. Shugoshin: the tension sensor Shugoshin: the tension sensor Ever since Nicklas’ elegant micromanipulation experi-
ments (Nicklas and Koch 1969) showed that tension
across centromeres stabilizes kinetochore attachments,
the mechanistic basis of this stabilization has been
pondered. More recent evidence has suggested that ten-
sion stabilizes attachments both directly (Akiyoshi et al. 2010) and, through opposition of the destabilizing kinase
Aurora B, indirectly (Lampson and Cheeseman 2011). However, it has remained unclear how the state of
tension at sister kinetochores is read so that the response
to a lack of tension can be silenced. Tension-dependent
changes in shugoshin localization have been observed in
mouse spermatocytes and oocytes and human somatic
cells (Gomez et al. 2007; Lee et al. 2008; Liu et al. 2013a). In these systems, shugoshin relocates from the inner
centromere to the kinetochore once sister kinetochore
biorientation is established. Similarly, here, we showed
that budding yeast shugoshin associates with the peri- 1303 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Western blotting Samples were prepared for Western blotting as described in Clift
et al. (2009) except that some antibodies were detected using the
fluorescence-based Li-Cor Odyssey system. Antibodies used
were mouse anti-HA 12CA5 (Roche), mouse anti-PK(V5) (AbD
Serotec), mouse anti-aid (Cosmo Bio Co.), and rabbit or mouse
anti-Pgk1 (laboratory stock and Life Technologies, respectively). Sgo1 regulated by tension Yamagishi et al. 2012). We speculate that moving kineto-
chores away from the reach of Aurora B, which is known to
antagonize PP1 (Pinsky et al. 2006a; Liu et al. 2010;
Rosenberg et al. 2011), will be key for Bub1 dissociation
from the kinetochore (Funabiki and Wynne 2013). This
reciprocal kinetochore–pericentromere phosphorylation
model provides an attractive framework for sensing inter-
kinetochore tension and raises additional questions for
future studies. Interestingly, a recent report in somatic
cells showed that reversal of the CDK-dependent phos-
phorylation of shugoshin triggers its relocation onto the
Bub1-dependent phospho-H2A receptor in the kinetochore
(Liu et al. 2013a). This suggests that shugoshins might
undergo phospho-regulation by multiple kinases. Further-
more, the spectrum of phospho-regulated substrates is
likely to be broad and, at a minimum, include shugoshin
itself and histones (Kawashima et al. 2010; Liu et al. 2013b;
Ng et al. 2013). While H2A-S121-P is required for Sgo1
localization within the pericentromere, we found that
regulated phosphorylation of this site does not underlie
Sgo1 behavior in response to tension. Unraveling the
important enzymes and substrates in the phospho-regula-
tion of shugoshin will be an important priority for the
future. Shugoshins have been found to be misregulated in
human cancers. This suggests that exquisite control of this
fundamental tension sensor is likely to be essential in
protecting against aneuploidy and its associated diseases. methionine (SC/Met/D) with a factor (4 or 5 mg/mL). Cells were
then washed with rich medium lacking glucose (YEP) and released
into rich medium containing 8 mM methionine (YPDA/Met). Methionine was readded to 4 mM every hour. To achieve a meta-
phase arrest in the absence of microtubules, 15 mg/mL nocodazole
was added immediately after release into YPDA/Met and readded
to 7.5 mg/mL every hour. To inhibit Ipl1-as5, 1 NA-PP1 was added
to a final concentration of 50 mM. The stu2-277 allele was
inactivated by shifting to 37°C. Doxycycline was used at 5 mg/
mL. Meiosis was performed as described in Marston et al. (2003). For meiotic prophase I block–release experiments using strains
carrying pGAL-NDT80 and GAL4-ER, prophase release was in-
duced by addition of b-estradiol to 1 mM (Carlile and Amon 2008). Benomyl was added to 90 mg/mL 30 min before harvesting. Copper
sulfate was used at 50 mM. ChIP ChIP was performed as described in Fernius et al. (2013) using
mouse anti-HA (12CA5, Roche Diagnostics), mouse anti-PK(V5)
(AbD Serotec), or rabbit anti-GFP (a kind gift of Dr. Eric
Schirmer, University of Edinburgh) antibodies. For experiments
in Supplemental Figure S2, A–C, qPCR was performed using
a Bio-Rad iCycler machine and the protocol described in Fernius
and Marston (2009). For all other experiments shown, qPCR was
performed on a Roche LightCycler. Immunofluorescence Indirect immunofluorescence was performed as described in
Visintin et al. (1999). Tubulin was visualized using a rat anti-
tubulin antibody (AbD Serotec) at a dilution of 1:50 and an anti-
rat FITC-conjugated antibody (Jackson ImmunoResearch) at
a dilution of 1:100. For detection of Sgo1-6HA, a mouse HA.11
antibody (Covance) at a dilution of 1:500 and an anti-mouse Cy3-
conjugated antibody (Jackson ImmunoResearch) at a dilution of
1:100 were used. Chromosome spreads were performed as de-
scribed in Bizzari and Marston (2011). Yeast strains and plasmids Yeast strains and plasmids All yeast strains were derivatives of W303 or SK1 and are listed in
Supplemental Table S1. SCC1-6HA was described in Megee and
Koshland (1999). A PCR-based approach was used to tag Bub1,
Mtw1, Bir1, and Ndc10 with 6HA; Mtw1 with tdTomato; replace
the CLB3 promoter with pCUP1; and generate null alleles (Longtine
et al. 1998; Knop et al. 1999). SGO1-yeGFP, BUB1-yeGFP, and IPL1-
yeGFP were also generated by PCR-based epitope tagging (Sheff and
Thorn 2004). Auxin-inducible degron tagging was performed as
described (Nishimura et al. 2009). pMET3-CDC20 was described in
Clift et al. (2009). SGO1-6HA, IPL1-6HA, BRN1-6HA, RTS1-3PK,
and SGO1-TetR-GFP were described in Verzijlbergen et al. (2014). The ipl1-as5 and stu2-277 alleles were described in Pinsky et al. (2006b) and He et al. (2001), respectively. pCLB2-CDC20 was
described in Lee and Amon (2003). REC8-13Myc and SGO1-9Myc
were described in Marston et al. (2004). REC8-GFP, MTW1-
tdTomato, PDS1-tdTomato, and CNM67-3mCherry were de-
scribed in Matos et al. (2008). CEN5-GFP and NDC10-6HA were
described in Toth et al. (2000). To label chromosome III close to
the centromere with GFP, a ;700-base-pair (bp) fragment adjacent
to CEN3 was cloned into pRS306-112xtetO (Michaelis et al. 1997)
to generate plasmid AMp679, which was integrated in a strain
producing TetR-GFP. Plasmid pER1 (CEN6-TRP1-HTA1-HTB1)
was a kind gift from Dr. F. van Leeuwen (Netherlands Cancer
Institute). Plasmids AMp920 (H2A-S121D) and AMp921 (H2A-
S121A) were generated by site-directed mutagenesis of pER1
using a QuikChange II XL kit (Agilent Technologies). Microscopy methods Fluorescent microscopy analysis of fixed cells was performed
using a Zeiss Axioplan 2 microscope. Images were taken using
a Hamamatsu camera operated through Axiovision software
and processed using ImageJ software (National Institutes of
Health). For live-cell imaging, the ONIX microfluidic perfusion plat-
form by CellASIC was used within a heated chamber set to 30°C,
with the exception of the experiment shown in Figure 1, E and F,
where an Attofluor (Life Technologies) chamber heated to 25°C
was used. The microfluidics system was set up on a DeltaVision
Core system with an Olympus IX-71 microscope with ultimate
focus, and a 1003 Plan Apochromat/1.4 NA (oil) lens was used
for taking images. For imaging vegetative cells, G1-arrested cells
were loaded onto the plate, and we began imaging (15-min
intervals) immediately upon release from the arrest; six to eight
Z-sections 0.6–0.7 mm apart were taken for each field, with the Opposing kinases and phosphatases trigger shugoshin
redistribution under tension What are the molecular events that lead to Sgo1 re-
distribution? Although the detailed tension-dependent
mechanism is yet to be worked out, it is clear that
dephosphorylation is key to this process (Supplemental
Fig. S6). We showed that PP2A-Rts1 negatively regulates
Sgo1 levels at the centromere. We propose that Sgo1-bound
PP2A, and possibly other phosphatases too, promote de-
phosphorylation of as yet unknown chromatin-associated
substrates, the phosphorylation of which is required for
Sgo1 association with the pericentromere. In the absence
of tension, Sgo1 remains pericentromere-bound because
of the proximity of the kinetochore-bound kinase Bub1. Spindle tension leads to the spatial separation of Bub1 from
the chromatin, leading to the reversal of phosphorylation
of its chromatin-bound substrates by PP2A-Rts1, releasing
Sgo1. Eventually, upon stable biorientation, Bub1 kinase
itself dissociates from its Spc105/Spc7/KNL1 receptor in
the kinetochore due to reversal of Mps1-dependent phos-
phorylation by PP1, which also binds to Spc105/Spc7/
KNL1 (Pinsky et al. 2009; Vanoosthuyse and Hardwick
2009; Meadows et al. 2011; Rosenberg et al. 2011; Espeut
et al. 2012; London et al. 2012; Shepperd et al. 2012; In contrast to mitosis, during meiosis I, sister kineto-
chores are mono-oriented. It has been suggested that the
suppression of sister kinetochore biorientation in meiosis
I ensures the protection of pericentromeric cohesin (Vaur
et al. 2005; Gomez et al. 2007; Lee et al. 2008). Fission
yeast cells defective in sister kinetochore mono-orienta-
tion fail to properly protect pericentromeric cohesin (Vaur
et al. 2005; Yokobayashi and Watanabe 2005). This was not
initially thought to be the case in budding yeast, as
monopolin mutants retain pericentromeric Rec8 during
anaphase I, and sister chromatids remain cohesed after
securin degradation in meiosis I (Toth et al. 2000). However, our data indicate that sister kinetochore bio-
rientation is not complete during meiosis I in monopolin
mutants. By additionally removing chiasmata, we were
able to increase the frequency of cells with sister kinet-
ochores under tension. Analysis of cells lacking monop-
olin and chiasmata showed that the suppression of sister
kinetochore biorientation during meiosis I helps to retain
shugoshin at the pericentromere and contributes to the
maintenance of pericentromeric cohesion during meiosis
I. This indicates that the state of sister kinetochore GENES & DEVELOPMENT 1304 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension Nerusheva et al. Carmena M, Wheelock M, Funabiki H, Earnshaw WC. 2012. The
chromosomal passenger complex (CPC): from easy rider to the
godfather of mitosis. Nat Rev Mol Cell Biol 13: 789–803. exception of the experiment shown in Supplemental Figure S1I,
where cycling cells were loaded onto the plate and filmed as
above. For imaging of meiotic samples, cells were induced to
sporulate by resuspension in sporulation medium in flasks for
2.5 h before transferring to a microfluidics plate, and we began
imaging ;1 h later at 15-min intervals. For each image, six
Z-sections 1 mm apart were grabbed at 10% T for the green
channel and 5% T for the red channel with exposure times of 0.3
sec (Rec8-GFP), 0.2 sec (CENV-GFP), and 0.2 sec (red channel). ONIX software was used to control the microfluidics system, and
SoftWoRx software was used for the control of the DeltaVision
microscopy system and taking images. Image analysis was per-
formed using Image-Pro and ImageJ programs, and final images
were assembled using Adobe Photoshop. A custom-written plug
in for Image J was used to generate V plots. Line scans were
manually drawn across Mtw1-tdTomato kinetochore foci/focus
of 100 single cell images. The center point between the two
brightest pixels was chosen as a reference for alignment, and line
scans were ordered according to their length. Details are avail-
able on request. Chambon J-P, Touati SA, Berneau S, Cladie`re D, Hebras C,
Groeme R, McDougall A, Wassmann K. 2013. The PP2A
inhibitor I2PP2A is essential for sister chromatid segregation
in oocyte meiosis II. Curr Biol 23: 485–490. Clift D, Bizzari F, Marston AL. 2009. Shugoshin prevents
cohesin cleavage by PP2A(Cdc55)-dependent inhibition of
separase. Genes Dev 23: 766–780. Eckert CA, Gravdahl DJ, Megee PC. 2007. The enhancement of
pericentric cohesin association by conserved kinetochore
components promotes high-fidelity chromosome segregation
and is sensitive to microtubule-based tension. Genes Dev 21:
278–291. Espeut J, Cheerambathur DK, Krenning L, Oegema K, Desai A. 2012. Microtubule binding by KNL-1 contributes to spindle
checkpoint silencing at the kinetochore. J Cell Biol 196: 469–
482. Fernius J, Hardwick KG. 2007. Bub1 kinase targets Sgo1 to
ensure efficient chromsoome biorientation in budding yeast
mitosis. PLoS Genet 3: e213. For fluorescence intensity measurements of kinetochore/
microtubule-associated Sgo1-GFP signal, we used the ‘‘box in
box’’ method described in Hoffman et al. Growth conditions To arrest cells in metaphase by Cdc20 depletion, strains carrying
pMET3-CDC20 were arrested in G1 in synthetic medium lacking 1305 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Pre
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Acknowledgments We are grateful to Fred van Leeuwen and Wolfgang Zachariae
for yeast strains and plasmids. We thank Eric Schirmer for the
anti-GFP antibody, Colette Connor for the anti-Pgk1 anti-
body, and Dzmitry Batrakou for help with bean plots. We are
grateful to Kevin Hardwick, Julie Welburn, and Kitty Verzijlbergen
for comments on the manuscript. This work was supported
by the Wellcome Trust (090903, 092076, 096994) and the
Scottish University Life Sciences Alliance. O.O.N. grate-
fully acknowledges a studentship from the Darwin Trust of
Edinburgh. Funabiki H, Wynne DJ. 2013. Making an effective switch at the
kinetochore by phosphorylation and dephosphorylation. Chromosoma 122: 135–158. Gillett ES, Espelin CW, Sorger PK. 2004. Spindle checkpoint
proteins and chromosome-microtubule attachment in bud-
ding yeast. J Cell Biol 164: 535–546. Gomez R, Valdeolmillos A, Parra MT, Viera A, Carreiro C,
Roncal F, Rufas JS, Barbero JL, Suja JA. 2007. Mammalian
SGO2 appears at the inner centromere domain and redistrib-
utes depending on tension across centromeres during meio-
sis II and mitosis. EMBO Rep 8: 173–180. Gutie´rrez-Caballero C, Cebollero LR, Penda´s AM. 2012. Shugoshins: from protectors of cohesion to versatile adaptors
at the centromere. Trends Genet 28: 351–360. Nerusheva et al. (2001), with the
modification that two ellipses were used, as this allowed better
isolation of the kinetochore and spindle area from the nuclear
area (see Supplemental Fig. S1J). Fernius J, Marston AL. 2009. Establishment of cohesion at the
pericentromere by the Ctf19 kinetochore subcomplex and
the replication fork-associated factor, Csm3. PLoS Genet 5:
e1000629. Fernius J, Nerusheva OO, Galander S, Alves F de L, Rappsilber J,
Marston AL. 2013. Cohesin-dependent association of scc2/4
with the centromere initiates pericentromeric cohesion
establishment. Curr Biol 23: 599–606. References Keeney S, Giroux CN, Kleckner N. 1997. Meiosis-specific DNA
double-strand breaks are catalyzed by Spo11, a member of
a widely conserved protein family. Cell 88: 375–384. Marston AL. 2014. Chromosome segregation in budding yeast:
sister chromatid cohesion and related mechanisms. Genetics
196: 31–63. Kerrebrock AW, Miyazaki WY, Birnby D, Orr-Weaver TL. 1992. The Drosophila mei-S332 gene promotes sister-chromatid
cohesion in meiosis following kinetochore differentiation. Genetics 130: 827–841. Marston AL, Amon A. 2004. Meiosis: cell-cycle controls shuffle
and deal. Nat Rev Mol Cell Biol 5: 983–997. Marston AL, Lee BH, Amon A. 2003. The Cdc14 phosphatase
and the FEAR network control meiotic spindle disassembly
and chromosome segregation. Dev Cell 4: 711–726. Kiburz BM, Reynolds DB, Megee PC, Marston AL, Lee BH, Lee
TI, Levine SS, Young RA, Amon A. 2005. The core centromere
and Sgo1 establish a 50-kb cohesin-protected domain around
centromeres during meiosis I. Genes Dev 19: 3017–3030. Marston AL, Tham WH, Shah H, Amon A. 2004. A genome-
wide screen identifies genes required for centromeric co-
hesion. Science 303: 1367–1370. Kiburz BM, Amon A, Marston AL. 2008. Shugoshin promotes
sister kinetochore biorientation in Saccharomyces cerevi-
siae. Mol Biol Cell 19: 1199–1209. Matos J, Lipp JJ, Bogdanova A, Guillot S, Okaz E, Junqueira M,
Shevchenko A, Zachariae W. 2008. Dbf4-dependent cdc7
kinase links DNA replication to the segregation of homolo-
gous chromosomes in meiosis I. Cell 135: 662–678. Kitajima TS, Kawashima SA, Watanabe Y. 2004. The conserved
kinetochore protein shugoshin protects centromeric cohe-
sion during meiosis. Nature 427: 510–517. Mc Intyre J, Muller EGD, Weitzer S, Snydsman BE, Davis TN,
Uhlmann F. 2007. In vivo analysis of cohesin architecture
using FRET in the budding yeast Saccharomyces cerevisiae. EMBO J 26: 3783–3793. Kitajima TS, Sakuno T, Ishiguro K, Iemura S, Natsume T,
Kawashima SA, Watanabe Y. 2006. Shugoshin collaborates
with protein phosphatase 2A to protect cohesin. Nature 441:
46–52. Meadows JC, Shepperd LA, Vanoosthuyse V, Lancaster TC,
Sochaj AM, Buttrick GJ, Hardwick KG, Millar JBA. 2011. Spindle checkpoint silencing requires association of PP1 to
both Spc7 and kinesin-8 motors. Dev Cell 20: 739–750. Knop M, Siegers K, Pereira G, Zachariae W, Winsor B, Nasmyth
K, Schiebel E. 1999. Epitope tagging of yeast genes using
a PCR-based strategy: more tags and improved practical
routines. Yeast 15: 963–972. Megee PC, Koshland D. 1999. A functional assay for centromere-
associated sister chromatid cohesion. Science 285: 254–257. References The centromeric
protein Sgo1 is required to sense lack of tension on mitotic
chromosomes. Science 307: 130–133. Liu D, Vader G, Vromans MJM, Lampson MA, Lens SMA. 2009. Sensing chromosome bi-orientation by spatial separation of
aurora B kinase from kinetochore substrates. Science 323:
1350–1353. Ishiguro T, Tanaka K, Sakuno T, Watanabe Y. 2010. Shugoshin-
PP2A counteracts casein-kinase-1-dependent cleavage of
Rec8 by separase. Nat Cell Biol 12: 500–506. Liu D, Vleugel M, Backer CB, Hori T, Fukagawa T, Cheeseman
IM, Lampson MA. 2010. Regulated targeting of protein
phosphatase 1 to the outer kinetochore by KNL1 opposes
Aurora B kinase. J Cell Biol 188: 809–820. Katis VL, Galova M, Rabitsch KP, Gregan J, Nasmyth K. 2004. Maintenance of cohesin at centromeres after meiosis I in
budding yeast requires a kinetochore-associated protein re-
lated to MEI-S332. Curr Biol 14: 560–572. Liu H, Jia L, Yu H. 2013a. Phospho-H2A and cohesin specify
distinct tension-regulated Sgo1 pools at kinetochores and
inner centromeres. Curr Biol 23: 1927–1933. Katis VL, Lipp JJ, Imre R, Bogdanova A, Okaz E, Habermann B,
Mechtler K, Nasmyth K, Zachariae W. 2010. Rec8 phosphor-
ylation by casein kinase 1 and Cdc7-Dbf4 kinase regulates
cohesin cleavage by separase during meiosis. Dev Cell 18:
397–409. Liu H, Rankin S, Yu H. 2013b. Phosphorylation-enabled binding
of SGO1-PP2A to cohesin protects sororin and centromeric
cohesion during mitosis. Nat Cell Biol 15: 40–49. Kawashima SA, Tsukahara T, Langegger M, Hauf S, Kitajima TS,
Watanabe Y. 2007. Shugoshin enables tension-generating
attachment of kinetochores by loading Aurora to centro-
meres. Genes Dev 21: 420–435. London N, Ceto S, Ranish JA, Biggins S. 2012. Phosphoregula-
tion of Spc105 by Mps1 and PP1 regulates Bub1 localization
to kinetochores. Curr Biol 22: 900–906. Kawashima SA, Yamagishi Y, Honda T, Ishiguro K-I, Watanabe
Y. 2010. Phosphorylation of H2A by Bub1 prevents chromo-
somal instability through localizing shugoshin. Science 327:
172–177. Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A,
Brachat A, Philippsen P, Pringle JR. 1998. Additional mod-
ules for versatile and economical PCR-based gene deletion
and modification in Saccharomyces cerevisiae. Yeast 14:
953–961. Keating P, Rachidi N, Tanaka TU, Stark MJR. 2009. Ipl1-
dependent phosphorylation of Dam1 is reduced by tension
applied on kinetochores. J Cell Sci 122: 4375–4382. Marco E, Dorn JF, Hsu P-H, Jaqaman K, Sorger PK, Danuser G. 2013. S. cerevisiae chromosomes biorient via gradual resolution
of syntely between S phase and anaphase. Cell 154: 1127–1139. References Akiyoshi B, Sarangapani KK, Powers AF, Nelson CR, Reichow
SL, Arellano-Santoyo H, Gonen T, Ranish JA, Asbury CL,
Biggins S. 2010. Tension directly stabilizes reconstituted
kinetochore-microtubule attachments. Nature 468: 576–
579. Haase J, Stephens A, Verdaasdonk J, Yeh E, Bloom K. 2012. Bub1 kinase and Sgo1 modulate pericentric chromatin in
response to altered microtubule dynamics. Curr Biol 22:
471–481. Attner MA, Miller MP, Ee L-S, Elkin SK, Amon A. 2013. Polo
kinase Cdc5 is a central regulator of meiosis I. Proc Natl
Acad Sci 110: 14278–14283. Hauf S, Roitinger E, Koch B, Dittrich CM, Mechtler K, Peters
JM. 2005. Dissociation of cohesin from chromosome arms
and loss of arm cohesion during early mitosis depends on
phosphorylation of SA2. PLoS Biol 3: e69. Bizzari F, Marston AL. 2011. Cdc55 coordinates spindle assem-
bly and chromosome disjunction during meiosis. J Cell Biol
193: 1213–1228. He X, Asthana S, Sorger PK. 2000. Transient sister chromatid
separation and elastic deformation of chromosomes during
mitosis in budding yeast. Cell 101: 763–775. Brar GA, Kiburz BM, Zhang Y, Kim JE, White F, Amon A. 2006. Rec8 phosphorylation and recombination promote the step-
wise loss of cohesins in meiosis. Nature 441: 532–536. He X, Rines DR, Espelin CW, Sorger PK. 2001. Molecular
analysis of kinetochore-microtubule attachment in budding
yeast. Cell 106: 195–206. Buvelot S, Tatsutani SY, Vermaak D, Biggins S. 2003. The
budding yeast Ipl1/Aurora protein kinase regulates mitotic
spindle disassembly. J Cell Biol 160: 329–339. Hoffman DB, Pearson CG, Yen TJ, Howell BJ, Salmon ED. 2001. Microtubule-dependent changes in assembly of microtubule
motor proteins and mitotic spindle checkpoint proteins at
PtK1 kinetochores. Mol Biol Cell 12: 1995–2009. Campbell CS, Desai A. 2013. Tension sensing by Aurora B
kinase is independent of survivin-based centromere locali-
zation. Nature 497: 118–121. Huang H, Feng J, Famulski J, Rattner JB, Liu ST, Kao GD,
Muschel R, Chan GK, Yen TJ. 2007. Tripin/hSgo2 recruits
MCAK to the inner centromere to correct defective kineto-
chore attachments. J Cell Biol 177: 413–424. Carlile TM, Amon A. 2008. Meiosis I is established through
division-specific translational control of a cyclin. Cell 133:
280–291. 1306 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Pre
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension protection by shugoshin in mammalian oocytes and somatic
cells. Nat Cell Biol 10: 42–52. Indjeian VB, Stern BM, Murray AW. 2005. References Pinsky BA, Nelson CR, Biggins S. 2009. Protein phosphatase 1
regulates exit from the spindle checkpoint in budding yeast. Curr Biol 19: 1182–1187. Vanoosthuyse V, Prykhozhij S, Hardwick KG. 2007. Shugoshin 2
regulates localization of the chromosomal passenger proteins
in fission yeast mitosis. Mol Biol Cell 18: 1657–1669. Rabitsch KP, Petronczki M, Javerzat JP, Genier S, Chwalla B,
Schleiffer A, Tanaka TU, Nasmyth K. 2003. Kinetochore
recruitment of two nucleolar proteins is required for homo-
log segregation in meiosis I. Dev Cell 4: 535–548. Vaur S, Cubizolles F, Plane G, Genier S, Rabitsch PK, Gregan J,
Nasmyth K, Vanoosthuyse V, Hardwick KG, Javerzat JP. 2005. Control of Shugoshin function during fission-yeast
meiosis. Curr Biol 15: 2263–2270. Rabitsch KP, Gregan J, Schleiffer A, Javerzat JP, Eisenhaber F,
Nasmyth K. 2004. Two fission yeast homologs of Drosophila
Mei-S332 are required for chromosome segregation during
meiosis I and II. Curr Biol 14: 287–301. Verzijlbergen KF, Nerusheva OO, Kelly D, Kerr A, Clift D, de
Lima Alves F, Rappsilber J, Marston AL. 2014. Shugoshin
biases chromosomes for biorientation through condensin
recruitment to the pericentromere. eLife 3: e01374. Rattani A, Wolna M, Ploquin M, Helmhart W, Morrone S,
Mayer B, Godwin J, Xu W, Stemmann O, Pendas A, et al. 2013. Sgol2 provides a regulatory platform that coordinates
essential cell cycle processes during meiosis I in oocytes. eLife 2: e01133. Visintin R, Hwang ES, Amon A. 1999. Cfi1 prevents premature
exit from mitosis by anchoring Cdc14 phosphatase in the
nucleolus. Nature 398: 818–823. Riedel CG, Katis VL, Katou Y, Mori S, Itoh T, Helmhart W,
Galova M, Petronczki M, Gregan J, Cetin B, et al. 2006. Protein phosphatase 2A protects centromeric sister chroma-
tid cohesion during meiosis I. Nature 441: 53–61. Waizenegger IC, Hauf S, Meinke A, Peters JM. 2000. Two
distinct pathways remove mammalian cohesin from chro-
mosome arms in prophase and from centromeres in ana-
phase. Cell 103: 399–410. Rivera T, Ghenoiu C, Rodrı´guez-Corsino M, Mochida S, Funabiki
H, Losada A. 2012. Xenopus Shugoshin 2 regulates the spindle
assembly pathway mediated by the chromosomal passenger
complex. EMBO J 31: 1467–1479. Welburn JPI, Vleugel M, Liu D, Yates JR, Lampson MA,
Fukagawa T, Cheeseman IM. 2010. Aurora B phosphorylates
spatially distinct targets to differentially regulate the kinet-
ochore-microtubule interface. Mol Cell 38: 383–392. Rosenberg JS, Cross FR, Funabiki H. 2011. KNL1/Spc105 re-
cruits PP1 to silence the spindle assembly checkpoint. Curr
Biol 21: 942–947. References Kogut I, Wang J, Guacci V, Mistry RK, Megee PC. 2009. The
Scc2/Scc4 cohesin loader determines the distribution of
cohesin on budding yeast chromosomes. Genes Dev 23:
2345–2357. Michaelis C, Ciosk R, Nasmyth K. 1997. Cohesins: chromo-
somal proteins that prevent premature separation of sister
chromatids. Cell 91: 35–45. Kueng S, Hegemann B, Peters BH, Lipp JJ, Schleiffer A, Mechtler
K, Peters JM. 2006. Wapl controls the dynamic association of
cohesin with chromatin. Cell 127: 955–967. Miller MP, Unal E, Brar GA, Amon A. 2012. Meiosis I chromosome
segregation is established through regulation of microtubule-
kinetochore interactions. eLife 1: e00117. Lampson MA, Cheeseman IM. 2011. Sensing centromere ten-
sion: Aurora B and the regulation of kinetochore function. Trends Cell Biol 21: 133–140. Nakajima Y, Cormier A, Tyers RG, Pigula A, Peng Y, Drubin
DG, Barnes G. 2011. Ipl1/Aurora-dependent phosphorylation
of Sli15/INCENP regulates CPC-spindle interaction to en-
sure proper microtubule dynamics. J Cell Biol 194: 137–153. Lee BH, Amon A. 2003. Role of Polo-like kinase CDC5 in
programming meiosis I chromosome segregation. Science
300: 482–486. Ng TM, Lenstra TL, Duggan N, Jiang S, Ceto S, Holstege FCP,
Dai J, Boeke JD, Biggins S. 2013. Kinetochore function and
chromosome segregation rely on critical residues in histones
H3 and H4 in budding yeast. Genetics 195: 795–807. Lee J, Kitajima TS, Tanno Y, Yoshida K, Morita T, Miyano T,
Miyake M, Watanabe Y. 2008. Unified mode of centromeric 1307 GENES & DEVELOPMENT Nerusheva et al. Sheff MA, Thorn KS. 2004. Optimized cassettes for fluorescent
protein tagging in Saccharomyces cerevisiae. Yeast 21: 661–
670. Nicklas RB, Koch CA. 1969. Chromosome micromanipulation. 3. spindle fiber tension and the reorientation of mal-oriented
chromosomes. J Cell Biol 43: 40–50. Shepperd LA, Meadows JC, Sochaj AM, Lancaster TC, Zou J,
Buttrick GJ, Rappsilber J, Hardwick KG, Millar JBA. 2012. Phosphodependent recruitment of Bub1 and Bub3 to Spc7/
KNL1 by Mph1 kinase maintains the spindle checkpoint. Curr Biol 22: 891–899. Nicklas RB, Ward SC. 1994. Elements of error correction in
mitosis: microtubule capture, release, and tension. J Cell
Biol 126: 1241–1253. Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki
M. 2009. An auxin-based degron system for the rapid de-
pletion of proteins in nonplant cells. Nat Methods 6: 917–922. Shi Y. 2009. Serine/threonine phosphatases: mechanism through
structure. Cell 139: 468–484. References Nishiyama T, Ladurner R, Schmitz J, Kreidl E, Schleiffer A,
Bhaskara V, Bando M, Shirahige K, Hyman AA, Mechtler K,
et al. 2010. Sororin mediates sister chromatid cohesion by
antagonizing Wapl. Cell 143: 737–749. Shintomi K, Hirano T. 2009. Releasing cohesin from chromo-
some arms in early mitosis: opposing actions of Wapl-Pds5
and Sgo1. Genes Dev 23: 2224–2236. Ocampo-Hafalla MT, Katou Y, Shirahige K, Uhlmann F. 2007. Displacement and re-accumulation of centromeric cohesin
during transient pre-anaphase centromere splitting. Chro-
mosoma 116: 531–544. Shonn MA, McCarroll R, Murray AW. 2000. Requirement of the
spindle checkpoint for proper chromosome segregation in
budding yeast meiosis. Science 289: 300–303. Tanaka TU. 2010. Kinetochore-microtubule interactions: steps
towards bi-orientation. EMBO J 29: 4070–4082. Pearson CG, Maddox PS, Zarzar TR, Salmon ED, Bloom K. 2003. Yeast kinetochores do not stabilize Stu2p-dependent spindle
microtubule dynamics. Mol Biol Cell 14: 4181–4195. Tanaka T, Fuchs J, Loidl J, Nasmyth K. 2000. Cohesin ensures
bipolar attachment of microtubules to sister centromeres
and resists their precocious separation. Nat Cell Biol 2: 492–
499. Pereira G, Schiebel E. 2003. Separase regulates INCENP–Aurora
B anaphase spindle function through Cdc14. Science 302:
2120–2124. Tanaka TU, Rachidi N, Janke C, Pereira G, Galova M, Schiebel
E, Stark MJ, Nasmyth K. 2002. Evidence that the Ipl1–Sli15
(Aurora kinase–INCENP) complex promotes chromosome
bi-orientation by altering kinetochore–spindle pole connec-
tions. Cell 108: 317–329. Petronczki M, Matos J, Mori S, Gregan J, Bogdanova A,
Schwickart M, Mechtler K, Shirahige K, Zachariae W,
Nasmyth K. 2006. Monopolar attachment of sister kineto-
chores at meiosis I requires casein kinase 1. Cell 126: 1049–
1064. Tang Z, Shu H, Qi W, Mahmood NA, Mumby MC, Yu H. 2006. PP2A is required for centromeric localization of Sgo1 and
proper chromosome segregation. Dev Cell 10: 575–585. Pinsky BA, Kotwaliwale CV, Tatsutani SY, Breed CA, Biggins S. 2006a. Glc7/protein phosphatase 1 regulatory subunits can
oppose the Ipl1/aurora protein kinase by redistributing Glc7. Mol Cell Biol 26: 2648–2660. Toth A, Rabitsch KP, Galova M, Schleiffer A, Buonomo SB,
Nasmyth K. 2000. Functional genomics identifies monopo-
lin: a kinetochore protein required for segregation of homo-
logs during meiosis I. Cell 103: 1155–1168. Pinsky BA, Kung C, Shokat KM, Biggins S. 2006b. The Ipl1–
Aurora protein kinase activates the spindle checkpoint by
creating unattached kinetochores. Nat Cell Biol 8: 78–83. Vanoosthuyse V, Hardwick KG. 2009. A novel protein phospha-
tase 1-dependent spindle checkpoint silencing mechanism. Curr Biol 19: 1176–1181. Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from References Woodruff JB, Drubin DG, Barnes G. 2010. Mitotic spindle
disassembly occurs via distinct subprocesses driven by the
anaphase-promoting complex, Aurora B kinase, and kinesin-
8. J Cell Biol 191: 795–808. Rowland BD, Roig MB, Nishino T, Kurze A, Uluocak P, Mishra
A, Beckoue¨t F, Underwood P, Metson J, Imre R, et al. 2009. Building sister chromatid cohesion: smc3 acetylation coun-
teracts an antiestablishment activity. Mol Cell 33: 763–774. Xu Z, Cetin B, Anger M, Cho US, Helmhart W, Nasmyth K, Xu
W. 2009. Structure and function of the PP2A-shugoshin
interaction. Mol Cell 35: 426–441. 1308 GENES & DEVELOPMENT Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from Sgo1 regulated by tension Sgo1 regulated by tension Yaakov G, Thorn K, Morgan DO. 2012. Separase biosensor
reveals that cohesin cleavage timing depends on phosphatase
PP2A(Cdc55) regulation. Dev Cell 23: 124–136. Yamagishi Y, Honda T, Tanno Y, Watanabe Y. 2010. Two histone
marks establish the inner centromere and chromosome bi-
orientation. Science 330: 239–243. Yamagishi Y, Yang C-H, Tanno Y, Watanabe Y. 2012. MPS1/
Mph1 phosphorylates the kinetochore protein KNL1/Spc7 to
recruit SAC components. Nat Cell Biol 14: 746–752. Yeh E, Haase J, Paliulis LV, Joglekar A, Bond L, Bouck D, Salmon
ED, Bloom KS. 2008. Pericentric chromatin is organized into
an intramolecular loop in mitosis. Curr Biol 18: 81–90. Yokobayashi S, Watanabe Y. 2005. The kinetochore protein
Moa1 enables cohesion-mediated monopolar attachment at
meiosis I. Cell 123: 803–817. Zimniak T, Fitz V, Zhou H, Lampert F, Opravil S, Mechtler K,
Stolt-Bergner P, Westermann S. 2012. Spatiotemporal regu-
lation of Ipl1/Aurora activity by direct Cdk1 phosphoryla-
tion. Curr Biol 22: 787–793. GENES & DEVELOPMENT GENES & DEVELOPMENT 1309 Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
oaded from indicator of sister chromosome biorientation
Tension-dependent removal of pericentromeric shugoshin is an
Cold Spring Harbor Laboratory Press
on September 7, 2018 - Published by
genesdev.cshlp.org
Downloaded from indicator of sister chromosome biorientation
Tension-dependent removal of pericentromeric shugoshin is an Olga O. Nerusheva, Stefan Galander, Josefin Fernius, et al. 10.1101/gad.240291.114
Access the most recent version at doi:
28:
2014,
Genes Dev. Material
Supplemental
http://genesdev.cshlp.org/content/suppl/2014/06/17/28.12.1291.DC1
References
http://genesdev.cshlp.org/content/28/12/1291.full.html#ref-list-1
This article cites 106 articles, 41 of which can be accessed free at:
License
Commons
Creative
. http://creativecommons.org/licenses/by/4.0
License (Attribution 4.0 International), as described at
, is available under a Creative Commons
Genes & Development
This article, published in
Service
Email Alerting
click here. right corner of the article or
Receive free email alerts when new articles cite this article - sign up in the box at the top Material
Supplemental © 2014 Nerusheva et al.; Published by Cold Spring Harbor Laboratory Press © 2014 Nerusheva et al.; Published by Cold Spring Harbor Laboratory Press © 2014 Nerusheva et al.; Published by Cold Spring Harbor Laboratory Press
|
https://openalex.org/W4297872453
|
https://www.research-collection.ethz.ch/bitstream/20.500.11850/574547/2/fninf-16-971231.pdf
|
English
| null |
Machine learning reveals interhemispheric somatosensory coherence as indicator of anesthetic depth
|
Frontiers in neuroinformatics
| 2,022
|
cc-by
| 9,969
|
ETH Library Author(s): Originally published in: g
y p
Frontiers in Neuroinformatics 16, https://doi.org/10.3389/fninf.2022.971231 Funding acknowledgement: 172962 - Deviant Signals in the Thalamocortical Loop: Circuitry and Perception (SNF) This page was generated automatically upon download from the ETH Zurich Research Collection. For more information, please consult the Terms of use. TYPE Original Research
PUBLISHED 12 September 2022
DOI 10.3389/fninf.2022.971231 TYPE Original Research
PUBLISHED 12 September 2022
DOI 10.3389/fninf.2022.971231 KEYWORDS
depth
of
anesthesia,
gradient
boosting,
cortico-cortical
coherence,
mouse,
somatosensory cortex (S1) Machine learning reveals
interhemispheric
somatosensory coherence as
indicator of anesthetic depth OPEN ACCESS
EDITED BY
Alexandros Tzallas,
University of Ioannina, Greece
REVIEWED BY
Eleni Daskalaki,
Australian National University, Australia
Alastair John Loutit,
Neuroscience Research Australia,
Australia
*CORRESPONDENCE
Wolfger von der Behrens
wolfger@ini.uzh.ch
†These authors have contributed
equally to this work
RECEIVED 16 June 2022
ACCEPTED 12 August 2022
PUBLISHED 12 September 2022
CITATION
Schmidt D, English G, Gent TC,
Yanik MF and von der Behrens W
(2022) Machine learning reveals OPEN ACCESS Dominik Schmidt1†, Gwendolyn English1,2†, Thomas C. Gent1,3,
Mehmet Fatih Yanik1,2 and Wolfger von der Behrens1,2* 1Institute of Neuroinformatics, Department of Information Technology and Electrical Engineering
(D-ITET), ETH Zurich, University of Zurich, Zurich, Switzerland, 2Neuroscience Center Zurich (ZNZ),
Eidgenössische Technische Hochschule Zürich (ETH), University of Zurich, Zurich, Switzerland,
3Anaesthesiology Section, Vetsuisse Faculty, Department of Clinical Diagnostics and Services,
University of Zurich, Zurich, Switzerland The goal of this study was to identify features in mouse electrocorticogram
recordings that indicate the depth of anesthesia as approximated by the
administered anesthetic dosage. Anesthetic depth in laboratory animals must
be precisely monitored and controlled. However, for the most common lab
species (mice) few indicators useful for monitoring anesthetic depth have been
established. We used electrocorticogram recordings in mice, coupled with
peripheral stimulation, in order to identify features of brain activity modulated
by
isoflurane
anesthesia
and
explored
their
usefulness
in
monitoring
anesthetic depth through machine learning techniques. Using a gradient
boosting regressor framework we identified interhemispheric somatosensory
coherence as the most informative and reliable electrocorticogram feature for
determining anesthetic depth, yielding good generalization and performance
over many subjects. Knowing that interhemispheric somatosensory coherence
indicates the efectively administered isoflurane concentration is an important
step for establishing better anesthetic monitoring protocols and closed-loop
systems for animal surgeries. Schmidt D, English G, Gent TC,
Yanik MF and von der Behrens W
(2022) Machine learning reveals
interhemispheric somatosensory
coherence as indicator of anesthetic
depth. Front. Neuroinform. 16:971231. doi: 10.3389/fninf.2022.971231 © 2022 Schmidt, English, Gent, Yanik
and von der Behrens. This is an
open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which
does not comply with these terms. KEYWORDS
depth
of
anesthesia,
gradient
boosting,
cortico-cortical
coherence,
mouse,
somatosensory cortex (S1) KEYWORDS
depth
of
anesthesia,
gradient
boosting,
cortico-cortical
coherence,
mouse,
somatosensory cortex (S1) 2. Results Acute experiments were conducted on eleven adult female
mice. Two epidural electrocorticogram (ECoG) electrodes were
placed above the right- and left- hemisphere somatosensory
cortices (see Section 4). Throughout the experiment, stimulation
of the right whisker pad was used to evoke somatosensory
responses, yielding the right- and left- hemispheres as the
ipsi- and contra- lateral hemispheres to simulation, respectively. Concurrent to the ECoG recordings and somatosensory
stimulation, animals were subjected to an anesthesia protocol
that varied the concentration of administered isoflurane in
consecutive blocks of approximately 15 minute duration
(Figures 1A,B). Signal features were extracted from both ECoG
channels, tested for anesthetic modulation, and ultimately
provided as input to a gradient boosting regressor trained
to estimate two proxies of anesthetic depth, namely the
administered isoflurane concentration and the evoked response
amplitude. The regressor was trained and evaluated across data
collected from all experimental animals in a leave-one-out cross-
validation scheme, showing good generalization errors across
the entire population. While methods to estimate DoA in humans are well-
developed and validated, for laboratory animals, and in
particular mice, the most commonly used animal model
in biomedical research, specific techniques and findings
remain sparse (Hickman et al., 2017). Previous work has
investigated closed-loop anesthetic delivery in rats to control
the
electroencephalogram-determined
burst
suppression,
a signature of inactivated brain states (Ching et al., 2013;
Yang et al., 2019). Another study has linked human DoA
techniques
to
viable
methods
in
neonatal
mice
using
intracortical electrophysiology (Chini et al., 2019). More
recent research has helped to elucidate the signatures of
anesthesia induced effects on electrocorticogram recordings
in rats (Wang et al., 2022). Despite prior work, a gap remains
in the understanding of DoA monitoring in adult mice and
of the specific features of electroencephalographic signals
beyond burst suppression that are modulated by anesthesia. Further research has investigated alternative physiological
measures for their usefulness in monitoring anesthetic depth,
however heart rate and blood pressure have been shown to less
accurately assess DoA than the bispectral index, suggesting
that methods for monitoring anesthetic depth in mice based
upon electroencephalographic signals may be most effective
(Jaber et al., 2015). 1. Introduction General anesthesia is commonly used in surgical procedures and acute experiments
performed on laboratory animals in both fundamental and biomedical research. Exposure to general anesthetic agents strongly perturbs multiple brain networks and can
have profound, lasting effects on the physiology of exposed animals (Franks, 2008; Bajwa
et al., 2018; Pal et al., 2020). In order to minimize both the acute and chronic effects
of anesthesia while also safeguarding the welfare of laboratory animals during surgery,
the exposure to anesthetic agents should be expertly balanced. Namely, the administered
anesthesia should be sufficient to maintain the animal in an unconscious state, while
still minimally dosed to reduce the anesthetic’s acute effect on brain function and its
longitudinal effect on general physiology. 01 Frontiers in Neuroinformatics Frontiers in Neuroinformatics frontiersin.org Schmidt et al. Schmidt et al. 10.3389/fninf.2022.971231 10.3389/fninf.2022.971231 somatosensory coherence varies with the isoflurane anesthesia,
that it has good predictive capacity and is less subject to
interindividual variability. These properties of interhemispheric
somatosensory coherence make it a useful feature for designing
a future DoA monitoring systems in mouse models. These anesthetic constraints are well-known in human
medicine where, to prevent post-operative complications,
general anesthesia should be titrated to avoid detrimental
physiological effects (Eger et al., 1965; Dumont, 2012). To
facilitate an anesthetic delivery that balances the demands
of interoperative awareness and adverse effects, a significant
amount of research has focused on measuring the human depth
of anesthesia (DoA) (Nguyen-Ky et al., 2012; Ferdous and
Kiber, 2014; Sadrawi et al., 2015). Such work has prominently
led to the development of the proprietary Bispectral Index
Score (BIS), which makes use of several electroencephalographic
parameters to estimate DoA, and has been established as the
predominant anesthetic monitor used during human surgeries
(Dumont, 2012). Other published approaches for human
DoA estimation rely on non-linear features extracted from
electroencephalographic measurements or evoked potentials
(Al-Kadi et al., 2013), which are used as inputs to traditional
machine learning algorithms or artificial neural networks (Li
et al., 2020; Abel et al., 2021). Frontiers in Neuroinformatics 2.1. Electrocorticogram feature qualities Statistical and spectral features were extracted from the
ECoG signals and the effects of varying the administered
isoflurane concentration were evaluated (Figures 1C,D). An
exhaustive list of the tested features and their median values
across all administered isoflurane concentrations, as well as
the statistical significances of their anesthetic modulation,
can be found in Supplementary Tables 1, 2. Similar to results
in humans (Ching et al., 2012; Akeju et al., 2014; Liang
et al., 2015), several features showed significant dependency on
the administered isoflurane concentration, specifically: sample
entropy (p = 5.35 × 10−5), interhemispheric somatosensory
coherence (p
=
4.08 × 10−5), Lempel-Ziv complexity
(LZC) (p
=
1.93 × 10−3), and the burst suppression ratio
(p = 9.04 × 10−3). In the present study, we want to address this lack of robust
indicators for monitoring DoA in mice. We set out to identify
features of brain activity that are modulated by anesthetic
depth. While performing electrocorticogram recordings with
concurrent sensory stimulation in mice, we systematically varied
the anesthetic depth by changing the isoflurane concentration. Different features of the electrocorticograms were then provided
to a machine learning framework to estimate the instantaneous
anesthetic depth. Our findings elucidate that interhemispheric The interhemispheric somatosensory coherence between
the two somatosensory cortex channels within the spectrum
of 5 Hz to 40 Hz, comprising the theta, alpha, beta, and
low gamma bands, significantly increases with administered
isoflurane concentration (see Figure 2A). Important for DoA Frontiers in Neuroinformatics 02 frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 B A
B A
C
D
B
FIGURE 1
Electrocorticogram signals are extracted for estimation of anesthetic depth. (A) Schematic overview of the recording setup. Two electrodes over
the somatosensory cortices (CH1 and CH2) were measured against a common reference over cerebellum (Ref), while stimulating the right
whiskers and varying the isoflurane concentration. The gray shaded bars in the “Vaporizer” inset mark the first 5 min of each given isoflurane
concentration segment which were excluded from statistical analysis. (B) (Top) Craniotomy over barrel cortices. The Ag/AgCl electrodes were
placed directly on the dura, then covered in phosphate-bufered saline based agar and two component silicone. (Bottom) Partial craniotomy
over cerebellum for the reference electrode, drilled to 20% thickness, and covered as above. (C) Example ECoG traces recorded using the
OpenBCI during diferent administered isoflurane concentrations. Red shaded areas denote the [−0.2, 0.5 s] interval around a stimulus. 2.1. Electrocorticogram feature qualities (D) The
evoked responses averaged over all trials in each isoflurane concentration block, zero-aligned at t = 0. The shaded area indicates the 2σ-range
of the standard error of the mean. A A A B C C D D FIGURE 1
Electrocorticogram signals are extracted for estimation of anesthetic depth. (A) Schematic overview of the recording setup. Two electrodes over
the somatosensory cortices (CH1 and CH2) were measured against a common reference over cerebellum (Ref), while stimulating the right
whiskers and varying the isoflurane concentration. The gray shaded bars in the “Vaporizer” inset mark the first 5 min of each given isoflurane
concentration segment which were excluded from statistical analysis. (B) (Top) Craniotomy over barrel cortices. The Ag/AgCl electrodes were
placed directly on the dura, then covered in phosphate-bufered saline based agar and two component silicone. (Bottom) Partial craniotomy
over cerebellum for the reference electrode, drilled to 20% thickness, and covered as above. (C) Example ECoG traces recorded using the
OpenBCI during diferent administered isoflurane concentrations. Red shaded areas denote the [−0.2, 0.5 s] interval around a stimulus. (D) The
evoked responses averaged over all trials in each isoflurane concentration block, zero-aligned at t = 0. The shaded area indicates the 2σ-range
of the standard error of the mean. offtime measurement may be a more effective indicator of DoA
than the overall burst suppression ratio of on- to off- times. estimation, the interhemispheric somatosensory coherence
exhibits highly significant differences across all tested isoflurane
regimes, indicating a robust modulation across the spectrum of
tested dosages. Both LZC (i.e., the compressibility of the signal) and sample
entropy values (i.e., the randomness of the signal) peak for
the lowest administered isoflurane concentration of 1.0 %,
and are significantly different from the values observed at
the higher concentrations of 1.5 and 2.3 % (p ≤1.28 × 10−2
for LZC and p ≤2.91 × 10−3 for sample entropy, illustrated
in Figures 2C,D). The increase of almost 100 % for sample
entropy and 20 % to 30 % for LZC at lower anesthetic levels
is observed in the electrode channels both contralateral
and
ipsilateral
to
the
location
of
whisker
stimulation
(Figures 2C,D). As
depicted
in
Figure 2B,
increasing
administered
isoflurane concentration affects the burst suppression ratio
by inducing more extended off(i.e., suppression) times. Frontiers in Neuroinformatics 2.1. Electrocorticogram feature qualities Increased offtime corresponding to increased administered
isoflurane concentrations can be observed in both the contra-
and ipsilateral hemispheres, though the former shows a
higher statistical significance (p
= 4.31 × 10−3). Notably,
while the hemisphere contralateral to stimulation exhibits
a significant dynamic between offtime and administered
anesthetic concentration, the on (i.e., bursting) times are
not significantly effected by the administered anesthetic
concentration (see Supplementary Table 1), indicating that the The
distribution
of
all
four
of
these
features
are
appreciably
separated
across
different
administered
isoflurane concentrations, rendering them useful measures Frontiers in Neuroinformatics 03 frontiersin.org 10.3389/fninf.2022.971231 Schmidt et al. A
B
C
D
FIGURE 2
ECoG signal features display modulation to administered isoflurane concentrations. The leftmost column depicts representative examples of the
features found to be most modulated by changes in administered isoflurane concentrations. The middle column depicts violin plots of the
respective feature averages over animals across isoflurane concentrations. The rightmost column depicts violin plots of the respective standard
deviation within the diferent isoflurane segments, relative to the mean value in the segment. All violin plots display the minimum, maximum,
and median values of the distributions. The regions highlighted in red in the left panels depicts the data used in further analysis shown in the
right panels. (A) Interhemispheric somatosensory coherence based on the unfiltered raw-data. The traces illustrate median and 95 % confidence
interval calculated over all isoflurane blocks (right panels: 5–40 Hz). (B) Bursting Patterns. Shaded regions in representative example depict
signal portions classified as burst. (C) Lempel-Ziv Complexity. Representative examples of signal segments with maximum and minimum LZC. (D) Sample Entropy. Representative examples of signal segments with maximum and minimum sample entropy. Significance testing computed
with two-sided Mann–Whitney U-test across isoflurane concentrations with three Benjamini-Hochberg False Detection Rate controls
(Benjamini and Hochberg, 1995). Significant results are denoted with ∗p < 0.05, ∗∗p < 0.01. Precise p-values are listed in Supplementary Table 1. for robust estimation of anesthetic depth
However while
entropy LZC and the burst suppression ratio do not show A A B B C C D D FIGURE 2
ECoG signal features display modulation to administered isoflurane concentrations. The leftmost column depicts representative examples of the
features found to be most modulated by changes in administered isoflurane concentrations. The middle column depicts violin plots of the
respective feature averages over animals across isoflurane concentrations. 2.1. Electrocorticogram feature qualities The rightmost column depicts violin plots of the respective standard
deviation within the diferent isoflurane segments, relative to the mean value in the segment. All violin plots display the minimum, maximum,
and median values of the distributions. The regions highlighted in red in the left panels depicts the data used in further analysis shown in the
right panels. (A) Interhemispheric somatosensory coherence based on the unfiltered raw-data. The traces illustrate median and 95 % confidence
interval calculated over all isoflurane blocks (right panels: 5–40 Hz). (B) Bursting Patterns. Shaded regions in representative example depict
signal portions classified as burst. (C) Lempel-Ziv Complexity. Representative examples of signal segments with maximum and minimum LZC. (D) Sample Entropy. Representative examples of signal segments with maximum and minimum sample entropy. Significance testing computed
with two-sided Mann–Whitney U-test across isoflurane concentrations with three Benjamini-Hochberg False Detection Rate controls
(Benjamini and Hochberg, 1995). Significant results are denoted with ∗p < 0.05, ∗∗p < 0.01. Precise p-values are listed in Supplementary Table 1. entropy, LZC, and the burst suppression ratio do not show
significant differences between the two highest concentrations
of administered isoflurane (1.5 and 2.3 %). Although the for robust estimation of anesthetic depth. However, while
the interhemispheric somatosensory coherence values clearly
distinguish the three different isoflurane regimes, sample 04 Frontiers in Neuroinformatics frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 aforementioned
features
displayed
consistent
modulation
by the administered isoflurane concentration, others we
tested did not reliably vary with the anesthesia protocol
(Supplementary Tables 1, 2). Aligned with prior research in rats (Antunes et al., 2003), modulation of the spectral edge
frequency and 1/f -slope was not detected in our population of
mice. We observed that the absolute power density reduces with
increasing anesthesia (Supplementary Table 2), but is subject rats (Antunes et al., 2003), modulation of the spectral edge
frequency and 1/f -slope was not detected in our population of
mice. We observed that the absolute power density reduces with
increasing anesthesia (Supplementary Table 2), but is subject A
B
E
F
G
C
D
FIGURE 3
ECoG features serve as input to a gradient boosting regressor to successfully estimate DoA. Feature importances indicate interhemispheric
somatosensory coherence as a critical DoA readout. (A) Overview of feature extraction and estimator workflow. 2.1. Electrocorticogram feature qualities Notch and high-pass filtered
signal traces were extracted in 10 s blocks, signal features were calculated (FE), and then the three most recent consecutive blocks provided as
input to estimate a target variable ˆy via a gradient boosting ensemble. (B) Example of the estimation of isoflurane concentration. GT denotes the
measured ground-truth. The filled area under the predicted curve shows the standard error of the past minute. (C) Distribution of the Mean
Absolute estimation Error (MAE) over all animals in each isoflurane regime, corresponding to estimation of the isoflurane concentration. (D)
Feature importance over all folds. The interhemispheric somatosensory coherences are the most important features, over almost all folds,
corresponding to estimation of the isoflurane concentration. (E) Example estimation of the evoked response attenuation (ERA). GT denotes the
measured ground-truth. The filled area under the predicted curve shows the standard error of the past minute. (F) MAE over all folds or animals
(n = 11), corresponding to estimation of the ERA. (G) Distribution of the feature importances over all animals, corresponding to estimation of
the ERA. All the violin plots indicate the minimum, maximum and median values. A A A B C
D C B D D G F E F E G FIGURE 3
ECoG features serve as input to a gradient boosting regressor to successfully estimate DoA. Feature importances indicate interhemispheric
somatosensory coherence as a critical DoA readout. (A) Overview of feature extraction and estimator workflow. Notch and high-pass filtered
signal traces were extracted in 10 s blocks, signal features were calculated (FE), and then the three most recent consecutive blocks provided as
input to estimate a target variable ˆy via a gradient boosting ensemble. (B) Example of the estimation of isoflurane concentration. GT denotes the
measured ground-truth. The filled area under the predicted curve shows the standard error of the past minute. (C) Distribution of the Mean
Absolute estimation Error (MAE) over all animals in each isoflurane regime, corresponding to estimation of the isoflurane concentration. (D)
Feature importance over all folds. The interhemispheric somatosensory coherences are the most important features, over almost all folds,
corresponding to estimation of the isoflurane concentration. (E) Example estimation of the evoked response attenuation (ERA). GT denotes the
measured ground-truth. The filled area under the predicted curve shows the standard error of the past minute. (F) MAE over all folds or animals
(n = 11), corresponding to estimation of the ERA. 2.2. Estimator performance To further test the power of the ECoG features to estimate
the instantaneous anesthetic depth, the extracted values were
provided as input to a customized machine learning framework. A gradient boosting regressor with 100 boosting steps and
maximum tree-depth of three was trained with the values of
all of the previously described extracted features from the three
most recent 10 s recording windows (t-0: 0 s to −10 s, t-1:
−10 s to −20 s, t-2: −20 s to −30 s) as inputs and targeted
two measures of anesthetic depth, namely, the administered
isoflurane concentration and the evoked response amplitude
(depicted in Figure 3A). Gradient boosting was chosen in
a pre-trial review among Support Vector Regression (SVR)
with Gaussian kernels, SVR with linear kernels, K-nearest
neighbors and standard linear regression after exhibiting the best
performance. Estimators for each anesthetic depth target were
each trained eleven times, using a leave-one-out cross-validation
scheme (i.e., for each iteration, one animal was removed
from the whole dataset before training and the estimation
performance evaluated against that animal). Individual feature
importances (measured by the Gini gain, i.e., the total estimation
improvement achieved by inclusion of the feature) were then
extracted and their statistics over all 11-folds were considered. Stable estimation should yield similar feature importances
over all folds. Such stability was observed when estimating
the administered isoflurane concentration, however, estimation
across folds was less reliable when the regressor was trained on
evoked response amplitude, as shown in Figure 3G. Analysis of the feature importances using the Gini gain
shows that the interhemispheric somatosensory coherence
between the two barrel cortex electrodes is critical for estimation,
with the interhemispheric somatosensory coherence from the
oldest time block (i.e., t −2, representing the window 30 −20
s before current time, from Figure 3D) exhibiting the strongest
importance for estimation. Sample entropy in the barrel cortex
electrode located ipsilateral to the whisker stimulation was also
important for estimation, again with the oldest values (t −2)
displaying highest importance. Finally, for estimation of the
administered isoflurane concentration, the burst suppression
ratio for the ECoG channel contralateral to the whisker
stimulation hemisphere demonstrated to be useful. To rule out that the estimators use elapsed time as
a hidden variable and subsequently learn a static map
from elapsed time to isoflurane concentration, an identical
gradient-boosting regressor was trained to estimate elapsed
time. 2.1. Electrocorticogram feature qualities (G) Distribution of the feature importances over all animals, corresponding to estimation of
the ERA. All the violin plots indicate the minimum, maximum and median values. 05 Frontiers in Neuroinformatics frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 10.3389/fninf.2022.971231 allows for evaluation of standard multi-class classification
metrics. Averaging over all One-vs.-All pairs yields an accuracy
of 0.715 ± 0.134 (mean ± std), an F1-score of 0.699 ±
0.156, a precision of 0.780 ± 0.088 and recall of 0.714 ±
0.134. Figure 3B depicts the regression results over time for an
example animal and demonstrates that the estimation quickly
captures changes in administered isoflurane concentration. The
stability of estimating this variable is further demonstrated
by the low variance in estimation performance over all 11-
folds, particularly for administered isoflurane concentrations
of 1.0 and 1.5 %, depicted in Figure 3C. Figure 3D displays
the feature importance extracted from all 11-folds. The high
accuracy achieved for estimating the administered isoflurane
concentration establishes our methodology is a proof-of-concept
depth of anesthesia monitoring system for mice. to large inter-animal variations (a standard deviation larger
than 62 % of the mean), likely rendering these frequency related
features ineffective in identifying consistent trends across
animals without prior baseline knowledge. allows for evaluation of standard multi-class classification
metrics. Averaging over all One-vs.-All pairs yields an accuracy
of 0.715 ± 0.134 (mean ± std), an F1-score of 0.699 ±
0.156, a precision of 0.780 ± 0.088 and recall of 0.714 ±
0.134. Figure 3B depicts the regression results over time for an
example animal and demonstrates that the estimation quickly
captures changes in administered isoflurane concentration. The
stability of estimating this variable is further demonstrated
by the low variance in estimation performance over all 11-
folds, particularly for administered isoflurane concentrations
of 1.0 and 1.5 %, depicted in Figure 3C. Figure 3D displays
the feature importance extracted from all 11-folds. The high
accuracy achieved for estimating the administered isoflurane
concentration establishes our methodology is a proof-of-concept
depth of anesthesia monitoring system for mice. Frontiers in Neuroinformatics 3. Discussion Here
we
have
investigated
the
effect
of
varying
concentrations
of
administered
isoflurane
anesthesia
on
electrocorticogram features and explored the capacity of
these features to estimate the instantaneous DoA in mice. Our results show that many of the important features
previously identified in human models translate also to mouse
models, suggesting that elements of techniques developed for
monitoring of human DoA may further warrant incorporation
into
systems
targeting
laboratory
animals. Our
analysis
confirms the modulation of interhemispheric somatosensory
coherence (Michelson and Kozai, 2018), burst suppression
ratio (Tonner and Bein, 2006), Lempel-Ziv complexity, and
sample entropy (Liang et al., 2015) through administered
isoflurane concentration. However, we observed a number
of features that do not appear to be significantly effected by
anesthesia. Notably, the spectral measures of 1/f -slope (Antunes
et al., 2003; Barter et al., 2005), spectral edge frequencies and
absolute power distributions either show no modulation
or high inter-animal variability, rendering them unsuitable
for estimation. Using all of the aforementioned features as input to
the gradient boosting regressor yielded poor performance
when the estimation of the evoked response amplitude
was targeted. This poor performance could be explained
by the intra- and inter-animal variability observed in the
somatosensory evoked response. Previous research investigating
the effects of anesthesia in mouse models identified that
the average amplitudes of visual evoked potentials were not
significantly affected by variations in administered isoflurane
concentration and that evoked responses exhibited large
trial-by-trial variability within delivered concentration blocks
(Aggarwal et al., 2019). Our results, coupled with these previous
findings, indicate that evoked responses recorded from primary
sensory cortices across sensory modalities may not be useful
readouts for DoA. Isoflurane is one of the most commonly used inhalable
anesthetic agents for laboratory animals and has an inhibitory
effect on excitatory neurons via a number of molecular
mechanisms (de Sousa et al., 2000; Franks, 2008). This reduction
in excitation results in a reduced stimulation of inhibitory
interneurons which later causes a depletion of endogenous
inhibition (Ferron et al., 2009). Such cycles of increased and
reduced inhibition are manifest in the burst suppression ratio,
where dose-dependent isoflurane delivery modulates the ratio of
on- and off-times in mice (Brown et al., 2018). Our experiments
also reveal a dependence between administered isoflurane
concentrations and the burst suppression ratio, however the
computed ratio values exhibit significant differences only
between the lowest administered isoflurane concentration and
the higher two concentrations (e.g., 1 vs. 2.2. Estimator performance The performance-metrics of both estimators were then
tested for correlation, yielding no significant correlation (see
Supplementary Figures 4, 5). Evoked response amplitude was selected as an alternative
target variable for DoA estimation in order to test for properties
of anesthetic depth systematically reflected in the neural
response to peripheral stimulation. To perform this estimation,
rather than using the raw maximum evoked response amplitude
(as would be measured in volts), a unit-less ratio of the
evoked response amplitude, as recorded from the ECoG channel
contralateral to peripheral stimulation, was calculated. The unit-
less ratio was computed using a moving-window average of the
stimulus-by-stimulus maximum evoked response amplitude and
dividing these averaged values across isoflurane concentration
blocks by those computed during all 1.0 % isoflurane blocks. If the evoked response amplitude would scale similarly across
varied anesthetic depths between animals, an accurate, stable
estimation could be achieved. The administered isoflurane concentration was selected as
the first target variable for DoA estimation, yielding estimators
capable of inferring the effective isoflurane concentration. Similar to the concepts underlying the bispectral index score
used in human surgeries, training the gradient boosting
regressor to estimate the administered isoflurane concentration
over a population of mice results in an estimate reflective
of the ECoG feature values expressed by the average mouse
at the specified isoflurane concentration. Furthermore, under
certain assumptions, estimating the administered isoflurane
concentration is similar to estimating the true depth of
anesthesia (see Section 4 for mathematical derivations). Estimation of the administered isoflurane concentration
performed robustly over all animals [mean absolute error over
folds: 0.238 ± 0.076 (mean ± std), R2-score: 0.576 ± 0.289]. Since the test set target variable is binned into three discrete
isoflurane values, treating the isoflurane regression results as
classification via nearest-neighbor quantization additionally Training the gradient boosting regressor to estimate the
evoked response amplitude proved less accurate and robust
[mean absolute error over folds: 0.446 ± 0.252 (mean ± std), 06 frontiersin.org 10.3389/fninf.2022.971231 Schmidt et al. R2-score: −1.103 ± 2.009, mean absolute errors depicted in
Figure 3F]. While the estimation was accurate for several folds,
or several animals, the results did not generalize well across
all folds (example regression results for one fold depicted in
Figure 3E). This irregularity can be observed in Figure 3G, which
depicts highly variable feature importance. yielding their values less useful in distinguishing between higher
levels of anesthetic depth. 2.2. Estimator performance As indicated by the feature importances in Figure 3D,
interhemispheric somatosensory coherence is a robust measure
for estimating depth of anesthesia, also between higher
concentrations
of
administered
isoflurane. The
variation
observed in interhemispheric somatosensory coherence from
5 to 40 Hz, comprising the theta, alpha, beta, and low gamma
bands, across different anesthetic depths has, to the best of our
knowledge, not been reported in recordings made from the
somatosensory cortices. Previous reports in humans and rats
have identified modulation of alpha coherence in recordings
made from somatosensory and frontal cortices under propofol
anesthesia (Cimenser et al., 2011; Supp et al., 2011; Baker et al.,
2014). Further studies using isoflurane in rats have indicated
that coherence between primary motor and visual cortices
during peripheral sensory stimulation declined as delivered
anesthetic concentrations increased (Imas et al., 2006). Our
results in recordings made from both hemispheres of the
somatosensory cortex may reflect that increased isoflurane
administration
enforces
more
phase-coherence
between
thalamocortical projections to the two sensory hemispheres
(Ching et al., 2010). While our experiments cannot reveal the
precise mechanisms of this isoflurane induced coherence, the
resulting effect proves to be a critical component for estimating
DoA across all depths. Frontiers in Neuroinformatics 3. Discussion 1.5 and 2.3 %). Our
data indicates that the burst suppression ratio is not a strong
indicator of anesthetic depth at higher DoA. The mechanisms
of isoflurane that contribute to the burst suppression ratio
similarly impact the Lempel Ziv Complexity and Sample
Entropy features, where induced extended offtimes create a
more stable, compressible signal. Similar to our observation in
the burst suppression ratio, both of these complexity features
exhibit stronger significance between 1 vs. 1.5 and 2.3 %, again The
administered
isoflurane
concentration
estimator
performs reliably over all animals as shown in Figures 3B,C. All
of the identified features and estimators can be evaluated with a
low latency of 10 s. This evaluation latency time makes the use of
the identified features suitable for integration into closed-loop
anesthetic delivery (CLAD) systems (Ching et al., 2013; Yang
et al., 2019), which could target specific variables reflective of
the DoA. Further enhancements could be made to estimator
performance via a more exhaustive exploration of possible
signal features, such as the bispectral coherence (Li et al., 2012),
or by the implementation of neural networks to replace manual
feature extraction (Sadrawi et al., 2015; Li et al., 2020). Frontiers in Neuroinformatics frontiersin.org 07 Schmidt et al. Schmidt et al. 10.3389/fninf.2022.971231 Communities
Council
Directive
of
November
24,
1986
(86/609/EEC). This study is reported in accordance with
ARRIVE guidelines. Communities
Council
Directive
of
November
24,
1986
(86/609/EEC). This study is reported in accordance with
ARRIVE guidelines. The use of the identified electrocorticogram signal features
exhibiting modulation by anesthesia in DoA monitoring systems
for mice could provide numerous improvements to animal
welfare and biomedical research, ultimately allowing for more
precise experimental control and informed adjustment of the
administered anesthesia. A potential future scenario would be
an (clinical) anesthesia monitoring systems that continuously
measures the effectively administered isoflurane anesthesia that
is independent of the individual subject. This may be particular
useful in scenarios where the drug dosage administration is
not well controlled or prone to miscalculation. However, such
a monitoring system certainly can go beyond and predict the
actual anesthetic depth when validated with other physiological
parameters. The development of such a monitoring system
would require testing the suitability of the identified ECoG signal
features obtained through less invasive electroencephalography
(EEG) recording methods and testing whether these results
generalize to different experimental paradigms (e.g., without
whisker stimuli or using a different anesthetic agent). 3. Discussion Further,
we validated our system using isoflurane anesthesia, the most
common and recommended modality for acute recordings and
recovery procedures in experimental mice (Gargiulo et al.,
2012). A valuable next step would be to extend this to other
anesthetic regimens, a finding which would not only allow
standardization of anesthetic depth across experiments, but also
shed light upon the neuronal mechanisms involved in anesthetic
induced unconsciousness (Franks, 2008). Finally, a translation
of our results into a clinical system would require to test if
interhemispheric somatosensory coherence is a predictive DoA
feature for different pathological conditions as well. Such a
validation and new training of the network may be, in particular,
necessary for brain disorders which are known to affect the
electrocorticogram and EEG including neurodegenerative (e.g.,
in different mouse models of Alzheimer’s diseases; Kent et al.,
2018), neurological (e.g., after striatal stroke in mice; Baumann
et al., 2006), and neuropsychiatric conditions (e.g., SAPAP3-/-
mice which are a model for anxiety and obsessive compulsive
disorders; Lei et al., 2019). Mice were briefly induced with 3 % isoflurane in oxygen
anesthesia and injected with 1 mg/kg Meloxicam (Boehringer
Ingelheim, Ingelheim am Rhein, Germany) as analgesic. Remaining surgical procedures were completed under 2 %
isoflurane in oxygen supplied at a 1 L/s flow rate. Body
temperature was regulated at 37 ◦C with a homeothermic
blanket
control
unit
(Harvard
Apparatus,
Holliston,
Massachusetts). Animals were mounted in a stereotactic
frame and the scalp was removed to expose the skull. Three silver electrodes were then positioned onto the
skull and affixed using dental cement (Dentsply Sirona, York,
Pennsylvania). Electrodes were manufactured with 250 µm
thin Teflon (PTFE) coated silver wire (Goodfellow Cambridge
Limited, Huntingdon, England). Teflon coating was removed to
expose the wire end, which was subsequently molten to a sphere
(diameter 500 µm) and chlorided to improve electrochemical
stability (Geddes et al., 1969). Two
craniotomies
over
the
barrel
field
of
primary
somatosensory cortex of both hemispheres (3.5 mm lateral
and 1.5 mm caudal of bregma) were performed to expose an
1 mm diameter circular region of intact dura. A third partial
craniotomy was drilled over the cerebellar region (2 mm lateral
and caudal of lambda) until 80 % of the skull was removed. All three rounded electrode tips were then placed into the
craniotomies, with both barrel cortex electrodes making contact
with the dura and the reference electrode contacting the thinned
skull above cerebellum. 3. Discussion Craniotomies and electrode tips were
covered with a phosphate buffered saline agar (PBS) mixture
(2 % PBS) (Sigma-Aldrich, St. Louis, Missouri). The agar was
subsequently covered with a two-component silicone (World
Precision Instruments, Sarasota, Florida) to prevent drying
of the electrode sites. Silicone deposits over each craniotomy
were isolated from one another to avoid electrical connectivity
between recording sites. The skull surface was finally rinsed
with de-ionized water to prevent parallel resistances that
could interfere with the individual biological signals. Electrode
impedances were typically around 10 k to 20 k and usually
increased slightly during the recordings. 4. Methods Multiple whiskers from the right hemisphere vibrissal field
(rows B-D, arcs 1-3) were then secured around a glass capillary
that was then positioned immediately tangential to the whisker
pads. The capillary was affixed to a piezo-bending actuator
(Piezo Systems, Woburn, Massachusetts) driven by a controller
with a maximum output voltage of 150 V (Thorlabs, Newton,
New Jersey). Whisker stimulation sequences were generated
using custom LabVIEW code (National Instruments, Austin,
Texas), which produced an analog waveform with a sample
rate of 200 kHz and a resolution of 16 bits. The mechanical
waveforms at the capillary tip were single 120 Hz raised cosine
pulses (8.3 ms duration) with an amplitude of 300 µm (1.72◦) Frontiers in Neuroinformatics 4.1. Animal experiments In this study, eleven adult female C57BL/6J mice (age
98.0 ± 19.3 days, weight 21.00 ± 1.83 g), housed in enriched
cages of four mice in husbandry facilities with a 12-h inverted
light/dark cycle, supplied by Charles River Laboratories
were used for acute experiments. All experimental and
surgical procedures were approved by the local veterinary
authorities
and
corresponding
ethics
committees
of
the
Canton
Zurich,
Switzerland,
and
were
carried
out
in
accordance with the guidelines published in the European Frontiers in Neuroinformatics 08 frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 10.3389/fninf.2022.971231 and a peak velocity of 113.1 mm s−1 (648.8 ◦s−1) (Musall et al.,
2017), confirmed using a 0.1 µm resolution laser displacement
sensor (Micro-Epsilon, Ortenburg, Germany). Throughout the
recording, whiskers were repeatedly deflected with a 1 Hz
train containing 2 s stimulus-on and stimulus-offperiods
(Figure 1A). Four of the eleven mice received tail pinches
delivered via Hoffman clamp to produce data used in subsequent
experiments. All time periods containing tail pinch stimulation
were removed from the analyzed dataset. quality factor Q = 30 and a forward-backward first order
Butterworth high-pass filter above 0.1 Hz to eliminate signal
drift. Both raw ECoG signals were extracted from each 15min
isoflurane concentration block. The first 5 min of each block
were excluded from analysis such as to ignore the transient
effects of the previous concentration block. The following
spectral and time domain features of interest were then
extracted from consecutive, non-overlapping 10 s sequences
from each signal block. quality factor Q = 30 and a forward-backward first order
Butterworth high-pass filter above 0.1 Hz to eliminate signal
drift. Both raw ECoG signals were extracted from each 15min
isoflurane concentration block. The first 5 min of each block
were excluded from analysis such as to ignore the transient
effects of the previous concentration block. The following
spectral and time domain features of interest were then
extracted from consecutive, non-overlapping 10 s sequences
from each signal block. Contemporaneous
with
the
whisker
stimulation
and
electrocorticogram recording, an anesthesia protocol was
applied to achieve stable conditions at a variety of isoflurane
concentrations. The protocol consisted of 15 min segments
of isoflurane concentration percentages delivered in the
following sequence: starting from 1.5%, then transitioning
to the following segments: 2.3, 1.0, 1.5, 1.0, 2.3, and 1.5%. 4.1. Animal experiments Isoflurane concentrations were selected to span a broad range of
depths while remaining below dosages of 1.5 minimum alveolar
concentrations as determined in mice (Cesarovic et al., 2010)
and represent values of inhalable isoflurane recommended for
maintenance of an anesthetized state in mice (Gargiulo et al.,
2012). We used the inhalable isoflurane anesthesia, as it allows
maximum temporal control of anesthetic depth over other
injectable alternatives (Tremoleda et al., 2012). From each 15
min segment of a given isoflurane concentration, the first 5 min
were excluded from the statistical analysis, yielding from each
animal a dataset of six segments of 10 min duration (see gray
bars in the inset “Vaporizer” of Figure 1A). Spectral features extracted from both electrophysiology
channels included the spectral edge frequency below which 95 %
of the spectral power was contained (SEF 95) and the 1/f -
slope fit over the frequency range 20 Hz to 40 Hz. Additionally,
the interhemispheric somatosensory coherence was calculated
between the two electrophysiology channels and averaged over
the frequency range 5 Hz to 40 Hz. Finally, spectral power
content in multiple EEG bands (Newson and Thiagarajan, 2019)
were extracted, namely in the δ (0.1 Hz to 4 Hz), θ (4 Hz to
8 Hz), α (8 Hz to 13 Hz), β (13 Hz to 30 Hz), and γ (above
30 Hz) bands. All spectral features are based on Welch’s power
spectral density estimation calculated independently over each
10 s window. To complement the spectral features, a variety of time
domain features were identified for further use in the
algorithmic determination of anesthetic depth. The sample
entropy was computed over signal subsequences of 80 ms
length. As a measure of compressibility of the electrophysiology
channels, a binary sequence was extracted from the voltage
traces by thresholding them against the median value in every
signal block, and its Lempel-Ziv complexity calculated. The
burst suppression ratio was calculated over the entire isoflurane
concentration block to quantify the ratio of periods of high and
low signal activity. The completed setup was then enclosed in grounded
aluminum foil for improved shielding against electromagnetic
influences (e.g., from the proximal piezoelectric stimulators). Recordings were made using an open-source neural data
acquisition platform (OpenBCI, Brooklyn, New York) with a
gain of 24x and sample rate of 250 Hz. For all subsequent
analysis, the gain factor was removed from the raw data,
consequently all data reported is input referred. 4.1. Animal experiments Electrode signals
and whisker stimulation onsets were communicated to the
recording computer via Bluetooth connection. The described features were tested for modulation by
isoflurane through averaging them over segments of equal
isoflurane concentration for each mouse. The significance of
this modulation was quantified by executing a Mann–Whitney
U-Test for every pair of unequal isoflurane concentrations
for each feature. To correct for multiple comparisons, a
Benjamini-Hochberg False Detection Rate control (Benjamini
and Hochberg, 1995) has been applied. Further details on feature
calculations can be found in Supplementary material. Data collected from all animals was considered to be in the
experimental group. In order to best capture the unique inter-
animal dynamics induced by the dosage of isoflurane anesthesia,
data collected from all animals was a priori selected for inclusion
in analysis. 4.2. Feature extraction As anesthetic depth is a value on a continuous spectrum, we
perform a regression analysis to identify the current anesthetic
state of the animal based on the extracted features. All extracted
features were provided as inputs to a gradient boosting regressor
implemented by the python scikit-learn package (Pedregosa
et al., 2011). Such gradient boosting algorithms combine weak The
recording
setup
yielded
two
electrocorticogram
channels obtained from the barrel cortex electrodes located
both ipsilateral and contralateral to the hemisphere of whisker
stimulation. Signal traces were preprocessed using a forward-
backward notch filter to remove line noise at 50 Hz with a 09 Frontiers in Neuroinformatics frontiersin.org 10.3389/fninf.2022.971231 Schmidt et al. Dtrain,i =
x0,0
x0,1
x0,2
y0,2
x0,1
x0,2
x0,3
y0,3
... ... ... ... x0,N0−2
x0,N0−1
x0,N0,
y0,N0
x1,0
x1,1
x1,2
y1,2
... ... ... ... x1,N1−2
x1,N1−1
x1,N1,
y1,N1
... ... ... ... xM,NM−2 xM,NM−1 xM,NM, yM,NM
\ Deval,i
(4) Dtrain,i =
x0,0
x0,1
x0,2
y0,2
x0,1
x0,2
x0,3
y0,3
... ... ... ... x0,N0−2
x0,N0−1
x0,N0,
y0,N0
x1,0
x1,1
x1,2
y1,2
... ... ... ... x1,N1−2
x1,N1−1
x1,N1,
y1,N1
... ... ... ... xM,NM−2 xM,NM−1 xM,NM, yM,NM
\ Deval,i
(4) estimators gm into a strong estimator Gm via superposition: GM(x) =
M
X
m=1
wmgm(x)
(1) (1) with w ∈Rm weighting the individual estimators. Here the
individual estimators are chosen to be shallow decision trees
(with a maximum depth of three). A decision tree is a binary tree whose leaf nodes are assigned
a real valued number—the estimation. On every non-leaf node,
a decision is made what child node to traverse to, based on
a single feature and a threshold for that feature. Estimation
using decision trees is thus a simple tree traversal, returning the
value assigned to the leaf. In the training process, the optimal
feature and threshold is determined for every node by iterating
through all possible features, determining the optimal threshold
(which can be done computationally efficient), and greedily
choosing the feature and threshold that minimizes the error
(Hastie et al., 2009). The decrease in error after the split based on
this feature is tracked globally, which (summed over all decision
trees and normalized over all features) is represented by the Gini
gain computed in feature importance calculations (Breiman
et al., 1983). 4.2. Feature extraction In gradient boosting regression, every successive
estimator is trained on the residual error of the superposition
of all previous estimators, resulting in successively improved
overall loss (Hastie et al., 2009). (4) xm,n denotes the feature vector of the n-th block from the m-th
mouse, ym,n likewise the target variable on block n of mouse
m. Nm denotes the number of 10 s blocks in the recording of
mouse m. While the administered isoflurane concentration was
binned into the discrete values 1.0,1.5,2.3 in our experimental
protocol, a regression approach was nevertheless preferred to
classification, in order to capture the continuous nature of
anesthetic depth and to better gauge the promptness of response
without delays due to the quantization inherent to classification. Therefore, for estimation of the administered isoflurane, a
surrogate classification metric has been used, by binning the
estimated effective isoflurane concentration into the three bins
1.0, 1.5, 2.3 via nearest-neighbor quantization and calculating
the 3-class classification scores with averaging over all One-vs.-
All pairs. Two target variables for the estimators were used, namely the
administered isoflurane concentration and the evoked response
amplitude. As input, the estimator was provided the values of
each feature x in the set of all extracted features x described
above at three consecutive 10 s sub-sequences, yielding a
dataset of To rule out that the regression framework is merely
estimating the elapsed time as a hidden variable, and from that
a linear map to the isoflurane curve, the following approach
has been employed: an identical estimator is trained not on
isoflurane or Evoked Response Attenuation (ERA), but on the
elapsed time as ground-truth. Its R2-Score is then correlated
using Spearmans R, to see whether good performance on time-
estimation will also yield good estimation of isoflurane or ERA. With this approach an estimator is established, capable of
making anesthetic depth estimations by requiring a total of only
30 s of feature data for estimation, with a minimal latency of 10 s. (2) D =
([x0, x1, x2], y2), ..., ([xN−2, xN−1, xN], yN)
(2) where xn represents the feature vector at the n-th 10 s sub-
sequence, and y represents the anesthetic depth, given as either
the administered isoflurane concentration of evoked response
amplitude, in the same sub-sequence. For analysis, the datasets
of all n
=
11 mice were typically concatenated into a
single dataset. 4.2. Feature extraction Using the datasets acquired from the n
=
11 mice,
leave-one-out cross-validation was performed, with each fold
excluding the dataset recorded from one animal. The estimator
was trained on the remaining n = 10 datasets, and evaluated
on the excluded test animal, yielding an estimation of the
generalization error. The training and evaluation set for the
estimator for the i-th fold is thus: Acknowledgments =
Z
d
Ec|d[c|d] · f (d|⃗F)dd
(7)
= Ed|⃗F
Ec|d[c|d]
⃗F
(8) (7) The
authors
would
like
to
thank
Markus
Marks
for
his
valuable
help
with
machine
learning
methods. This
document
was
published
on
bioRxiv
(Schmidt et al., 2021). (8) We can see that what separates this estimator from the
optimal one is a transformation from anesthetic depth to the
average isoflurane concentration h : d 7→c, h(d) = E[c|d]. Assuming that h is sufficiently linear where f (d|⃗F) has most of
its support, we can apply the linearity of the expectation and get
the following approximation: Publisher’s note We estimate that the local linearity of E[c|d] should be
fulfilled for reasonable values of d, i.e., between very high
anesthetic depths and awake state. 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. Funding This can be expanded if we marginalize over the hidden
variable d, and by applying the Bayes rule we get: This work was funded by the Swiss National Science
Foundation (Project Grant No. 310030_172962). TCG was
supported by the University of Zürich Forschungskredit (FK-
017-64) and the Federal Food Safety and Veterinary Office
of Switzerland (2.20.02). Open access funding provided by
ETH Zurich. =
Z
c
Z
d
c · f (c|d)f (d|⃗F)dddc
(6) (6) Swapping the integrals results in an inner expectation: Swapping the integrals results in an inner expectation: Conflict of interest The authors declare that the research was conducted in
the absence of any commercial or financial relationships
that
could
be
construed
as
a
potential
conflict
of interest. = Ed|⃗F
h(d)
⃗F
(9)
≈h
Ed|⃗F
d|⃗F
(10) (9) (10) It is thus approximately a linear transformation of the
optimal estimation. Data availability statement The raw data supporting the conclusions of this article will
be made available by the authors, without undue reservation. 4.3.1. Estimation for true depth of anesthesia Estimating the administered isoflurane concentration can
be understood as an anesthetic depth estimation similar to the
bispectral index score. A population of mice has an average
reaction to any given isoflurane concentration. Training over
enough data yields an estimator that assigns the most probable
isoflurane concentration at which the average mouse (over the
training population) would have the observed reaction. We
can further show that, with certain assumptions, estimating
isoflurane concentration is equivalent to estimating the true
depth of anesthesia. We do this by modeling the DoA problem
as a Bayesian network, with probability density function Deval,i =
xi,0
xi,1
xi,2
yi,2
xi,1
xi,2
xi,3
yi,3
... ... ... ... xi,Ni−2 xi,Ni−1 xi,Ni, yi,Ni
(3) Deval,i =
xi,0
xi,1
xi,2
yi,2
xi,1
xi,2
xi,3
yi,3
... ... ... ... xi,Ni−2 xi,Ni−1 xi,Ni, yi,Ni
(3) (3) Frontiers in Neuroinformatics
1 10 10 frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 Author contributions f (⃗F, d, c) = f (⃗F|d) · f (d|c) · f (c) where ⃗F is the extracted features,
d the true depth of anesthesia (which is a hidden variable), and c
the administered isoflurane concentration. GE
and
WB
conceived
the
study. GE
and
DS
designed
the
experiments,
recording
techniques,
and
wrote
the
manuscript
with
review
from
WB,
MFY,
and
TCG. DS
conceived
of
and
conducted
all
data
analysis. GE, TCG, and DS performed the experiments. All authors contributed to the article and approved the
submitted version. An optimal estimator (minimizing a square loss), which has
perfect knowledge of the density f (⃗F, d, c), will estimate E[d|⃗F]
(Hastie et al., 2009). Since d is a hidden variable, we have no way
to train on it, and thus settle on estimating E[c|⃗F]. How does this
surrogate compare to the optimal estimation? E[c|⃗F] =
Z
c
c · f (c|⃗F)dc
(5) (5) References doi: 10.1002/jnr.24343 Hastie, T., Tibshirani, R., and Friedman, J. (2009). The Elements of Statistical
Learning, 2nd Edn. Springer Series in Statistics. New York, NY: Springer New York
Inc. doi: 10.1007/978-0-387-84858-7 Baker, R., Gent, T. C., Yang, Q., Parker, S., Vyssotski, A. L., Wisden, W., et al. (2014). Altered activity in the central medial thalamus precedes changes in the
neocortex during transitions into both sleep and propofol anesthesia. J. Neurosci. 34, 13326–13335. doi: 10.1523/JNEUROSCI.1519-14.2014 Hickman, DL., Johnson, J., Vemulapalli, TH., Crisler, J., and Shepherd, R. (2017). “Commonly used animal models,” in Principles of Animal Research for Graduate
and Undergraduate Students, eds M. A. Suckow and K. L. Stewart (Boston, MA:
Academic Press), 117–175. Barter, L., Dominguez, C. L., Carstens, E., and Antognini, J. F. (2005). The effect of isoflurane and halothane on electroencephalographic activation
elicited by repetitive noxious c-fiber stimulation. Neurosci. Lett. 382, 242–247. doi: 10.1016/j.neulet.2005.03.017 Imas, O., Ropella, K., Wood, J., and Hudetz, A. (2006). Isoflurane disrupts
anterio-posterior phase synchronization of flash-induced field potentials in the rat. Neurosci. Lett. 402, 216–221. doi: 10.1016/j.neulet.2006.04.003 Baumann, C. R., Kilic, E., Petit, B., Werth, E., Hermann, D. M., Tafti, M., and
Bassetti, C. L. (2006). Sleep EEG changes after middle cerebral artery infarcts
in mice: different effects of striatal and cortical lesions. Sleep 29, 1339–1344. doi: 10.1093/sleep/29.10.1339 Jaber, S. M., Sullivan, S., Hankenson, F. C., Kilbaugh, T. J., and Margulies, S. S. (2015). Comparison of heart rate and blood pressure with toe pinch and bispectral
index for monitoring the depth of anesthesia in piglets. J. Am. Assoc. Lab. Anim. Sci. 54, 536–544. Benjamini, Y., and Hochberg, Y. (1995). Controlling the false discovery rate:
a practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 57,
289–300. doi: 10.1111/j.2517-6161.1995.tb02031.x Kent, B. A., Strittmatter, S. M., and Nygaard, H. B. (2018). Sleep and
eeg power spectral analysis in three transgenic mouse models of Alzheimer’s
disease: APP/PS1, 3xtgad, and tg2576. J. Alzheimer’s Dis. 64, 1325–1336. doi: 10.3233/JAD-180260 Breiman, L., Friedman, J., Stone, C. J., and Olshen, R. A. (1983). Classification
and Regression Trees. Boca Raton, FL: CRC Press. Lei, H., Lai, J., Sun, X., Xu, Q., and Feng, G. (2019). Lateral orbitofrontal
dysfunction in the SAPAP3 knockout mouse model of obsessive-compulsive
disorder. J. Psychiatry Neurosci. 44, 120–131. doi: 10.1503/jpn.180032 Brown, P. L., Zanos, P., Wang, L., Elmer, G. I., Gould, T. D., and Shepard,
P. D. (2018). References Isoflurane but not halothane prevents and reverses helpless
behavior: a role for EEG burst suppression? J. Neuropsychophramacol. 21, 777–785. doi: 10.1093/ijnp/pyy029 Li, D., Li, X., Hagihira, S., and Sleigh, J. W. (2012). Cross-frequency coupling
during isoflurane anaesthesia as revealed by electroencephalographic harmonic
wavelet bicoherence. Br. J. Anaesth. 110, 409–419. doi: 10.1093/bja/aes397 Cesarovic, N., Nicholls, F., Rettich, A., Kronen, P., Hässig, M., Jirkof, P.,
et al. (2010). Isoflurane and sevoflurane provide equally effective anaesthesia in
laboratory mice. Lab. Anim. 44, 329–36. doi: 10.1258/la.2010.009085 Li, R., Wu, Q., Liu, J., Wu, Q., Li, C., and Zhao, Q. (2020). Monitoring depth of
anesthesia based on hybrid features and recurrent neural network. Front. Neurosci. 14, 26. doi: 10.3389/fnins.2020.00026 Ching, S., Cimenser, A., Purdon, P. L., Brown, E. N., and Kopell, N. J. (2010). Thalamocortical model for a propofol-induced α-rhythm associated
with loss of consciousness. Proc. Natl. Acad. Sci. U.S.A. 107, 22665–22670. doi: 10.1073/pnas.1017069108 Liang, Z., Wang, Y., Sun, X., Li, D., Voss, L. J., Sleigh, J. W., et al. (2015). EEG entropy measures in anesthesia. Front. Comput. Neurosci. 9, 16. doi: 10.3389/fncom.2015.00016 Michelson, N. J., and Kozai, T. D. Y. (2018). Isoflurane and ketamine
differentially influence spontaneous and evoked laminar electrophysiology in
mouse v1. J. Neurophysiol. 120, 2232–2245. doi: 10.1152/jn.00299.2018 Ching, S., Liberman, M. Y., Chemali, J. J., Westover, M. B., Kenny, J. D.,
Solt, K., et al. (2013). Real-time closed-loop control in a rodent model of
medically induced coma using burst suppression. Anesthesiology 119, 848–860. doi: 10.1097/ALN.0b013e31829d4ab4 Musall, S., Haiss, F., Weber, B., and von der Behrens, W. (2017). Deviant
processing in the primary somatosensory cortex. Cereb. Cortex 27, 863–876. doi: 10.1093/cercor/bhv283 Ching, S., Purdon, P. L., Vijayan, S., Kopell, N. J., and Brown, E. N. (2012). A
neurophysiological-metabolic model for burst suppression. Proc. Natl. Acad. Sci. U.S.A. 109, 3095–3100. doi: 10.1073/pnas.1121461109 Newson, J. J., and Thiagarajan, T. C. (2019). EEG frequency bands in
psychiatric disorders: A review of resting state studies. Front. Hum. Neurosci. 12:521. doi: 10.3389/fnhum.2018.00521 Chini, M., Gretenkord, S., Kostka, J. K., Pöpplau, J. A., Cornelissen, L., Berde,
C. B., et al. (2019). Neural correlates of anesthesia in newborn mice and humans. Front. Neural Circuits 13, 38. doi: 10.3389/fncir.2019.00038 Nguyen-Ky, T., Wen, P., Li, Y., and Malan, M. (2012). Measuring the hypnotic
depth of anaesthesia based on the EEG signal using combined wavelet transform,
eigenvector and normalisation techniques. Comput. Biol. Med. 42, 680–691. doi: 10.1016/j.compbiomed.2012.03.004 Cimenser, A., Purdon, P. References Dumont, G. A. (2012). “Closed-loop control of anesthesia - A review,” in IFAC
Proceedings Volumes 45:373-378. 8th IFAC Symposium on Biological and Medical
Systems, August 29-31, Vol. 45 (Budapest), 373–378. Abel, J. H., Badgeley, M. A., Meschede-Krasa, B., Schamberg, G., Garwood,
I. C., Lecamwasam, K., et al. (2021). Machine learning of EEG spectra
classifies unconsciousness during gabaergic anesthesia. PLoS ONE 16, e0246165. doi: 10.1371/journal.pone.0246165 Eger,
E. I.,
Saidman,
L. J.,
and
Brandstater,
B. (1965). Minimum
alveolar
anesthetic
concentration:
A
standard
of
anesthetic
potency. Anesthesiology
26,
756–763. doi:
10.1097/00000542-196511000-
00010 Aggarwal, A., Brennan, C., Shortal, B., Contreras, D., Kelz, M. B., and Proekt,
A. (2019). Coherence of visual-evoked gamma oscillations is disrupted by propofol
but preserved under equipotent doses of isoflurane. Front. Syst. Neurosci. 13, 19. doi: 10.3389/fnsys.2019.00019 Ferdous, N., and Kiber, M. A. (2014). Estimating depth of anesthesia
from eeg signals using wavelet transform. Int. J. Intell. Inform. Syst. 3, 40–44. doi: 10.11648/j.ijiis.20140304.12 Akeju, O., Westover, M. B., Pavone, K. J., Sampson, A. L., Hartnack,
K. E., Brown, E. N., et al. (2014). Effects of sevoflurane and propofol on
frontal electroencephalogram power and coherence. Anesthesiology 121, 990–998. doi: 10.1097/ALN.0000000000000436 Ferron, J.-F., Kroeger, D., Chever, O., and Amzica, F. (2009). Cortical inhibition
during burst suppression induced with isoflurane anesthesia. J. Neurosci. 29,
9850–9860. doi: 10.1523/JNEUROSCI.5176-08.2009 Al-Kadi, M. I., Reaz, M. B. I., and Ali, M. A. M. (2013). Evolution of
electroencephalogram signal analysis techniques during anesthesia. Sensors 13,
6605–6635. doi: 10.3390/s130506605 Franks, N. P. (2008). General anaesthesia: from molecular targets to neuronal
pathways of sleep and arousal. Nat. Rev. Neurosci. 9, 370–386. doi: 10.1038/nrn2372 Antunes, L. M., Golledge, H. D., Roughan, J. V., and Flecknell, P. A. (2003). Comparison of electroencephalogram activity and auditory evoked responses
during isoflurane and halothane anaesthesia in the rat. Vet. Anaesth. Anal. 30,
15–23. doi: 10.1046/j.1467-2995.2003.00085.x Gargiulo, S., Greco, A., Gramanzini, M., Esposito, S., Affuso, A., Brunetti, A.,
et al. (2012). Mice anesthesia, analgesia, and care, part i: anesthetic considerations
in preclinical research. ILAR J. 53, E55–E69. doi: 10.1093/ilar.53.1.55 Geddes, L. A., Baker, L. E., and Moore, A. G. (1969). Optimum electrolytic
chloriding of silver electrodes. Med. Biol. Eng. 7, 49–56. doi: 10.1007/BF02474669 Bajwa, N. M., Lee, J. B., Halavi, S., Hartman, R. E., and Obenaus, A. (2018). Repeated isoflurane in adult male mice leads to acute and persistent motor
decrements with long-term modifications in corpus callosum microstructural
integrity. J. Neurosci. Res. 97, 332–345. Ethics statement The Supplementary Material for this article can be
found online at: https://www.frontiersin.org/articles/10.3389/
fninf.2022.971231/full#supplementary-material The animal study was reviewed and approved by Veterinary
office of the Canton of Zurich. 11 Frontiers in Neuroinformatics frontiersin.org frontiersin.org 10.3389/fninf.2022.971231 10.3389/fninf.2022.971231 Schmidt et al. Schmidt, D., English, G., Gent, T., Yanik, M. F., and von der Behrens, W. (2021).
Electrocorticography based monitoring of anaesthetic depth in mice. bioRxiv. 2021,
1–14. doi: 10.1101/2021.07.12.452032 Sadrawi, M., Fan, S.-Z., Abbod, M. F., Jen, K.-K., and Shieh, J.-S. (2015).
Computational depth of anesthesia via multiple vital signs based on artificial neural
networks. BioMed Res. Int. 2015, 536863. doi: 10.1155/2015/536863 Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O.,
et al. (2011). Scikit-learn: machine learning in Python. J. Mach. Learn. Res. 12,
2825–2830. Supp, G. G., Siegel, M., Hipp, J. F., and Engel, A. K. (2011). Cortical
hypersynchrony
predicts
breakdown
of
sensory
processing
during
loss
of
consciousness.
Curr.
Biol.
21,
1988–1993.
doi:
10.1016/j.cub.2011.
10.017 Yang, Y., Lee, J. T., Guidera, J. A., Vlasoz, K. Y., Pei, J., Brown, E. N., et al. (2019).
Developing a personalized closed-loop controller of medically-induced coma in a
rodent model. J. Neural Eng. 16, 036022. doi: 10.1088/1741-2552/ab0ea4 Schmidt et al. References L., Pierce, E. T., Walsh, J. L., Salazar-Gomez, A. F., Harrell, P. G., et al. (2011). Tracking brain states under general anesthesia
by using global coherence analysis. Proc. Natl. Acad. Sci. U.S.A. 108, 8832–8837. doi: 10.1073/pnas.1017041108 Pal, D., Li, D., Dean, J. G., Brito, M. A., Liu, T., Fryzel, A. M., et al. (2020). Level of consciousness is dissociable from electroencephalographic measures of
cortical connectivity, slow oscillations, and complexity. J. Neurosci. 40, 605–618. doi: 10.1523/JNEUROSCI.1910-19.2019 de Sousa, S. L., Dickinson, R., Lieb, W. R., and Franks, N. P. (2000). Contrasting synaptic actions of the inhalation gneral anesthetics isoflurane and
xenon. Anesthesiology 92, 1055–1066. doi: 10.1097/00000542-200004000-00024 Frontiers in Neuroinformatics 12 frontiersin.org Schmidt et al. 10.3389/fninf.2022.971231 10.3389/fninf.2022.971231 Tonner, P., and Bein, B. (2006). Classic electroencephalographic parameters:
Median frequency, spectral edge frequency etc. Best Pract. Res. Clin. Anaesthesiol. 20, 147–159. doi: 10.1016/j.bpa.2005.08.008 Tonner, P., and Bein, B. (2006). Classic electroencephalographic parameters:
Median frequency, spectral edge frequency etc. Best Pract. Res. Clin. Anaesthesiol. 20, 147–159. doi: 10.1016/j.bpa.2005.08.008 Tremoleda, J. L., Kerton, A., and Gsell, W. (2012). Anaesthesia and
physiological monitoring during in vivo imaging of laboratory rodents:
considerations on experimental outcomes and animal welfare. EJNMMI Res. 2, 44. doi: 10.1186/2191-219X-2-44 Schmidt, D., English, G., Gent, T., Yanik, M. F., and von der Behrens, W. (2021). Electrocorticography based monitoring of anaesthetic depth in mice. bioRxiv. 2021,
1–14. doi: 10.1101/2021.07.12.452032 Wang, Z., Zhang, F., Yue, L., Hu, L., Li, X., Xu, B., et al. (2022). Cortical
complexity and connectivity during isoflurane-induced general anesthesia: a rat
study. J. Neural Eng. 19, 1–22. doi: 10.1088/1741-2552/ac6a7b 13 13 frontiersin.org Frontiers in Neuroinformatics
|
https://openalex.org/W2993510023
|
https://periodicos.ufjf.br/index.php/RPDE/article/download/31437/22048
|
Portuguese
| null |
O compartilhamento de conhecimentos por intermédio de vídeos
|
Pesquisa e Debate em Educação
| 2,020
|
cc-by
| 6,780
|
Artigo de pesquisa
https://periodicos.ufjf.br/index.php/RPDE Artigo de pesquisa Arthur Gualberto Bacelar da Cruz Urpia Universidade Cesumar, Vice-coordenador do Programa de Pós-graduação
Conhecimento nas Organizações, Maringá, Paraná, Brasil arthur.urpia@unicesumar.edu.br | https://orcid.org/0000-0002-5273-6373 Bruna Hernandes Scarabelli Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
b ll b
l
| h
//
d
/ Fernanda Crocetta Schraiber Fernanda Crocetta Schraiber Instituto Federal de Educação, Ciência e Tecnologia do Paraná, Ivaiporã, Paraná, Brasil
fernanda.schraiber@ifpr.edu.br | https://orcid.org/0000-0002-8653-868 Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
luismarchi@bol com br | https://orcid org/0000-0003-2198-1648 Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
luismarchi@bol.com.br | https://orcid.org/0000-0003-2198-1648 Fabrício Ricardo Tomaz Bernardelli Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil Bruna Hernandes Scarabelli Esta revista está licenciada sob a licença Creative Commons Attribution 4.0 International License (CC BY 4.0). O compartilhamento de conhecimentos por intermédio de vídeos: um estudo
de caso em uma turma de pós-graduação stricto sensu
Knowledge sharing through video: a case study in a stricto sensu graduate class O compartilhamento de conhecimentos por intermédio de vídeos: um estudo
de caso em uma turma de pós-graduação stricto sensu
Knowledge sharing through video: a case study in a stricto sensu graduate class Felipe Pereira de Melo
Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
felipedemelo.esc@gmail.com | https://orcid.org/0000-0002-3513-9884
Fabrício Ricardo Tomaz Bernardelli
Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
bernardelliwolf@gmail.com | https://orcid.org/0000-0002-1492-2554
Bruna Hernandes Scarabelli
Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
scarabellibruna@gmail.com | https://orcid.org/0000-0001-7285-2876
Fernanda Crocetta Schraiber
Instituto Federal de Educação, Ciência e Tecnologia do Paraná, Ivaiporã, Paraná, Brasil
fernanda.schraiber@ifpr.edu.br | https://orcid.org/0000-0002-8653-868
Luis Augusto Sautchuk Marchi
Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil
luismarchi@bol.com.br | https://orcid.org/0000-0003-2198-1648
Arthur Gualberto Bacelar da Cruz Urpia
Universidade Cesumar, Vice-coordenador do Programa de Pós-graduação em Gestão do
Conhecimento nas Organizações, Maringá, Paraná, Brasil
arthur.urpia@unicesumar.edu.br | https://orcid.org/0000-0002-5273-6373 Universidade Cesumar, Programa de Pós-graduação em Gestão do Conhecimento nas
Organizações, Maringá, Paraná, Brasil felipedemelo.esc@gmail.com | https://orcid.org/0000-0002-3513-9884 Resumo As constantes transformações no cenário social, proporcionaram um grande acúmulo de conhecimentos e informações
por parte dos indivíduos, não se trata apenas de possuir a informação, mas saber de que forma torná-la valor e
potencializar em oportunidades. Assim sendo, utilizar os recursos tecnológicos disponíveis para o compartilhamento do
conhecimento torna-se essencial para a ampliação do conhecimento como um todo, diante da perspectiva de que a
troca de conhecimentos gera crescimento. Diante disso, o objetivo geral deste trabalho é analisar o processo de
compartilhamento do conhecimento por intermédio de vídeos em uma turma de Pós-graduação stricto sensu de uma
Instituição de Ensino Superior (IES) do norte do Paraná. Para tal, do ponto de vista metodológico, esta pesquisa aplicada
se enquadra, quanto ao procedimento, como um estudo de caso. Como principal resultado, observa-se que, com base
nas análises das interpretações pessoais dos integrantes do grupo de trabalho utilizado na pesquisa, o exercício das
práticas de compartilhamento da Gestão do Conhecimento empregados na preparação e elaboração dos vídeos
resumos facilitaram no processo de aprendizagem de forma a garantir que o conhecimento transpassasse muito além
do que estava apenas nos textos, trazendo ricas contribuições advindas das experiências e das discussões, gerindo o
conhecimento por meio da Gestão do Conhecimento e proporcionando a transformação do conhecimento tácito em
explícito. chave: Gestão do conhecimento. Compartilhamento do conhecimento. Vídeos resumos. Aprendizagem. Palavras-chave: Gestão do conhecimento. Compartilhamento do conhecimento. Vídeos resumo Esta revista está licenciada sob a licença Creative Commons Attribution 4.0 International License (CC BY 4.0). Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Abstract The constant changes in the social scenario, provided a great accumulation of knowledge and information on the part
of individuals, it is not just about having the information, but knowing how to make it value and potentialize
opportunities. Therefore, using the technological resources available for knowledge sharing becomes essential for the
expansion of knowledge as a whole, given the perspective that the exchange of knowledge generates growth. In view of
this, the general objective of this work is to analyze the knowledge sharing process through videos in a stricto sensu
graduate class of a Higher Education Institution (HEI) in northern Paraná. To this end, from a methodological point of
view, this applied research fits, as to the procedure, as a case study. As a main result, it is observed that, based on the
analysis of the personal interpretations of the members of the work group used in the research, the exercise of the
Knowledge Management sharing practices employed in the preparation and elaboration of the summary videos
facilitated the learning process of in order to ensure that knowledge goes far beyond what was only in the texts, bringing
rich contributions from experiences and discussions, managing knowledge through Knowledge Management and
providing the transformation of tacit knowledge into explicit. p
g
f
f
g
p
Keywords: Knowledge management. Knowledge sharing. Video summaries. Learning. Keywords: Knowledge management. Knowledge sharing. Video summaries. Learning. Pesquisa e Debate em Educação, Juiz de Fora, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. Universidade Federal de Juiz de
Fora. ISSN 2237-9444. 1 Introdução Em uma sociedade de rápidas transformações, de incertezas, de grande
competitividade, com grande volume de dados e informações, a Gestão do
Conhecimento tem se destacado como uma estratégia para qualquer organização
que queira sobreviver nesta nova economia. Isto porque, diante deste cenário, o
capital intelectual se tornou o maior diferencial, a maior vantagem competitiva das
organizações (STEWART, 2002). Os ativos intangíveis, a exemplo do conhecimento,
passaram a ter tanto, ou até mais, valor do que qualquer outro. Assim, a Gestão do Conhecimento surge para gerenciar este capital
intelectual nas organizações. Sveiby (1998, p. 1) define a Gestão do Conhecimento
como “a arte de gerar valor a partir de bens intangíveis da organização”. Stewart
(2002, p. 172) complementa ao afirmar que a Gestão do Conhecimento é
“identificar o que se sabe, captar e organizar esse conhecimento e utilizá-lo de
modo a gerar retornos”. Já Mattera (2014) explica que a Gestão do Conhecimento
é um processo com objetivos definidos, onde são agregadas metodologias e
ferramentas
que
proporcionam
um
ambiente
de
aprendizagem
e
compartilhamento de informações gerando eficiência e competitividade para as
organizações. Para que a Gestão do Conhecimento aconteça de forma eficiente, práticas
e ferramentas estratégicas são utilizadas neste processo. Possoli (2012) considera
que as ferramentas de GC promovem inovação nos processos: [As ferramentas] resgatam os
elementos tácitos do
conhecimento – que, naturalmente, são subjetivos e estão
presentes de modo horizontal nas empresas – e os põem a
disposição dos diferentes níveis de tomada de decisão, o que
engloba desde as decisões cotidianas até grandes diretrizes
corporativas. Por meio disso, os elementos são transformados
em mais um ativo dos capitais diferentes e complementares da
empresa (POSSOLI, 2012, p.46). Para Davenport e Prusak (1998), as ferramentas de Gestão do
Conhecimento possuem a função de reproduzir parte do conhecimento que existe
na mente das pessoas, nos documentos e rotinas organizacionais para, na
sequência, disponibilizar este conhecimento para todo o órgão. Se o conhecimento
não fica disponível para ser utilizado, ele não agrega valor para as pessoas e
organizações. Neste caso, as ferramentas seriam um suporte, na maior parte das
vezes tecnológico, para que este conhecimento realmente possa fluir e se
transformar em vantagem competitiva. Dutra et al. (2014, p.1198) complementa
ao afirmar que as ferramentas de Gestão do Conhecimento “são aquelas que
possibilitam tratar, de modo sistemático e intencional, o conhecimento”. Como citar MELO, Felipe Pereira de; BERNARDELLI, Fabrício Ricardo Tomaz; SCARABELLI, Bruna Hernandes; SCHRAIBER, Fernanda Crocetta;
MARCHI, Luis Augusto Sautchuk; URPIA, Arthur Gualberto Bacelar da Cruz. O compartilhamento de conhecimentos por intermédio
de vídeos: um estudo de caso em uma turma de pós-graduação stricto sensu. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. DOI: https://doi.org/10.34019/2237-9444.2020.v10.31437. Pesquisa e Debate em Educação, Juiz de Fora, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. Universidade Federal de Juiz de
Fora. ISSN 2237-9444. 1384 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 1 Introdução Diante da necessidade constante de se potencializar as ações, são inúmeras
as organizações que buscam lapidar seu ativo intangível, até mesmo no meio
educacional, no qual a busca pelo conhecimento é um desafio constante dos
professores (DIOGO et al., 2015). Nesse sentido, utilizar os recursos tecnológicos
disponíveis para o compartilhamento torna-se essencial para a ampliação do
conhecimento organizacional, inclusive em organizações escolares, pois “pensar no
processo de ensino e aprendizagem em pleno século XXI sem o uso constante dos
diversos instrumentos tecnológicos é deixar de acompanhar a evolução que está na
essência da humanidade” (SILVA; CORREA, 2014, p. 26). Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Dentre as tecnologias para o compartilhamento de conhecimentos, é
perceptível que o número de pessoas que utilizam celulares e smartphones vem
crescendo de maneira intensa. Essa utilização ocorre nos mais diversos segmentos,
que vai desde o uso pessoal, corporativo e até mesmo institucional. Assim, no meio
educacional, o uso de smartphones é considerado por muitos como prejudicial à
aprendizagem, uma vez que dispersa a atenção de alguns alunos, que muitas vezes
preferem acessar as redes sociais através de seus celulares a prestar a atenção em
conteúdos elencados como importantes para sua formação (SILVA; CORREA, 2014). Todavia, muitos recursos desses aparelhos podem ser utilizados a favor desse
processo, como, por exemplo, o compartilhamento de conteúdos educativos. Bartol e Srivastava (2002) definem compartilhamento de conhecimentos
como sendo subdivisão de informações, ideias, sugestões e experiências
organizacionalmente relevantes do indivíduo com outros, afirmando que o
compartilhamento de conhecimentos é um elemento essencial dos sistemas de
Gestão do Conhecimento. Dyer e Nobeoka (2000) reiteram que para ocorrer o
compartilhamento do conhecimento nos ambientes, além das motivações, a
interação social é de suma importância. Conforme
Nonaka e Takeuchi
(1997)
o
compartilhamento
de
conhecimentos com o mundo exterior é a última fase do processo envolvendo a
criação do conhecimento, construindo, assim redes de conhecimentos. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 1 Introdução O
Compartilhamento do conhecimento está relacionado com o início do próprio
conhecimento (NONAKA; TAKEUCHI, 1997), efetuando, assim, um papel
fundamental para a modificação nas organizações, pois o conhecimento
compartilhado auxilia em ideias novas, produtos e serviços (ORDAZ; CRUZ; GINEL,
2010). Um sistema de viabilizar esses conhecimentos é aplicando metodologias de
compartilhamento de conhecimentos entre indivíduos, departamentos e setores
de empresas. Desta forma, é perceptível que o compartilhar vai muito além de apenas
fornecer ou disponibilizar conhecimentos, pois o compartilhar possibilidade a
criação de novos conhecimentos, além disso favorece a socialização e o
desenvolvimento do grupo, facilita o processo de aprendizagem e aperfeiçoa a
capacidade estratégica, trazendo como consequência melhores resultados (FREIRE;
FURLAN; SILVEIRA, 2018). Atribui-se o compartilhamento de conhecimentos ao know-how e às
referências ao trabalho, como auxílio na colaborarão de deliberação de problemas,
expandindo ou realizando novas políticas ou estratégias (CUMMINGS, 2003;
PULAKOS;
DORSEY;
BORMAN,
2003). Compreende-se,
então,
que
o
compartilhamento de conhecimentos foi articulado como procedimento
colaborativo. Sendo assim, a metodologia necessitaria ser afável, em que a origem
e o receptor do compartilhamento se auxiliariam e seriam favorecidos com os
excelentes resultados. De acordo com Davenport e Prusak (2000), o modo de compartilhar
conhecimento no encadeamento organizacional não é simples como expressado
nas observações expostas. Conforme os indivíduos compreendem um interesse
expressivo nos conhecimentos já possuídos, os processos de compartilhamento de
conhecimentos com seus parceiros podem ser dificultados por impasses neste
processo (HONG; SUH; KOO, 2011). Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Entretanto, os indivíduos apresentam inúmeros conceitos do que é
significativo
compartilhar
e
outros
elementos
também
afetam
no
compartilhamento do conhecimento (HONG; SUH; KOO, 2011), como costumes
crenças e valores individuais que compõem a cultura organizacional (DI CHIARA;
ALCARA; TOMAEL, 2010). Consequentemente, a formação de conhecimento se dá
em uma interpretação diferente daquela orientada meramente à aquisição de
conhecimento. 1 Introdução Ainda, a criatividade, expertise, conhecimento, habilidades e métodos
utilizados na rotina do trabalho são itens vistos como vantagens que o indivíduo
pode proporcionar para os ambientes em que está inserido, uma vez que são
características individuais que estão principalmente na mente de cada um,
portanto, não passíveis de cópia por qualquer outra pessoa ou organização e,
assim, um ativo que pode fazer a diferença e agregar valor e inovação tecnológica
e organizacional (POSSOLI, 2012). O manual da Asian Productivity Organization (APO) (2010) classifica o
compartilhamento de vídeo como uma ferramenta de Gestão do conhecimento
que pode ser usada para criar, armazenar e compartilhar o conhecimento. O uso
do vídeo como compartilhamento do conhecimento na educação já é uma
realidade, com a pulverização dos cursos técnicos e graduação a distância, que é
uma modalidade de ensino considerada flexível e eficiente para aqueles que
buscam uma alternativa ao método tradicional. Segundo Nunes (2013), a técnica
de compartilhamento de vídeo consiste em publicar conteúdo no formato de vídeo
para um público específico ou para todos. Dentre os benefícios do uso do vídeo na educação, pode-se mencionar a
gestão do tempo, pois os alunos podem se organizar de maneira flexível (assim
como em um ensino a distância) para se beneficiar do conteúdo, que já está
previamente
registrado,
além
da
facilidade
do
compartilhamento
do
conhecimento, independentemente da localização ou horário. Além disso, Nunes
(2013) também atribui como benefício de utilização dessa técnica pelo vídeo o fato
dela ser um meio propicio para a captura, compartilhamento e consumo de
conhecimento. O compartilhamento do conteúdo através de vídeo é facilitado pelo uso de
smartphones, visto que esses dispositivos oferecem importantes recursos para
gravação, edição e compartilhamento desse material. O Manual de Ferramentas e
Técnicas em Gestão do Conhecimento da Asian Productivity Organization (APO,
2010), traz em seu conteúdo a perspectiva de compartilhamento por vídeos,
conceituando como a capacidade de publicar conteúdo de vídeos, seja para
públicos específicos ou todo o mundo. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 1 Introdução Vale ressaltar que, embora todos os
benefícios mencionados, o manual da APO discorre algumas situações que o uso do
vídeo não é recomendado, sendo elas: I) Quando não há uma boa conexão de internet, pois os arquivos em vídeo
são mais “pesados” que arquivos em áudio, por exemplo; I) Quando não há uma boa conexão de internet, pois os arquivos em vídeo
são mais “pesados” que arquivos em áudio, por exemplo; II) Se o público-alvo precisar ter as informações de maneira mais facilitada,
recomenda-se o uso de texto, pois o vídeo é um meio serial; Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 III) A criação de vídeo é um processo demorado, portanto se a informação
repassada é algo susceptível a muitas mudanças, a melhor forma de compartilhar
o conteúdo é na forma textual (APO, 2010). Diante disso, o objetivo geral deste trabalho é analisar como ocorre o
compartilhamento do conhecimento por intermédio de vídeos em uma turma de
Pós-graduação stricto sensu de uma Instituição de Ensino Superior (IES) do norte
do Paraná. Faz-se importante destacar que a utilização de vídeos para o
compartilhamento do conhecimento, além dos benefícios já citados, também tem
como vantagem tornar os alunos expectadores de si mesmo, promovendo uma
maior interação entre si e entre o conteúdo. Além disso, possibilita uma
interpretação do conteúdo pelos próprios acadêmicos, diferente da metodologia
tradicional que apresenta boa parte da interpretação do conteúdo pelo professor,
resultando em maior produtividade. 2 Materiais e método O trabalho, quanto sua natureza, caracteriza-se como sendo uma pesquisa
aplicada, sendo considerada como a pesquisa que tem por objetivo gerar
conhecimentos para aplicação prática dirigidos à problemas específicos
(PRODANOV; DE FREITAS, 2013), sendo que, durante a realização do referido
estudo, foi selecionada uma turma de Pós-graduação stricto sensu, de determinada
instituição de ensino superior (IES) da região norte do Paraná, que compartilha
conhecimentos através do uso de vídeos. Com isto, quanto ao procedimento, esta
pesquisa se enquadra como um estudo de caso, que é visto como o método que
visa compreender fenômenos sociais complexos, preservando as características
holísticas e significativas dos eventos da vida real (YIN, 2015). Já quanto aos
objetivos, ela é exploratória, pois houve o envolvimento direto dos pesquisadores
para a familiarização com universo investigado e levantamento de informações que
subsidiaram a análise dos dados e entendimento. Tem-se ainda que a pesquisa
exploratória visa maior familiaridade com os problemas propostos, tornado
explícitos e possibilitando o desenvolvimento de hipóteses sobre eles (PRODANOV;
DE FREITAS, 2013). Quanto à abordagem, a pesquisa é qualitativa, sendo esta
considerada a percepção entre uma relação dinâmica entre o mundo real e o
sujeito, um vínculo entre a objetividade e a subjetividade que não pode ser
traduzido em números (PRODANOV; DE FREITAS, 2013). Para a sua
operacionalização, a pesquisa foi dividida em quatro fases, conforme a Figura 1. Figura 1: Fases do processo de compartilhamento de vídeos resumos
Fonte: dos autores, 2020. Figura 1: Fases do processo de compartilhamento de vídeos resumos Figura 1: Fases do processo de compartilhamento de vídeos resumos Fonte: dos autores, 2020. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 1- Na primeira fase, 24 alunos foram divididos em 4 grupos de trabalho,
sendo realizada a proposta de leitura de textos, de forma integral e tradicional
pelos alunos, com posterior debate em sala de aula, nesta fase a proposta que os
alunos compreendam a forma tradicional de leitura de textos de parte dos
programas de Pós-graduação; 2- Na segunda fase foi selecionado um dos grupos de trabalho, contendo 6
alunos, e proposto o modelo de resumos em vídeos, de forma que os alunos
realizassem a leitura prévia de textos diferenciados da primeira fase e
confeccionassem vídeos resumos para compartilhamento com seu grupo de
trabalho. Nesta segunda fase é criado o grupo no aplicativo em uma plataforma de
comunicação de mensagens instantâneas e de comunicação por voz, com o
credenciamento de todos os membros como administradores do grupo, e criação
da pasta para compartilhamento em um Drive de armazenamento em nuvem, o
qual se tornará a Biblioteca Virtual; 3- Na terceira fase aos alunos participantes realizaram a gravação de vídeos
resumos e de forma consequente o compartilhamento dos vídeos, disponibilizando
tanto no drive, quanto no aplicativo de comunicação com posterior debate sobre
os conteúdos assimilados e apresentados nos vídeos resumos; 4- Na quarta fase os alunos pertencentes ao grupo de trabalho realizaram
a avaliação dos resultados obtidos e dissertaram sobre suas impressões positivas e
críticas acerca de sua participação, visando compartilhar suas expectativas e
posicionamentos com relação à proposta. Evidente que, durante a realização do estudo, os alunos foram orientados
que não se tratava de uma substituição da leitura dos textos pela mera visualização
dos vídeos, mas sim a utilização destes como uma ferramenta de apoio no processo
de aprendizagem. Neste sentido, todos os participantes foram orientados que as
leituras dos textos deveriam ser integrais, ampliando a capacidade de assimilação
e de discussão dos conteúdos apresentados Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 3 Resultados e discussão Para obtenção dos resultados, os alunos pertencentes ao grupo de trabalho
selecionado, conforme a 4° fase, foram convidados a dissertar suas considerações
acerca da proposta, visando obter-se as impressões positivas e críticas. De acordo
com os feedbacks dos membros integrantes, quanto as suas impressões na
participação da pesquisa, é perceptível que informações e discussões sobre os
textos estudados geraram uma maior aderência do conhecimento. Liu e Phillips (2011) destacam que incentivar os indivíduos a
compartilharem conhecimentos úteis pode assegurar vantagem competitiva para
as organizações e melhorar o seu desempenho, a sua produtividade e a sua
capacidade de inovação. Os vídeos gravados e produzidos pelos alunos, além de arquivados em uma
biblioteca virtual compartilhada, foram enviados para serem compartilhados em
um grupo do aplicativo de mensagens instantâneas e comunicação por voz e
imagem, grupo este criado pelos próprios alunos, no qual todos os participantes
são administradores, tornando o conhecimento compartilhado disponível em um
espaço livre para discussões e inferências. Destaca-se que o compartilhamento via
aplicativo de comunicação por mensagens instantâneas facilitou a discussão e Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 visibilidade dos conteúdos, permitindo que abaixo de cada vídeo compartilhado as
discussões primárias acontecessem de uma forma ampla e abrangente. Outro
ponto de destaque é o fato da utilização de palavras chaves com a utilização de
hashtags (#), visando facilitar a busca no grupo em caso de posterior pesquisa. Diante dos resultados tem-se: O pós-graduando A destacou positivamente o fácil manuseio através do
celular, gerando maior facilidade de expressão pela fala do que pela escrita e uma
melhor memorização do conteúdo. Outro ponto positivo relatado pelo discente é
que a prática auxiliou na sua desinibição no contato com a câmera e na sua oratória
em seminários. Quanto aos aspectos críticos, ele relatou também que foi preciso
muita preparação (dedicação de tempo para leitura) para uma compreensão dos
textos a ponto de poder explicar. Outra dificuldade relatada foi em relação ao
tamanho de alguns arquivos, que na maioria das vezes necessitava de um aplicativo
para compactar o tamanho da mídia. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 3 Resultados e discussão 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 primeira impressão apontada quanto a utilização da ferramenta durante o
processo, foi promoção de benefícios aos pesquisadores, tanto na aprendizagem
dos conteúdos quanto na construção de novos conhecimentos. Além disso,
considerou a percepção sobre as mudanças nas relações interpessoais entre os
participantes do grupo, afirmando que a pesquisa e a gestão do conhecimento
facilitaram a integração dos mesmos, estreitando os laços, o que otimizou o
trabalho em equipe. O Quadro 1 traz uma síntese dos pontos positivos e negativos do
compartilhamento do conhecimento por intermédio de vídeos por parte de alunos
de uma Pós-Graduação stricto sensu de uma IES do norte do Paraná. Quadro 1: Síntese dos pontos positivos e negativos do compartilhamento do
conhecimento por intermédio de vídeos
Pontos positivos
Pontos negativos
• Fácil manuseio através de smartphones;
• Maior facilidade de expressão pela fala
do que pela escrita;
• Ampliação da memorização do conteúdo;
• Melhora da oratória;
•
Maior
facilidade
para
acessar
o
conhecimento;
• Maior compreensão dos conteúdos
debatidos;
• Melhoria das relações pessoais entre os
participantes;
• Necessidade de dedicação de tempo para
compreensão dos textos;
• Necessidade de smartphones para a
gravação dos vídeos;
•
Necessidade
de
qualidade
na
comunicação de rede;
• Tamanho dos arquivos dos vídeos;
Fonte: dos autores, 2020. Quadro 1: Síntese dos pontos positivos e negativos do compartilhamento do
conhecimento por intermédio de vídeos
Pontos positivos
Pontos negativos
• Fácil manuseio através de smartphones;
• Maior facilidade de expressão pela fala
do que pela escrita;
• Ampliação da memorização do conteúdo;
• Melhora da oratória;
•
Maior
facilidade
para
acessar
o
conhecimento;
• Maior compreensão dos conteúdos
debatidos;
• Melhoria das relações pessoais entre os
participantes;
• Necessidade de dedicação de tempo para
compreensão dos textos;
• Necessidade de smartphones para a
gravação dos vídeos;
•
Necessidade
de
qualidade
na
comunicação de rede;
• Tamanho dos arquivos dos vídeos;
Fonte: dos autores, 2020. Quadro 1: Síntese dos pontos positivos e negativos do compartilhamento do
conhecimento por intermédio de vídeos Fonte: dos autores, 2020. 3 Resultados e discussão O pós-graduando B, salienta que a experiência de participação do
compartilhamento só tem pontos positivos. Segundo ele, pessoalmente o
conhecimento ficou muito mais acessível e fácil para que ele absorvesse as
informações. Com isto, ele teve uma compreensão maior sobre os conteúdos
debatidos. Corroborando com o posicionamento do referido aluno, Lin (2007)
discute que o compartilhamento do conhecimento pode ser definido como uma
cultura de interação social em que ocorre a troca de conhecimentos, experiência e
habilidades. O pós-graduando C, defende que o compartilhamento dos vídeos
promoveu maior dinâmica no processo de aprendizagem, contribuindo para o
enriquecimento das discussões, visto que os alunos compartilharam informações
que foram além dos textos, expondo suas opiniões, experiências, análises e
impressões, sendo que os vídeos resumos serviram como um meio de integração
entre os membros do grupo. O pós-graduando D, expressou que o compartilhamento de conhecimentos
por intermédio de vídeo resumo facilitou o entendimento em questões em que eu
não tinha muito domínio, pois por meio de uma linguagem mais próxima e acessível
aliada à expressão facial, gesticulações e a entonação da voz, possibilitou que temas
complexos fossem assimilados de forma mais rápida e agradável. Além disso,
considerou que a confiança e proximidade com quem está transmitindo o conteúdo
também foi essencial para despertar o interesse no que estava sendo
compartilhado. Destacou que o conhecimento compartilhado por intermédio de
vídeo não substituiu as demais formas de ensino-aprendizagem, mas serviu como
complemento eficaz na compreensão de conteúdos complexos, extenuantes e de
linguajar técnico e/ou específico. O pós-graduando E, em suas considerações, afirmou que o vídeo resumo
compartilhado foi essencial para o aprendizado, pois, além dele possuir muita
dificuldade em resumir por escrito, os longos textos de estudo, o vídeo resumo
realizado, por meio de um celular com câmera, facilitou todo o processo de estudo
e fixação do conteúdo, assim como a dos vídeos compartilhados entre os colegas,
visto que ele poderia se expressar com as próprias palavras. O pós-graduando F, afirmou que durante o processo de compartilhamento
do conhecimento por meio dos vídeos resumos, adotado pelo grupo de estudo, a Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. 3 Resultados e discussão Assim sendo, pode-se afirmar que cada elemento do grupo apresentou
uma melhora no processo de aprendizagem, ainda que de forma distinta, sendo
perceptível que dentre os pontos positivos, é perceptível a facilidade no manuseio
e gravação dos vídeos por meio da utilização de smartphones; Maior facilidade na
explicitação dos conteúdos por meio da fala do que pela escrita; a ampliação da
memorização do conteúdo trabalhado; melhoria na oratória dos comunicadores;
facilidade para acessar o conhecimento armazenado; maior compreensão dos
conteúdos discutidos entre os alunos. Dentre os pontos negativos explorados tem-
se o acesso ao recurso tecnológico, visto que nem todos os alunos podem deter o
equipamento de smartphone; necessidade de tempo para compreensão dos textos,
além de qualidade na comunicação de rede e tamanho de arquivos para
compartilhamento. Conforme Schwartzman (1999), a aprendizagem de cada aluno submete-
se, entre outras vertentes, da heterogenia agregada aos sistemas neurológicos e de
atribuições intrínsecas do seu sistema motor, como a linguagem, a percepção, o
esquema corporal, a temporalidade e a lateralidade. Nesse sentido, pode-se
concluir que os vídeos resumos são uma prática que estrategicamente comtemplou
as diferenças e características dos pós-graduandos na aderência do conhecimento,
facilidade a comunicação e discussão dos textos, trazendo resultados significativos
para o aprendizado dos alunos participantes. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 4 Considerações finais Com base nas análises das interpretações pessoais dos integrantes do grupo de
trabalho utilizado na pesquisa, pode-se afirmar que o exercício das práticas de
compartilhamento da Gestão do Conhecimento empregados na preparação e
elaboração dos vídeos resumos facilitaram no processo de aprendizagem de forma
a garantir que o conhecimento transpassasse muito além do que estava apenas nos
textos, trazendo ricas contribuições advindas das experiências e das discussões,
gerindo o conhecimento por meio da Gestão do Conhecimento e proporcionando
a transformação do conhecimento tácito em explícito. A pesquisa demonstrou que a proposta de vídeo resumos resultou na
melhoria da comunicação dos alunos participantes, e no estreitamento dos laços
de cooperação, ampliando sua capacidade oratória, aumentando a dinâmica de
desinibição na gravação e facilidade na externalização do conteúdo aprendido. Verificou-se que a gravação de vídeo resumos trouxe maior facilidade no acesso ao
conteúdo, além de facilitar a recuperação do material, conforme a necessidade de
cada participante. O armazenamento em uma biblioteca virtual compartilhada possibilitou
ainda que os conteúdos explorados se tornassem repositório de conhecimento
para consultas futuras, se tornando conteúdo acessível a amplo universo. No que
diz respeito ao compartilhamento pelo aplicativo de mensagens instantâneas,
percebeu-se que os conteúdos se tornaram mais bem visualizados e trazendo os
debates no próprio grupo do aplicativo, além disso a utilização de hashtags facilitou
a recuperação dos conteúdos quando pesquisados. Explorou-se que os vídeos resumos trouxeram maior integração entre o
grupo, permitindo que os debates sobre os conteúdos se tornassem mais
acentuados, envolvendo além do conteúdo dos textos a perspectiva e
conhecimento individual de cada participante, trazendo contextualizações e
experiências vivenciadas ao longo da vida. Como sugestão para trabalhos futuros, traz-se a possibilidade de ampliação
da pesquisa com maior grupo de participantes a fim de que se possa aumentar as
perspectivas e propiciar maior qualidade no aprendizado de discentes. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. Referências APO. Knowledge management: facilitator´s guide. 2010. Disponível em: http://www.apo-
tokyo.org/00e-books/IS-39_APO-KM-FG.htm. Acesso em: 29 jul. 2019. BARTOL, Kathryn M.; SRIVASTAVA, Abhishek. Encouraging Knowledge Sharing: The Role of
Organizational Reward Systems. Journal of Leadership & Organizational Studies, 2002. BARTOL, Kathryn M.; SRIVASTAVA, Abhishek. Encouraging Knowledge Sharing: The Role of
Organizational Reward Systems. Journal of Leadership & Organizational Studies, 2002. CUMMINGS, Jeffrey. Knowledge sharing: A review of the literature. The World Bank, 2003. DAVENPORT, Thomas H.; PRUSAK, Laurence; Working Knowledge. Harvard Business School
Press, 2000. DAVENPORT, Thomas H.; PRUSAK, Laurence. Conhecimento empresarial: como as
organizações gerenciam o seu capital intelectual. Trad. de Lenke Peres. 8. ed. Rio de Janeiro:
Campus, 1998. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 DI CHIARA, Ivone Guerreiro.; ALCARA, Adriana Rosecler; TOMAEL, Maria Inês. Tipos de
compartilhamento de informação e conhecimento no ambiente de P&D. Inf. & Soc. Est. João Pessoa, v. 20, n. 2, p. 105-118, maio/ago., 2010. DIOGO, Rodrigo Claudino et al. Tecnologias da informação e comunicação na sala de aula:
produção de vídeos por meio de smartphones como uma possibilidade viável para o ensino
e a aprendizagem. Anais da Semana de Licenciatura, v. 1, n. 6, p. 169-173, 2015. Disponível
em: http://www.jatai.ifgoias.edu.br/semlic/seer/index.php/anais/article/view/378/pdf_136. Acesso em: 28 jul. 2019. http://www.jatai.ifgoias.edu.br/semlic/seer/index.php/anais/article/view/378/pdf_136. Acesso em: 28 jul. 2019. DYER, Jeffrey H.; NOBEOKA, Kentauro. Creating and managing a high-performance
knowledge-sharing network: The Toyota case. Strategic Management Journal, 2000. DUTRA, Mitchell de Oliveira et al. Ferramentas da tecnologia da informação para a gestão
do conhecimento e inovação – taxonomia e oportunidades de pesquisa. Revista GEINTEC;
vol. 4,
nº
3,
p. 1195-1208;
São
Cristóvão:
2014. Disponível
em:
http://www.revistageintec.net/index.php/revista/article/view/349/623. Acesso em: 31 jul. 2019. FREIRE, Jocemar José. FURLAN, Sandra Aparecida. SILVEIRA, José L. Gonçalves. Gestão do
conhecimento na atividade de inteligência de segurança pública – uma abordagem prática
e tecnológica. Curitiba: Appris, 2018. HONG, Daegeun; SUH, Euiho; KOO, Choonghyo. Referências Developing strategies for overcoming
barriers to knowledge sharing based on conversational knowledge management: A case
study of a financial company. Expert systems with Applications, v. 38, n. 12, p. 14417-
14427, 2011. LIN, Hsiu-Fen. Effects of extrinsic and intrinsic motivation on employee knowledge sharing
intentions. Journal of Information Science, v. 33, n. 2, p. 135-149, 2007. LIU, Yuwen; PHILLIPS, James S. Examining the antecedents of knowledge sharing in
facilitating team innovativeness from a multilevel perspective. International Journal of
Information Management, v. 31, n. 1, p. 44–52, 2011. MATTERA, Tayane Cristina. Gestão do conhecimento na prática. IN: SOUTO, Leonardo
Fernandes (org.). Gestão da Informação e do conhecimento: práticas e reflexões. Rio de
Janeiro: Interciência, 2014. NONAKA, Ikujiro e TAKEUCHI, Hirotaka. Criação do Conhecimento na Empresa: como as
empresas geram a dinâmica da inovação. Rio de Janeiro: Campus,1997. NUNES, Carolina Schmitt et al. O compartilhamento de conhecimento entre os agentes de
um curso na modalidade EaD: um estudo de caso. 2013. Disponível em:
https://repositorio.ufsc.br/bitstream/handle/123456789/107591/318587.pdf?sequence=
1&isAllowed=y. Acesso em: 28 jul. 2019. ORDAZ, Carmen Camelo; CRUZ Joaquim Garcia; GINEL Elena Sousa. Facilitadores de los
procesos de compartir conocimiento y su influencia sobre la innovación, Cuadernos de
Economía y Dirección de la Empresa, n. 42, p. 113-150, 2010. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. POSSOLI, Gabriela Eyng. Gestão da Inovação e do conhecimento (livro eletrônico). Curitiba:
InterSaberes, 2012. (Coleção Gestão Empresarial; v.2). POSSOLI, Gabriela Eyng. Gestão da Inovação e do conhecimento (livro eletrônico). Curitiba:
InterSaberes, 2012. (Coleção Gestão Empresarial; v.2). PRODANOV, Cleber Cristiano; DE FREITAS, Ernani Cesar. Metodologia do trabalho
científico: métodos e técnicas da pesquisa e do trabalho acadêmico. 2. ed. Novo Hamburgo:
Editora Feevale, 2013. SCHWARTZAN, José S. Síndrome de Down. São Paulo: Mackenzie, 1999. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. 1395 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 SILVA, Franco Renildo; CORREA, Sena Emilce. Novas tecnologias e educação: a evolução do
processo de ensino e aprendizagem na sociedade contemporânea. Educação e Linguagem,
ano,
v. 1,
n. 1,
p. 23-25,
2014. Disponível
em:
https://fvj.br/revista/wp-
content/uploads/2014/12/2Artigo1.pdf. Acesso em: 28 jul. 2019. SILVA, Franco Renildo; CORREA, Sena Emilce. Novas tecnologias e educação: a evolução do
processo de ensino e aprendizagem na sociedade contemporânea. Educação e Linguagem,
ano,
v. 1,
n. 1,
p. 23-25,
2014. Disponível
em:
https://fvj.br/revista/wp-
content/uploads/2014/12/2Artigo1.pdf. Acesso em: 28 jul. 2019. STEWART, Thomas A. A riqueza do conhecimento: o capital intelectual e a nova
organização. Tradução de Afonso Celso Cunha Serra. Rio de Janeiro: Campus, 2002. SVEIBY, Karl Erick. A nova riqueza das organizações: gerenciando e avaliando patrimônios
de conhecimento. Rio de Janeiro: Campus, 1998. YIN, Robert K. Estudo de caso: planejamento e métodos. 5. ed. Porto Alegre: Bookman,
2015. YOUNG, Ronald. Knowledge management tools and techniques manual. Asian Productivity
Organization, v. 98, p. 1-98, 2010. Contribuição de autoria Concepção e elaboração do manuscrito: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli; Fernanda Crocetta Schraiber; Luis Augusto
Sautchuk Marchi. Coleta de dados: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Fernanda
Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Análise de dados: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Fernanda
Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Discussão dos resultados: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli;
Bruna Hernandes Scarabelli; Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Análise de dados: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Fernanda
Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Discussão dos resultados: Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli;
Bruna Hernandes Scarabelli; Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi. Revisão e aprovação: Arthur Gualberto Bacelar da Cruz Urpia; Felipe Pereira de Melo;
Fernanda Crocetta Schraiber. Conjunto de dados de pesquisa Conjunto de dados de pesquisa
Não há dados disponibilizados. Não há dados disponibilizados. Informações complementares Financiamento
Não se aplica. Preprint, originalidade e ineditismo p
,
g
Uma versão preliminar deste artigo foi publicada no XI Encontro Internacional de Produção
Científica. Acesse: http://rdu.unicesumar.edu.br/handle/123456789/3317. Conflito de interesse Conflito de interesse
Não há conflitos de interesse. Não há conflitos de interesse. Consentimento de uso de imagem Consentimento de uso de imagem Não se aplica. Aprovação de Comitê de Ética em Pesquisa Aprovação de Comitê de Ética em Pesquisa
Não se aplica. Publisher Universidade Federal de Juiz de Fora (UFJF), Faculdade de Educação (FACED), Centro de
Políticas Públicas e Avaliação da Educação (CAEd), Programa de Pós-Graduação Profissional
em Gestão e Avaliação da Educação Pública (PPGP). Publicação no Portal de Periódicos da
UFJF. As ideias expressadas neste artigo são de responsabilidade de seus autores, não
representando, necessariamente, a opinião dos editores ou da universidade. Licença de uso ç
As autoras cedem à Revista Pesquisa e Debate em Educação os direitos exclusivos de
primeira publicação, com o trabalho simultaneamente licenciado sob a Licença Creative Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. Felipe Pereira de Melo; Fabrício Ricardo Tomaz Bernardelli; Bruna Hernandes Scarabelli;
Fernanda Crocetta Schraiber; Luis Augusto Sautchuk Marchi; Arthur Gualberto Bacelar da Cruz Urpia
O compartilhamento de conhecimentos por intermédio de vídeos
https://doi.org/10.34019/2237-9444.2020.v10.31437 Commons Attribution (CC BY) 4.0 International. Esta licença permite que terceiros remixem,
adaptem e criem a partir do trabalho publicado, atribuindo o devido crédito de autoria e
publicação inicial neste periódico. Os autores têm autorização para assumir contratos
adicionais separadamente, para distribuição não exclusiva da versão do trabalho publicada
neste periódico (ex.: publicar em repositório institucional, em site pessoal, publicar uma
tradução, ou como capítulo de livro), com reconhecimento de autoria e publicação inicial
neste periódico. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020.
ISSN 2237-9444. Editores Frederico Braida; Liamara Scortegagna. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444. Pesquisa e Debate em Educação, Juiz de Fora: UFJF, v. 10, n. 2, p. 1383 - 1395, jul. - dez. 2020. ISSN 2237-9444.
|
https://openalex.org/W3126481757
|
https://publikationen.bibliothek.kit.edu/1000134228/118450568
|
English
| null |
Wide-blocky veins explained by dependency of crystal growth rate on fracture surface type: Insights from phase-field modeling
|
Geology
| 2,021
|
cc-by
| 5,859
|
Wide-blocky veins explained by dependency of crystal growth
rate on fracture surface type: Insights from phase-field
modeling Liene Spruženiece1, Michael Späth2, Janos L. Urai1, Estibalitz Ukar3, Michael Selzer2,4 and Britta Nestler2,4
1Institute of Structural Geology, Tectonics and Geomechanics, RWTH Aachen University, Lochnerstraße 4-20, 52056 Aachen,
Germany 2Institute for Applied Materials–Computational Materials Science, Karlsruhe Institute of Technology, Straße am Forum 7,
76131 Karlsruhe, Germany 3Bureau of Economic Geology, Jackson School of Geosciences, The University of Texas at Austin, 10100 Burnet Road,
Austin, Texas 78758, USA Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user ABSTRACT from both sides seal a syntaxial vein, and en
abling three-dimensional (3-D) modeling (e.g.,
Ankit et al., 2015; Wendler et al., 2016; Kling
et al., 2017; Prajapati et al., 2020). However,
up to now, the phase-field models have not
been quantitatively compared with natural vein
microstructures. Vein microstructures contain a wealth of information on coupled chemical and mechanical
processes of fracturing, fluid transport, and crystal growth. Numerical simulations have been
used for exploring the factors controlling the development of vein microstructures; however,
they have not been quantitatively validated against natural veins. Here we combined phase-
field modeling with microtextural analysis of previously unexplained wide-blocky calcite veins
in natural limestone and of the fresh fracture surface in this limestone. Results show that the
wide-blocky vein textures can only be reproduced if ∼10%–20% of crystals grow faster than
the rest. This fraction corresponds to the amount of transgranularly broken grains that were
observed on the experimental fracture surfaces, which are dominantly intergranular. We hy
pothesize that transgranular fractures allow faster growth of vein minerals due to the lack
of clay coatings and other nucleation discontinuities that are common along intergranular
cracks. Our simulation results show remarkable similarity to the natural veins and reproduce
the nonlinear relationship between vein crystal width and vein aperture. This allows accurate
simulations of crystal growth processes and related permeability evolution in fractured rocks. This study fills this gap by applying the phase-
field method to simulate enigmatic wide-blocky
microstructures in natural calcite veins from Som
erset, UK. We were able to replicate the natural
microstructures quantitatively and provide new
insights on vein formation mechanisms, showing
how variations in transgranular and intergranular
segments on fracture surfaces lead to heteroge
nous crystal growth, producing microstructures
that are not predicted by previous models. Manuscript received 4 October 2020
Revised manuscript received 30 November 2020
Manuscript accepted 1 December 2020 Manuscript received 4 October 2020
Revised manuscript received 30 November 2020
Manuscript accepted 1 December 2020 © 2021 The Authors. Gold Open Access: This paper is published under the terms of the CC-BY license. Published online 5 February 2021 INTRODUCTION the Elle numerical simulation platform (www. http://elle.ws/; Bons, 2001; Hilgers et al., 2001;
Nollet et al., 2005) and cellular automation–type
models (Lander et al., 2008; Lander and Lau
bach, 2015) explored anisotropic crystal growth
in antitaxial veins, where crystals can develop
facets during epitaxial growth from a rough
surface toward the inert vein wall. However,
the vast majority of veins in rocks at depths of
1–10 km and at 100–350 °C are syntaxial: crys
tals grow on both fracture sides (Durney and
Ramsay, 1973; Bons et al., 2012). More recently,
phase-field models were introduced to model
both syntaxial and antitaxial veins. Due to the
different approach to moving boundaries, these
models are more efficient, allowing simulations
against dynamic, moving interfaces as crystals the Elle numerical simulation platform (www. http://elle.ws/; Bons, 2001; Hilgers et al., 2001;
Nollet et al., 2005) and cellular automation–type
models (Lander et al., 2008; Lander and Lau
bach, 2015) explored anisotropic crystal growth
in antitaxial veins, where crystals can develop
facets during epitaxial growth from a rough
surface toward the inert vein wall. However,
the vast majority of veins in rocks at depths of
1–10 km and at 100–350 °C are syntaxial: crys
tals grow on both fracture sides (Durney and
Ramsay, 1973; Bons et al., 2012). More recently,
phase-field models were introduced to model
both syntaxial and antitaxial veins. Due to the
different approach to moving boundaries, these
models are more efficient, allowing simulations
against dynamic, moving interfaces as crystals Fractures provide important pathways for flu
id migration in Earth’s crust (Newhouse, 1942;
Cox et al., 1987; Nelson, 2001). Microstructures
of minerals precipitated in veins contain a wealth
of information on the mechanical, chemical, and
hydrothermal history of veins (e.g., Boullier and
Robert, 1992; Bons et al., 2012; Ukar and Lau
bach, 2016; Laubach et al., 2019). CITATION: Spruženiece, L., et al., 2021, Wide-blocky veins explained by dependency of crystal growth rate on fracture surface type: Insights from phase-field
modeling: Geology, v. 49, p. 641–646, https://doi.org/10.1130/G48472.1 1Supplemental Material. Analytical methods, description of numerical approach, image library of results, and video files of the 2-D and 3-D simulations. Please
visit https://doi.org/10.1130/GEOL.S.13584911 to access the supplemental material, and contact editing@geosociety.org with any questions. CITATION: Spruženiece, L., et al., 2021, Wide-blocky veins explained by dependency of crystal growth rate on fracture
odeling: Geology, v. 49, p. 641–646, https://doi.org/10.1130/G48472.1 hods, description of numerical approach, image library of results, and video files of the 2-D and 3-D simulations. Please
4911 to access the supplemental material, and contact editing@geosociety.org with any questions. SAMPLES AND METHODS We analyzed arrays of calcite microveins
in Liassic (Lower Jurassic) limestones near
Blue Anchor, Kilve, and Lilstock beaches in
Somerset, UK (Figs. 1A and 1B). The veins
are normal to bedding in the damage zone of
regional normal faults (Caputo and Hancock,
1999; Nixon et al., 2019). Double-polished thin
sections, cut perpendicular to the veins, were
imaged and studied in the PetroScan Virtual
Microscope (see Item S1 in the Supplemental
Material1) as well as by scanning electron mi
croscope (SEM)–based cathodoluminescence
(CL), secondary electron (SE), and backscat
tered electron (BSE) imaging (Item S1). A number of different approaches have been
presented for simulating crystal growth kinemat
ics in veins. Early models used geometric pro
jections for exploring kinematics of polycrystals
growing in a confined space (Urai et al., 1991;
Dickson, 1993) to explain fibrous and elongate-
blocky crystal growth. Subsequent numerical
methods including front-tracking algorithms of 1Supplemental Material. Analytical methods, description of numerical approach, image library of results, and video files of the 2-D and 3-D simulations. Please
visit https://doi.org/10.1130/GEOL.S.13584911 to access the supplemental material, and contact editing@geosociety.org with any questions. CITATION: Spruženiece, L., et al., 2021, Wide-blocky veins explained by dependency of crystal growth rate on fracture surface type: Insights from phase-field
modeling: Geology, v. 49, p. 641–646, https://doi.org/10.1130/G48472.1 1, Wide-blocky veins explained by dependency of crystal growth rate on fracture surface type: Insights from phase-field
s://doi.org/10.1130/G48472.1 Geological Society of America | GEOLOGY | Volume 49 | Number 6 | www.gsapubs.org 641 ed from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
he Institute of Technology, KIT Library user Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user A
B
C A
B
C
D
E
Figure 1. (A) Geologic setting and sampling locations in Somerset, UK (map adapted from Glen et al. [2005] and Peacock and Sanderson
[2018]). (B) Outcrop-scale relationships between veins and Liassic (Lower Jurassic) limestone and shale beds. (C,D) Optical micrographs
under crossed polarizers showing wide-blocky microstructures. WBC—wide-blocky crystal; red arrows mark host-rock grains in optical
continuity with vein crystals. (E) Scanning electron microscope–based cathodoluminescence image showing faceted growth zoning within
wide-blocky crystals. A B B D E D E Figure 1. (A) Geologic setting and sampling locations in Somerset, UK (map adapted from Glen et al. [2005] and Peacock and Sanderson
[2018]). (B) Outcrop-scale relationships between veins and Liassic (Lower Jurassic) limestone and shale beds. (C,D) Optical micrographs
under crossed polarizers showing wide-blocky microstructures. SAMPLES AND METHODS WBC—wide-blocky crystal; red arrows mark host-rock grains in optical
continuity with vein crystals. (E) Scanning electron microscope–based cathodoluminescence image showing faceted growth zoning within
wide-blocky crystals. Analysis of fresh fracture surface was per
formed on 4.5 × 2 × 1 cm blocks of the same
rocks broken by three-point loading to obtain
mode I fractures parallel to existing veins. Fracture surfaces were imaged in SEM-SE,
and transgranular fractures in these images
were quantified using Fiji software (Schindelin
et al., 2012). Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user MICROSTRUCTURAL RESULTS We focused
on microveins (aperture <1 mm) filled by lat
erally wide, blocky crystals whose formation
mechanism remains unexplained (Figs. 1C and
1D). These microveins lack host-rock inclu
sion bands, suggesting a single crack-seal cycle
(Ramsay, 1980). The vein crystals show optical
continuity with adjacent grains in the host rock. Smaller equidimensional crystals with euhedral
terminations occur along the vein walls, indi
cating syntaxial growth into an open fracture. SEM-CL images show growth zoning within
both small and large crystals, indicating fac
eted growth toward the vein interior (Figs. 1D
and 1E). grain diameter in the host rock, so that natural
microstructures can be compared with simulat
ed ones. In the narrowest veins, W and Dm are
similar: the vein crystal width increases linearly
with the increase of the vein aperture (Fig. 3A). For wider veins with Dm >∼4, W/Dm slowly
increases toward 2. At Dm >∼10, W becomes
independent of Dm. For Dm <∼4, vein crystals
are mostly equant, while for Dm >4, they can
reach aspect ratios of 10. formed along cleavage planes in the host-rock
calcite, and slower on intergranular cracks that
are coated by clay minerals (Lander et al., 2008;
Ajdukiewicz and Larese, 2012; Williams et al.,
2015). The host rock in the 2-D models was
generated with a Voronoi algorithm (Fig. 2E;
Item S2), where grains have random crystal
lographic orientations to mimic micrite. The
starting fracture shape was based on the ge
ometry of one of the natural veins, with nor
malized apertures (Dm) varied from 1 to 16 in
different simulations. Vein crystals were set to
grow epitaxially on host-rock grains into the
open fractures, as inferred from microstruc
tures in the natural veins (Figs. 1C and 1E). Crystals were assigned anisotropic surface en
ergy so they could develop facets and vertices
corresponding with calcite crystal symmetry. The relative growth rate differences for differ
ent fracture surfaces were incorporated as a di
mensionless factor ξ. Three types of surfaces
were distinguished based on the analysis of
natural samples and experimental fracture sur
faces (Figs. 2A–2E; Table S3): (1) inert acces
sory minerals with no epitaxial calcite growth Experimental fracture surfaces consist most
ly of intergranular, rather than transgranular,
segments that follow nano-porous fossil, mi
critic grain, and accessory mineral boundaries,
as recognized in SE images by their surface mor
phologies (Figs. 2B and 2C). MICROSTRUCTURAL RESULTS Phase-field vein growth simulations in two
and three dimensions (2-D and 3-D) were per
formed using a multiphase-field approach (e.g.,
Nestler et al., 2005) (Items S2–S4). Computa
tional fluid dynamics analysis of 3-D micro
structure was used to solve fluid flow and per
meability changes during the sealing process of
the fracture (e.g., Kling et al., 2017). The host rock is a micritic limestone with
∼95% calcite grains, carbonate fossils, and
∼5% accessory minerals (quartz, dolomite,
albite, clays, and pyrite) (Figs. 1C, 1D, and
2A). It contains sets of subparallel calcite veins
with apertures from a few micrometers to sev
eral centimeters. The veins that are wider than www.gsapubs.org | Volume 49 | Number 6 | GEOLOGY | Geological Society of America 642 Figure 2. (A) Backscattered electron image of sample cut perpendicular to the vein (Qz—quartz; Cal—calcite). (B,C) Secondary electron images
of experimentally broken samples, looking at the fracture surfaces (gb—grain boundary). (D) Distribution of calcite cleavage planes (black;
transgranular fractures) on an experimental fracture surface. Four profile lines were used for two-dimensional simulations in Figure 3A, where
percentages refer to the proportion of transgranular segments on each profile (see details in Item S2 [see footnote 1]). Rectangle marks the loca
tion of the chosen domain for the three-dimensional (3-D) models in Figure 4. (E) Setup for phase-field simulations along the 17.5% profile line
in D. “Broken” grains are mirrored and marked with black dots. Color scale shows crystal orientations with respect to the fracture orientation. Figure 2. (A) Backscattered electron image of sample cut perpendicular to the vein (Qz—quartz; Cal—calcite). (B,C) Secondary electron images
of experimentally broken samples, looking at the fracture surfaces (gb—grain boundary). (D) Distribution of calcite cleavage planes (black;
transgranular fractures) on an experimental fracture surface. Four profile lines were used for two-dimensional simulations in Figure 3A, where
percentages refer to the proportion of transgranular segments on each profile (see details in Item S2 [see footnote 1]). Rectangle marks the loca
tion of the chosen domain for the three-dimensional (3-D) models in Figure 4. (E) Setup for phase-field simulations along the 17.5% profile line
in D. “Broken” grains are mirrored and marked with black dots. Color scale shows crystal orientations with respect to the fracture orientation. 1 mm show elongate-blocky microstructures
(Fisher and Brantley, 1992; Bons, 2001) with
solid inclusion bands suggesting crack-seal
processes and growth competition. MICROSTRUCTURAL RESULTS In most cases, such
grain boundaries show clay-mineral coatings on
calcite. Only ∼10% of the fracture surface is
transgranular, exposing clean calcite cleavage
planes (Fig. 2D). A characteristic feature of the 59 analyzed
microveins is the relationship between mi
crovein aperture (Dm) and the average width of
the crystals (W) as measured along the median
line of the vein (Figs. 1D and 3A; Fig. S1 in
the Supplemental Material). Both parameters
are non-dimensional, scaled against the average Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user Geological Society of America | GEOLOGY | Volume 49 | Number 6 | www.gsapubs.org SETUP OF THE PHASE-FIELD MODEL Note that the
entire data set is scaled
logarithmically; in addi
tion, the inset shows
measurements from the
smallest veins on a linear
scale. (B) Vein microstruc
tures in two-dimensional
models for different
opening apertures (Dm),
using a setup with 17.5%
transgranular segments. ξ—dimensionless factor
of relative growth rate
differences
between
transgranularly versus
intergranularly fractured
grains. Colors illus
trate crystal orientations
according to the legend
in Figure 2E. (C) Optical
photomicrograph under
crossed polarizers of a
natural wide-blocky vein. (ξ = 0), (2) clay-covered intergranular micro
cracks in calcite with slow growth rates (ξ = 1),
and (3) clean transgranular microcracks along
calcite cleavage planes with fast growth rates
(ξ = 5–20). The proportions of these surfaces
were based on measurements along profile lines
on an experimental fracture surface (Fig. 2D). Only grains that underwent transgranular frac
ture are mirrored on both sides of the fracture
wall in the model. SIMULATION RESULTS
Curves of the W/Dm relationship prod
numerical models show good corresp
with our measurements performed on
veins (Fig. 3A). Models show that larg
Figure 3. (A) Relat
between vein a
(Dm) and crysta
(W) in wide-block
from the Somers
area and simu
(scaled against th
age diameter of ho
grains). Note th
entire data set is
logarithmically;
tion, the inset
measurements fr
smallest veins on
scale. (B) Vein mic
tures in two-dime
models for d
opening aperture
using a setup wit
transgranular seg
ξ—dimensionles
of relative grow
differences
be
transgranularly
intergranularly fr
grains. Colors
trate crystal orien
according to the
in Figure 2E. (C)
photomicrograph
crossed polarize
natural wide-bloc
B
C B
C B B ξ—dimensionless factor
of relative growth rate
differences
between
transgranularly versus
intergranularly fractured
grains. Colors illus
trate crystal orientations
according to the legend
in Figure 2E. (C) Optical
photomicrograph under
crossed polarizers of a
natural wide-blocky vein. C C SIMULATION RESULTS (ξ = 0), (2) clay-covered intergranular micro
cracks in calcite with slow growth rates (ξ = 1),
and (3) clean transgranular microcracks along
calcite cleavage planes with fast growth rates
(ξ = 5–20). The proportions of these surfaces were based on measurements along profile lines
on an experimental fracture surface (Fig. 2D). Only grains that underwent transgranular frac
ture are mirrored on both sides of the fracture
wall in the model. Curves of the W/Dm relationship produced by
numerical models show good correspondence
with our measurements performed on natural
veins (Fig. 3A). SETUP OF THE PHASE-FIELD MODEL Based on our microstructural observations,
we hypothesized that precipitation of vein cal
cite is faster on clean, transgranular microcracks Geological Society of America | GEOLOGY | Volume 49 | Number 6 | www.gsapubs.org 643 Figure 3. (A) Relationship
between vein aperture
(Dm) and crystal width
(W) in wide-blocky veins
from the Somerset, UK,
area and simulations
(scaled against the aver
age diameter of host-rock
grains). Note that the
entire data set is scaled
logarithmically; in addi
tion, the inset shows
measurements from the
smallest veins on a linear
scale. (B) Vein microstruc
tures in two-dimensional
models for different
opening apertures (Dm),
using a setup with 17.5%
transgranular segments. ξ—dimensionless factor
of relative growth rate
differences
between
transgranularly versus
intergranularly fractured
grains. Colors illus
trate crystal orientations
according to the legend
in Figure 2E. (C) Optical
photomicrograph under
crossed polarizers of a
natural wide-blocky vein. B
A
C Figure 3. (A) Relationship
between vein aperture
(Dm) and crystal width
(W) in wide-blocky veins
from the Somerset, UK,
area
and
simulations
A (ξ = 0), (2) clay-covered intergranular micro
cracks in calcite with slow growth rates (ξ = 1),
and (3) clean transgranular microcracks along
calcite cleavage planes with fast growth rates
(ξ = 5–20). The proportions of these surfaces
were based on measurements along profile lines
on an experimental fracture surface (Fig. 2D). Only grains that underwent transgranular frac
ture are mirrored on both sides of the fracture
wall in the model. SIMULATION R
Curves of the W
numerical models
with our measure
veins (Fig. 3A). M
B
C Figure 3. (A) Relationship
between vein aperture
(Dm) and crystal width
(W) in wide-blocky veins
from the Somerset, UK,
area and simulations
(scaled against the aver
age diameter of host-rock
grains). Note that the
entire data set is scaled
logarithmically; in addi
tion, the inset shows
measurements from the
smallest veins on a linear
scale. (B) Vein microstruc
tures in two-dimensional
models for different
opening apertures (Dm),
using a setup with 17.5%
transgranular segments. ξ—dimensionless factor
of relative growth rate
differences
between
transgranularly versus
intergranularly fractured
grains. Colors illus
trate crystal orientations
according to the legend
in Figure 2E. (C) Optical
photomicrograph under
crossed polarizers of a
natural wide-blocky vein. Figure 3. (A) Relationship
between vein aperture
(Dm) and crystal width
(W) in wide-blocky veins
from the Somerset, UK,
area and simulations
(scaled against the aver
age diameter of host-rock
grains). Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user SETUP OF THE PHASE-FIELD MODEL In nat
ural veins, intergranular fracture segments might
have different amounts of clay coatings, or sur
face defects and transgranular segments might
have cleavage steps, both of which affect the
precipitation rate of vein minerals. Despite that,
our results show a very good correspondence
between modeled and natural microstructures,
including the quantified trend of increasing crys
tal width with increasing vein aperture (Fig. 3A)
as well as the microstructural characteristics,
including distributions of bridge crystals, equi
dimensional crystals, and rims of small grains
along the vein wall (Figs. 1C, 1D, 3B, and 3C). fracture apertures result in wider crystals, but
there is a maximum width that vein crystals can
reach (plateau) that is dependent on the propor
tion of “fast-growing” grains in comparison to
“slow-growing” and “inert” grains on fracture
surfaces. Furthermore, the growth-rate differ
ence (ξ) determines the slope of W/Dm, so that
a larger ξ produces wider crystals. Most of the simulations were performed in
2-D to allow reasonable computational time
for setups with a large number of grains. How
ever, 3-D runs are also possible, and we show
the results of some such runs in Figure 4. Fig
ures 4B–4D show the evolution of porosity and
fluid flow at three stages of vein filling. At early
stages (step 1), the fracture is highly permeable
and fluid-flow rate is high. As filling progresses
(step 2), the two parts of the broken, fast-grow
ing crystals on transgranular fracture surfaces
touch near the median plane of the fracture and
form bridges, then continue to grow laterally. Fluid pathways and pores remain mostly inter
connected until >50% of the grains have crossed
the median line. After that, the porosity structure
changes and permeability decreases as pores be
come isolated (step 3). Figure 3B shows that, regardless of fracture
aperture, wide-blocky microstructures form only
in cases where ξ >1. If ξ = 1, the veins consist
of blocky crystals with typical growth compe
tition microstructures and a prominent median
line. Increasing the initial fracture aperture re
sults in fewer and wider grains at the median
line (as in Prajapati et al. [2018]). In addition to the studied location in the UK,
wide-blocky vein textures have been documented
in micritic limestones from Sestri Levante, Italy
(Bons et al., 2012), and the Oman Mountains
(Holland and Urai, 2010) but never explained in
detail. SETUP OF THE PHASE-FIELD MODEL Distribution of f
growing grains is bas
on the morphology
the experimental fract
(Fig. 2D). (B) Two-dim
sional sections across
center of vein show
grains that have reach
the median line at th
successive stages of v
filling. Yellow marks flu
filled parts of the ve
(C) Porosity (blue) evo
tion during fracture fill
(D) Streamlines show
changes in permeabi
and fluid flow rate dur
fracture filling. B-D sho
a rotated view of A, w
the long side perpendi
lar to the viewer. Velo
magnitude is non-dim
sional, expressed as g
spacing (Δx) versus t
increment (Δt). B
C
D B g
( )
vein microstructure i
three dimensions (3-D
Colors show the orien
tation of calcite c-axi
with respect to the frac
ture orientation; hos
rock is shown with fade
colors. Dm—vein aper
ture; ξ—dimensionles
factor of relative growt
rate differences betwee
transgranularly versu
intergranularly fracture
grains. Distribution of fast
growing grains is base
on the morphology o
the experimental fractur
(Fig. 2D). (B) Two-dimen
sional sections across th
center of vein showin
grains that have reache
the median line at thre
successive stages of vei
filling. Yellow marks fluid
filled parts of the vein
(C) Porosity (blue) evolu
tion during fracture filling
(D) Streamlines showin
changes in permeabilit
and fluid flow rate durin
fracture filling. B-D show
a rotated view of A, wit
the long side perpendicu
lar to the viewer. Velocit
magnitude is non-dimen
sional, expressed as gri
spacing (Δx) versus tim
increment (Δt). B
C
D three di
Colors s
tation o
with res
ture or
rock is s
colors. ture; ξ—
factor of
rate diffe
transgra
intergran
grains. D
growing
on the
the expe
(Fig. 2D)
sional se
center o
grains th
the med
success
filling. Ye
filled pa
(C) Poro
tion duri
(D) Strea
changes
and fluid
fracture f
a rotated
the long
lar to the
magnitud
sional, e
spacing
incremen
C
D B B C C C D D C B (C) Porosity (blue) evolu
tion during fracture filling. (D) Streamlines showing
changes in permeability
and fluid flow rate during
fracture filling. B-D shows
a rotated view of A, with
the long side perpendicu
lar to the viewer. Velocity
magnitude is non-dimen
sional, expressed as grid
spacing (Δx) versus time
increment (Δt). initial fracture aperture and/or further changes
in ξ (Item S3). classes (fast and slow) is a simplification. SETUP OF THE PHASE-FIELD MODEL We infer that wide-blocky textures might
be characteristic in clay-rich, micritic host rocks
with medium grain-boundary cohesion so that
fractures are mostly intergranular, with 25%–5%
transgranular segments. However, the heteroge
neity of the fracture surface, not the lithology or
grain size of the host rock, is the controlling fac
tor in these veins, therefore the observed crystal In simulations where ξ >1, fast-growing
grains on transgranular cleavage planes outgrow
slow-growing grains at early stages. However,
for low ξ values and small fracture apertures (Dm
<2), most slow-growing grains can still reach
the other side of the fracture wall, blocking the
lateral expansion of the fast-growing grains
(Item S3). If the growth rate difference is large
(ξ ∼20), slow-growing grains are outcompeted
almost immediately, and the width of the wide-
blocky grains remains constant regardless of the Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user SETUP OF THE PHASE-FIELD MODEL Models show that larger initial Curves of the W/Dm relationship produced by
numerical models show good correspondence
with our measurements performed on natural
veins (Fig. 3A). Models show that larger initial www.gsapubs.org | Volume 49 | Number 6 | GEOLOGY | Geological Society of America 644 A Figure 4. (A) Simulated
vein microstructure in
three dimensions (3-D). Colors show the orien
tation of calcite c-axis
with respect to the frac
ture orientation; host
rock is shown with faded
colors. Dm—vein aper
ture; ξ—dimensionless
factor of relative growth
rate differences between
transgranularly versus
intergranularly fractured
grains. Distribution of fast-
growing grains is based
on the morphology of
the experimental fracture
(Fig. 2D). (B) Two-dimen
sional sections across the
center of vein showing
grains that have reached
the median line at three
successive stages of vein
filling. Yellow marks fluid-
filled parts of the vein. (C) Porosity (blue) evolu
tion during fracture filling. (D) Streamlines showing
changes in permeability
and fluid flow rate during
fracture filling. B-D shows
a rotated view of A, with
the long side perpendicu
lar to the viewer. Velocity
magnitude is non-dimen
sional, expressed as grid
spacing (Δx) versus time
increment (Δt). Figure 4. (A) Simulated
vein microstructure in
three dimensions (3-D). Colors show the orien
tation of calcite c-axis
with respect to the frac
ture orientation; host
rock is shown with faded
colors. Dm—vein aper
ture; ξ—dimensionless
factor of relative growth
rate differences between
transgranularly versus
intergranularly fractured
grains. Distribution of fast-
growing grains is based
on the morphology of
the experimental fracture
(Fig. 2D). (B) Two-dimen
sional sections across the
center of vein showing
grains that have reached
the median line at three
successive stages of vein
filling. Yellow marks fluid-
filled parts of the vein. (C) Porosity (blue) evolu
tion during fracture filling. (D) Streamlines showing
changes in permeability
and fluid flow rate during
fracture filling. B-D shows
a rotated view of A, with
the long side perpendicu
lar to the viewer. Velocity
magnitude is non-dimen
sional, expressed as grid
spacing (Δx) versus time
increment (Δt). Figure 4. (A) Simula
vein microstructure
three dimensions (3
Colors show the ori
tation of calcite c-a
with respect to the fr
ture orientation; h
rock is shown with fad
colors. Dm—vein ap
ture; ξ—dimensionle
factor of relative grow
rate differences betwe
transgranularly vers
intergranularly fractu
grains. Geological Society of America | GEOLOGY | Volume 49 | Number 6 | www.gsapubs.org DISCUSSION Our models show that incorporation of dif
ferential crystal growth rates for transgranular
and intergranular fracture segments is a key for
simulating the evolution of wide-blocky veins. We recognize that distinction of only two growth 645 A phase-field study: Journal of Geophysical Re
search: Solid Earth, v. 120, p. 3096–3118, https://
doi.org/10.1002/2015JB011934. Geologists Bulletin, v. 92, p. 1537–1563, https://
doi.org/10.1306/07160808037. Geologists Bulletin, v. 92, p. 1537–1563, https://
doi.org/10.1306/07160808037. growth patterns are likely to operate in a wider
range of host rocks that are characterized by com
posite fracture paths and polymineralic compo
sitions. On the other hand, poorly consolidated
rocks with weak grain boundaries that form most
ly intergranular fractures are less likely to form
wide-blocky veins due to the absence of surface
type–dependent crystal growth rate variations. Similarly, wide-blocky textures are not predicted
in monomineralic rocks with strong grain bound
aries, where most of the fracture is transgranular. g
Laubach, S.E., et al., 2019, The role of chemistry in
fracture pattern development and opportunities
to advance interpretations of geological materi
als: Reviews of Geophysics, v. 57, p. 1065–1111,
https://doi.org/10.1029/2019RG000671. g
Bons, P.D., 2001, Development of crystal morphol
ogy during unitaxial growth in a progressively
widening vein: I. The numerical model: Journal
of Structural Geology, v. 23, p. 865–872, https://
doi.org/10.1016/S0191-8141(00)00159-0. Nelson, R.A., 2001, Geologic Analysis of Naturally
Fractured Reservoirs (second edition): Boston,
Gulf Professional Publishing, 352 p. Bons, P.D., Elburg, M.A., and Gomez-Rivas, E.,
2012, A review of the formation of tectonic veins
and their microstructures: Journal of Structural
Geology, v. 43, p. 33–62, https://doi.org/10.1016/
j.jsg.2012.07.005. Nestler, B., Garcke, H., and Stinner, B., 2005, Mul
ticomponent alloy solidification: Phase-field
modeling and simulations: Physical Review E:
Statistical, Nonlinear, and Soft Matter Phys
ics, v. 71, 041609, https://doi.org/10.1103/
PhysRevE.71.041609. Boullier, A.-M., and Robert, F., 1992, Palaeoseismic
events recorded in Archaean gold-quartz vein
networks, Val d’Or, Abitibi, Quebec, Canada:
Journal of Structural Geology, v. 14, p. 161–179,
https://doi.org/10.1016/0191-8141(92)90054-Z. The 3-D models in Figure 4 show how per
meability and fluid-flow regime in the wide-
blocky veins are strongly affected by the de
velopment of crystal bridges early in the filling
process. Although these bridges locally block
fluid pathways and increase the tortuosity of the
flow paths, high pore connectivity is maintained
until late stages in the sealing process (Figs. 4B–
4D). DISCUSSION This is very different from veins where
all crystals grow at the same rate and the flow
paths are more direct due to the lack of crystal
bridges, maintaining higher permeability in the
early stages of crystal growth, as was reported
by Kling et al. (2017) for quartz veins. However,
the early bridge formation in wide-blocky veins
might prevent fracture collapse, keeping veins
open between fluid pulses. y
Newhouse, W.H., 1942, Ore Deposits as Related to
Structural Features: Princeton, New Jersey, Princ
eton University Press, 280 p. Caputo, R., and Hancock, P.L., 1999, Crack-jump mech
anism of microvein formation and its implications
for stress cyclicity during extension fracturing:
Journal of Geodynamics, v. 27, p. 45–60, https://
doi.org/10.1016/S0264-3707(97)00029-X. Nixon, C.W., Vaagan, S., Sanderson, D.J., and Gawthor
pe, R.L., 2019, Spatial distribution of damage and
strain within a normal fault relay at Kilve, U.K.:
Journal of Structural Geology, v. 118, p. 194–209,
https://doi.org/10.1016/j.jsg.2018.10.016. Cox, S.F., Etheridge, M.A., and Wall, V.J., 1987, The
role of fluids in syntectonic mass transport, and
the localization of metamorphic vein-type ore
deposits: Ore Geology Reviews, v. 2, p. 65–86,
https://doi.org/10.1016/0169-1368(87)90024-2. Nollet, S., Urai, J.L., Bons, P.D., and Hilgers, C.,
2005, Numerical simulations of polycrystal
growth in veins: Journal of Structural Geol
ogy, v. 27, p. 217–230, https://doi.org/10.1016/
j.jsg.2004.10.003. Dickson, J.A.D., 1993, Crystal growth diagrams as
an aid to interpreting the fabrics of calcite ag
gregates: Journal of Sedimentary Petrology,
v. 63, p. 1–17, https://doi.org/10.1306/d4267a78-
2b26-11d7-8648000102c1865d. j j g
Peacock, D.C.P., and Sanderson, D.J., 2018, Struc
tural analyses and fracture network charac
terisation: Seven pillars of wisdom: Earth-
Science Reviews, v. 184, p. 13–28, https://doi
.org/10.1016/j.earscirev.2018.06.006. Durney, D.W., and Ramsay, J.G., 1973, Incremental
strains measured by syntectonic crystal growth,
in De Jong, K.A., and Scholten, R., eds., Gravity
and Tectonics: New York, Wiley, p. 67–96. Prajapati, N., Selzer, M., Nestler, B., Busch, B., and
Hilgers, C., 2018, Modeling fracture cementa
tion processes in calcite limestone: A phase-field
study: Geothermal Energy, v. 6, 7, https://doi
.org/10.1186/s40517-018-0093-4. We infer that once the veins become fully
sealed, they are mechanically stronger than the
micritic host rock due to the large, bridging crys
tals that connect both vein walls. Higher me
chanical strength implies that new fractures form
more easily in the host rock than by a failure of
an existing vein. ACKNOWLEDGMENTS Holland, M., and Urai, J.L., 2010, Evolution of anas
tomosing crack-seal vein networks in limestones:
Insight from an exhumed high-pressure cell, Ja
bal Shams, Oman Mountains: Journal of Struc
tural Geology, v. 32, p. 1279–1290, https://doi
.org/10.1016/j.jsg.2009.04.011. We thank the German Science Foundation (DFG) for
funding this project grants (NE 822/34-1, UR 64/17-
1). Ukar acknowledges grant DE-FG02-03ER15430
from the Chemical Sciences, Geosciences, and Bio
sciences (CSGB) Division, Office of Basic Energy
Sciences, U.S. Department of Energy, for financial
support, and Sara Elliott for assistance with SEM-CL
imaging and post-processing. We thank M. Elburg, S. Cox, Y.D. Kuiper, and an anonymous reviewer for their
constructive reviews. Urai, J.L., Williams, P.F., and van Roermund, H.L.M.,
1991, Kinematics of crystal growth in syntec
tonic fibrous veins: Journal of Structural Geology,
v. 13, p. 823–836, https://doi.org/10.1016/0191-
8141(91)90007-6. g
j j g
Kling, T., Schwarz, J.-O., Wendler, F., Enzmann, F.,
and Blum, P., 2017, Fracture flow due to hydro
thermally induced quartz growth: Advances in
Water Resources, v. 107, p. 93–107, https://doi
.org/10.1016/j.advwatres.2017.06.011. Wendler, F., Okamoto, A., and Blum, P., 2016, Phase-
field modeling of epitaxial growth of polycrys
talline quartz veins in hydrothermal experi
ments: Geofluids, v. 16, p. 211–230, https://doi
.org/10.1111/gfl.12144. Lander, R.H., and Laubach, S.E., 2015, Insights
into rates of fracture growth and sealing from a
model for quartz cementation in fractured sand
stones: Geological Society of America Bulle
tin, v. 127, p. 516–538, https://doi.org/10.1130/
B31092.1. g
gl
Williams, R.T., Farver, J.R., Onasch, C.M., and
Winslow, D.F., 2015, An experimental investi
gation of the role of microfracture surfaces in
controlling quartz precipitation rate: Applications
to fault zone diagenesis: Journal of Structural
Geology, v. 74, p. 24–30, https://doi.org/10.1016/
j.jsg.2015.02.011. DISCUSSION The observed abundance of sub
parallel arrays of microveins rather than larger
veins with crack-seal textures suggests that once
filled, the studied veins are rarely reactivated. Fisher, D.M., and Brantley, S.L., 1992, Models of
quartz overgrowth and vein formation: Defor
mation and episodic fluid flow in an ancient
subduction zone: Journal of Geophysical Re
search, v. 97, p. 20,043–20,061, https://doi
.org/10.1029/92jb01582. Prajapati, N., Abad Gonzalez, A., Selzer, M., Nes
tler, B., Busch, B., and Hilgers, C., 2020,
Quartz cementation in polycrystalline sand
stone: Insights from phase-field simula
tions: Journal of Geophysical Research: Solid
Earth, v. 125, e2019JB019137, https://doi
.org/10.1029/2019JB019137. g
j
Glen, R.A., Hancock, P.L., and Whittaker, A., 2005,
Basin inversion by distributed deformation:
The southern margin of the Bristol Channel
Basin, England: Journal of Structural Geolo
gy, v. 27, p. 2113–2134, https://doi.org/10.1016/
j.jsg.2005.08.006. Ramsay, J.G., 1980, The crack-seal mechanism of rock
deformation: Nature, v. 284, p. 135–139, https://
doi.org/10.1038/284135a0. This study demonstrates the power and ver
satility of the phase-field method to model the
evolution of veins. Its ability to incorporate
crystallography, host-rock composition, realis
tic fracture geometries, differential growth rates,
and thermodynamics allows the fine tuning of
models to natural vein microstructures. g
Schindelin, J., et al., 2012, Fiji: An open-source plat
form for biological-image analysis: Nature Meth
ods, v. 9, p. 676–682, https://doi.org/10.1038/
nmeth.2019. Hilgers, C., Koehn, D., Bons, P.D., and Urai, J.L.,
2001, Development of crystal morphology dur
ing unitaxial growth in a progressively widening
vein: II. Numerical simulations of the evolution of
antitaxial fibrous veins: Journal of Structural Ge
ology, v. 23, p. 873–885, https://doi.org/10.1016/
S0191-8141(00)00160-7. Ukar, E., and Laubach, S.E., 2016, Syn- and post
kinematic cement textures in fractured carbonate
rocks: Insights from advanced cathodolumines
cence imaging: Tectonophysics, v. 690, p. 190–
205, https://doi.org/10.1016/j.tecto.2016.05.001. REFERENCES CITED Ajdukiewicz, J.M., and Larese, R.E., 2012, How clay
grain coats inhibit quartz cement and preserve po
rosity in deeply buried sandstones: Observations
and experiments: American Association of Petro
leum Geologists Bulletin, v. 96, p. 2091–2119,
https://doi.org/10.1306/02211211075. Lander, R.H., Larese, R.E., and Bonnell, L.M., 2008,
Toward more accurate quartz cement models:
The importance of euhedral versus noneuhedral
growth rates: American Association of Petroleum Ankit, K., Urai, J.L., and Nestler, B., 2015, Micro
structural evolution in bitaxial crack-seal veins: Printed in USA www.gsapubs.org | Volume 49 | Number 6 | GEOLOGY | Geological Society of America 646 Downloaded from http://pubs.geoscienceworld.org/gsa/geology/article-pdf/49/6/641/5323300/g48472.1.pdf
by Karlsruhe Institute of Technology, KIT Library user
|
https://openalex.org/W2181476317
|
https://zenodo.org/record/3378127/files/101013896%20Edagbo%20et%20al.pdf
|
English
| null |
The Influence of African Mistletoe (Tapinanthus bangwensis) on the Conservation Status and Productivity of Irvingia gabonensis in Moor Plantation Area of Ibadan, Nigeria
|
Greener Journal of Agricultural Science
| 2,013
|
cc-by
| 3,967
|
The Influence of African Mistletoe
(Tapinanthus bangwensis) on the
Conservation Status and Productivity of
Irvingia gabonensis in Moor Plantation
Area of Ibadan, Nigeria Plant Genetic Resources Unit, National Centre for Genetic Resources and Biotechnology, Moor
Plantation, Apata, PMB 5382, Ibadan, Oyo State, Nigeria. A preliminary study of the host parasite relationship between Tapinanthus
bangwensis and its host, Irvingia gabonensis was carried out to gain an
understanding of the impact and outcome of their co-habitation on the host plant. Assessment of the physiological interaction was conducted via a study of some
of the physiognomy and reproductive capacity of infested and uninfested hosts. The parasitic infestation it was observed vary between adjoining locations of the
host stands because of differences in eco-habitat, physiognomy of susceptible
hosts and as well as the capacity of the interacting host and parasitic plants to
carry out their routine physiological activities. The incidence of the parasitic plant
population on the host plantation in this study indicated marginal loss in value of
the conserved host plants with correlated loss in productivity. Ultimately, the
presence of Tapinanthus on its Irvingia host in this study would serve not only as
a source of decline in the conservation status and productivity of the host plants
but could also lead to the death of many susceptible hosts if proliferation of the
parasite is not kept in check. Greener Journal of Agricultural Sciences
ISSN: 2276-7770; ICV: 6.15
Vol. 3 (10), pp. 743-747, October 2013
Copyright ©2017, the copyright of this article is retained by the author(s)
http://gjournals.org/GJAS Greener Journal of Agricultural Sciences
ISSN: 2276-7770; ICV: 6.15
Vol. 3 (10), pp. 743-747, October 2013
Copyright ©2017, the copyright of this article is retained by the author(s)
http://gjournals.org/GJAS MATERIALS AND METHODS The study was carried out in the Irvingia gabonensis
plantation of the National Centre for Genetic Resources
and
Biotechnology
(NACGRAB)
within the
Moor
Plantation
complex,
Ibadan,
South-West,
Nigeria
between November 2011 and June 2013. The collected
parasitic plant samples of the Tapinanthus species of
mistletoe were identified at the herbarium of NACGRAB. The Irvingia plantation was mapped out into two with 50
selected stands each from the two wings of the
plantation. Girth size of all the Irvingia trunks was
determined by measuring the diameter at height of
primary branches using a measuring tape. The number
of host trees infested and those uninfested at each
range of girth size was determined and percentage
infestation was calculated. The severity of infestation
was estimated according to the amount of plant crown
area infested by mistletoes on a visual scale of 1 to 4, by
standing at a distance of 3 to 6m to the host tree from
four different directions. Total numbers of fruits on the
infested and uninfested Irvingia were determined by
plucking and counting of the fruits. The extent of
impact/loss in the tree productivity was evaluated by
random sampling of three infested and three uninfested
trees from each range of girth size and counting the
number of fruits produced by each at maturity with the
mean of the productivity calculated for each group. Irvingia gabonensis has been used wholly or as
supplement in the treatment of type II diabetics and in
reducing obesity (Omoruyi et al., 1994; Judith et al.,
2005). Ofoefule et al., (1997) reported that dika fat, a
vegetable oil obtained from the kernel are also used in
the formulation of sustained released frusimide granules
and a highly gross energy is obtained from it compared
to other tropical seeds, this is as a result of its high fat
content. Leakey (1999) had also reported on the
nutritional value of I. gabonensis fruit and kernel. Other
services derived from the Irvingia tree species include:
fresh bark being considered to be a powerful antibiotic
against scabies, a cure for diarrhoea when mixed with
palm oil, a toothache remedy and intercropping with
other crops in farm systems for shelter (Asaah et al.,
2003). )
The parasitic plant family of the Loranthacean
mistletoe (including Tapinanthus bangwensis (Engl. and
K. Keywords: Keywords:
Host, infestation, parasite,
susceptibility, Girth size Edagbo et al / Greener Journal of Agricultural Sciences 744 INTRODUCTION nutrients) from the host into the parasite. It thus deprives
the host of essential nutrients that may be utilized for
photosynthetic and other metabolic activities (Benzing
1990; Polhill and Wiens, 1998). When one part of the
host
is
intensively
attacked
by
mistletoe,
the
reproductive and photosynthetic potential of the part
distal to the infestation declines leading to death of the
part. But the extent of damage caused to the host
depends on size of the parasite, the growth rate and
metabolic activity of the parasite, the degree of
dependency on the host for resources, and the stage of
development of the host (Aliero and Ismaila, 2002;
Davcota, 2005; Kwon-Ndung and Ismaila, 2009). Going
by the nature of the relationships between mistletoe and
its host tree(s), this study was therefore carried out to
examine the impact of the mistletoe (Tapinanthus
bangwensis) infestation on the conservation status and
productive output of the stands of Irvingia gabonensis. Irvingia gabonensis and Irvingia wombulu commonly
called bush mango or dika nut are indigenous fruit trees
in Africa that produce edible fruits and seeds (Harris,
1996; Atangana et al., 2001, 2002). The fruit of I. gabonensis has a sweet mesocarp which is eaten fresh
while that of I. wombulu is sour. The seed cotyledons
(edible kernels) from both are used for culinary
purposes. Nigerians distinguished between kernels from
I. gabonensis and I. wombulu, referring to the former as
‘ugiri’ in Igbo or “apon” in Yoruba (Ladipo et al., 1996),
“ujuru” in Idoma and the later as “ogbono” in Igbo
(Okafor, 1975), “upi” in Idoma. In Nigeria and some other
parts of West and Central Africa sub-region, the kernels
are used as a condiment and are highly valued for their
food thickening property and their commercial value
(Ndjouenekeu et al., 1996; Asaah et al., 2003). Some
studies conducted in the West and Central Africa
established that there exist substantial local and regional
markets for non-woody forest products of certain
indigenous trees, such as Irvingia species (Asaah et al.,
2003), and it was noted that the centre of diversity for I. gabonensis is in southern Cameroon and southeast
Nigeria, while that of I. wombolu is in southeast
Cameroon and western Nigeria (Lowe et al., 2000). MATERIALS AND METHODS Krause) Danser and other species which are known
to be of widespread occurrence even in Nigeria have
been reported attacking a number of many wild and
domesticated tree and shrub species of which I. gabonensis is one of the susceptible host plant (Bright
and Okusanya, 1998; Ayuba, 2000; Bako et al., 2001;
Wahab et al., 2010). Mistletoes grow attached to
branches and stems of host trees by means of
specialized absorbing organ called the haustorium,
which penetrates into host’s living tissues and functions
for translocation of various materials (water and mineral RESULTS The rate of infestation of the mistletoe, Tapinanthus
bangwensis on Irvingia in the study site (Table I) vary
sharply from a 58% high at the West to a modest 14%
level at the East. Edagbo et al / Greener Journal of Agricultural Sciences 745 Table I: Rate of infestation of Tapinanthus bangwensis on Irvingia gabonensis at the two studied wings of
the plantation
Location Total No. of Total No. of Trees Percentage (%)
Trees Surveyed Infested Infestation
West wing 50 29 58
East wing 50 07 14 Table I: Rate of infestation of Tapinanthus bangwensis on Irvingia gabonensis at th
the plantation n of Tapinanthus bangwensis on Irvingia gabonensis at the two studied wings of
the plantation anthus bangwensis on Irvingia gabonensis at the two studied wings of
h
l
i population. The scale of severity for the East Wing was
in sharp contrast to the prominent spread of the parasitic
infestation noticed at the West Wing. It had just a minute
fraction of its population infested with a 7 stand host
infestation, 5 stands were of low infestation and the
remainder 2 host trees on fairly high infestation regime. In Table II, the severity of the mistletoe infestation on the
Irvingia plantation showed a trend of gradual build up in
the intensity of the parasitism. At the West Wing, from
the aggregate population of 29 infested host trees; a
greater proportion of 15 stands were of low infestation,
10 stands fairly high infestation, 2 stands on high and
another 2 stands on very high infestation effect on the Table II: Severity of infestation of the infested Irvingia stands across its plantation
Location Total No. of Trees
Infested
EFFECT ON CROWN AREA AMONG INFESTED POPULATION
Low (1) Fairly high (2) High (3) Very high (4)
West wing 29 15 10 2 2
East wing 07 5 2 0 0 Table II: Severity of infestation of the infested Irvingia stands across
ocation
Total No of Trees
EFFECT ON CROWN AREA AMONG IN and 23.3% infestation rates respectively. The higher
girth range classification of 61-80cm and 81-100cm
offered greater infestation of 51.4% and 60% in tune with
the
sequence
of
the
incremental
size. The parasitic relationship between T. bangwensis and its
host I. gabonensis based on stem girth was presented in
Table III. RESULTS The stem girth at 0-20cm had 0% infestation, a
further 21-40cm and 41-60cm girth range had 22.2% Table III: Classification of Irvingia gabonensis based on the girth size and rate of Tapinanthus infestation
Girth size (cm) No of trees surveyed No. of trees infested Percentage (%)
infestation
0-20 03 00 00
21-40 09 02 22.2
41-60 43 10 23.3
61-80 35 18 51.4
81-100 10 06 60 rvingia gabonensis based on the girth size and rate of Tapinanthus infestation
No of trees surveyed
No of trees infested
Percentage (%) Table III: Classification of Irvingia gabonensis based on the girth size and rate of Tapinanthus infestation
Girth size (cm) No of trees surveyed No. of trees infested Percentage (%)
infestation
0-20 03 00 00
21-40 09 02 22.2
41-60 43 10 23.3
61-80 35 18 51.4
81-100 10 06 60
The mean of numbers of fruits produced by I. gabonensis based on the girth size and on the presence
or absence of infestation (Table IV) generally portrayed
a situation in which fruit production increased with
increases in girth size and decreased with infestation
except in the rare case of a deviation. Hence at 0-20cm,
an average of 2 fruits was produced by uninfested trees
while there was no sample population of infested trees at
that girth range. Uninfested Irvingia stands at 21-40cm
range had 36 fruits while infested stands yielded 8 fruits. However, for the 41-60cm girth range, the sample
population for the uninfested trees yielded 163 fruits to
the 46 fruits produced by the infested population. 61-
80cm had 119 fruit production receipt for the uninfested
and 255 fruits for infested while 81-100cm girth range
had an output of 468 fruits for uninfested and 149 fruits
for infested host trees. The cumulative total fruit
production for the uninfested sample population stood at
788 fruits and the infested was 458 fruits and thus gives
a distinction of over 20% output higher for the uninfested
as against the infested hosts. on of Irvingia gabonensis based on the girth size and rate of Tapinanthus infestation
No of trees surveyed No. of trees infested Percentage (%)
infestation population for the uninfested trees yielded 163 fruits to
the 46 fruits produced by the infested population. DISCUSSION The nature of the parasitic infestation of the African
Mistletoe, Tapinanthus bangwensis across the Irvingia
plantation presented a phase of advanced infestation by
the parasite at the West wing while the East Wing was
still at the phase of primary infestation; hence the sharp
differences in the percentage infestation rate. The
Irvingia trees on the plantation were of same age of
establishment but were at different phase of mistletoe
infestation because of prevailing ecological peculiarity of
each wing of the plantation. The plantation at the West
Wing had their edges in close proximity to a roadside
which transverse the Irvingia plantation and an older
Citrus orchard which harboured mistletoe population. By
and large, the dispersal agent of Tapinanthus spp, the
tinker birds (Pogoniulus spp.) would have introduced the
parasite early to Irvingia at the West axis because of
ease of accessibility (Norton and De Lange, 1999). Conversely, the edge of the plantation at the East axis
was shielded by the predominant presence of Dacryodes
edulis,
Penthaclethra
macrophylla
and
Spondias
mombin. These trees therefore offered some form of
cover at the East which minimized the exposure of the
Irvingia to the dispersal agent of the parasitic plant
hence much less infestation. p
y
The incidence rate of infestation by Tapinanthus
bangwensis on the Irvingia host trees across the select
range of girth sizes reflected correlated rise in the
percentage
infestation
rate;
thus
there
were
progressions from the least girth size (0-20cm) to the
highest (81-100cm). This observation corroborate the
findings of similar work carried out by other researchers
(Kwon-Ndung and Ismaila, 2009; Edagbo et al., 2012)
that plant parasitic infestation increase with increase in
girth size of host plants. This would imply that enlarged
stem (increases in girth size) provides a platform for
more nutrient flow, increased physiological activities and
structurally more surface medium for increased parasitic
infestation. The mean of the productivity for fruit yield in
consideration of different girth sizes and actual
infestation status of the susceptible host trees generally
tell of correlated increases in reproductive output in both
the infested and uninfested hosts with increase in girth
size. It was however observed that at the same category
of girth sizes for infested and uninfested host trees, the
output were much higher for the uninfested hosts. RESULTS 61-
80cm had 119 fruit production receipt for the uninfested
and 255 fruits for infested while 81-100cm girth range
had an output of 468 fruits for uninfested and 149 fruits
for infested host trees. The cumulative total fruit
production for the uninfested sample population stood at
788 fruits and the infested was 458 fruits and thus gives
a distinction of over 20% output higher for the uninfested
as against the infested hosts. The mean of numbers of fruits produced by I. gabonensis based on the girth size and on the presence
or absence of infestation (Table IV) generally portrayed
a situation in which fruit production increased with
increases in girth size and decreased with infestation
except in the rare case of a deviation. Hence at 0-20cm,
an average of 2 fruits was produced by uninfested trees
while there was no sample population of infested trees at
that girth range. Uninfested Irvingia stands at 21-40cm
range had 36 fruits while infested stands yielded 8 fruits. However, for the 41-60cm girth range, the sample Edagbo et al / Greener Journal of Agricultural Sciences 746 Table IV: The mean of the productivity of fruits produced by Irvingia gabonensis based on the girth size and
on the presence/absence of Tapinanthus infestation
Girth size (cm) No. of fruits produced No. of fruits produced
by uninfested trees by infested trees
(Mean) (Mean)
0-20 002 ̶
21-40 036 008
41-60 163 046
61-80 119 255
81 -100 468 149
Cumulative Total 788 458
(Mean), Mean of data of three random samples; ̶ , Absent
DISCUSSION
an accentuated parasitism may lead, prior to host death,
t
l
f
f li
d
i di
tl
d
d oductivity of fruits produced by Irvingia gabonensis based on the girth size and
on the presence/absence of Tapinanthus infestation an accentuated parasitism may lead, prior to host death,
to loss of foliage cover and indirectly reduced
photosynthetic rates. REFERENCES Ladipo D. O., Foudoun J. M., Ganga N., 1996. Domestication of bush
mango (Irvingia spp). Some exploitable intraspecific variations in
West and Central Africa. Food and Agriculture Organization,
Rome. Italy. Aliero B.L. and Samaila A., 2000. The occurrence of parasitic
mistletoe (Tapinanthus spp) on Parkia biglobosa (Jacq.) Benth
(African Locust Bean Tree) in Yauri Local Government Area,
Kebbi State, Nigerian Journal of Basic and Applied Sciences, 9:
5-10. Leakey, R. R. B., 1999. Potential for novel food products from
agroforestry trees: A review. Food-Chemistry, 66 ( 1): 1-14. Lowe A J, Gillies A C M, Wilson J, and Dawson I K., 2000. Conservation genetics of bush mango from central/west Africa:
implications from random amplified polymorphic DNA analysis,
Moleculary Ecology, 9: 831-841 Asaah, E. K., Tchoundjeu Z. and Atangana, A. R., 2003. Cultivation
and conservation status of Irvingia wombolu in humid lowland
forest of Cameroon. Food, Agriculture & Environment 1 (3&4):
251-256. Ndjouenekeu R., Goycoolea F. M., Morris E. R., Akingbala J. O., 1996. Rheology of Okra (Hibiscus esculentus) and dika nut (Irvingia
gabonensis) polysaccharides, Carbonhydrate Polymer, 29: 263-
269. Atangana A. R, Tchoundjeu Z, Foldout J. M, Asaah E, Dumb M,
Leakey R. R. B., 2001. Domestication of Irvingia gabonensis:
1.Phenotypic variation in fruit and kernels in two populations from
Cameroon. Agroforestry Syst. 53: 55-64. Norton, D.A. and De Lange, P.J., 1999. Host specificity in parasitic
mistletoes (Loranthaceae) in New Zealand. Functional Ecology,
13: 552-559. Atanngana A. R., Ukafor V., Anegbeh P. O., Asaah E., Tchoundjeu Z.,
Usoro C., Fondoun J. M., Ndoumbe M., Leakey R. R. B., 2002. Domestication of Irvingia gabonensis: 2. The selection of multiple
traits for potential cultivars from Cameroon and Nigeria,
Agroforestry Syst. 55: 221- 229. Ofoefule S. I., Chuckwu A., Okore U. C., Ugwali M. O., 1997. Use of
dika fat in the formulation of sustained released frusimide –
encapsulated granules.1: Boll. Chem farm. 136(10): 646-650. Okafor J. C., 1975. Varietal delimitation in Irvingia gabonensis
(Irvingiaceae): Bulletin du Jardin Botanique National de Belgique
45(1-2): 211-221. g
y
y
Ayuba, I., 2000. A Survey of Occurrence, Distribution and Effects of
African Mistletoe (Tapinanthus spp.) on Parkia biglobosa (Jacq)
and Butryospermum Paradoxum (Gaertn. F.) Hepper in Yauri
Local Government Area of Kebbi State. M.Sc. Dissertation, UDUS
Sokoto State. (
)
Omoruyi F. O., Adamson I., 1994. Effect of dika nut (Irvingia
gabonensis) and cellulose on plasma Lipid in streptozotocin
induced diabetic rat, Nutr. Res, 14: 537-544. CONCLUSION CONCLUSION Edagbo David, E., Ajiboye, T. O., Borokini Temitope, I., Ighere
Dickson, A., Alowonle Ahmed, A. and Michael Clement, 2012. The Influence of African Mistletoe (Tapinanthus bangwensis) on
the Conservation Status of Citrus sinensis in Moor Plantation
Area of Ibadan, Nigeria. International Journal of Current
Research, 4(12): 484-487. Parasitic plants such as Tapinanthus bangwensis when
left to blossom on susceptible host plants like Irvingia
gabonensis may alter the rates of survival and fecundity
of their hosts and hence modify the structure and
dynamics of their populations thereby becoming a
serious threat to their sustenance and conservation
(Press and Phoenix, 2005). Host plants with trunks of
larger girth size provide enlarged platform for increased
flow of nutrients and proportionally favourable and
enriched surface area for attachment and growth of the
infective parasite and are thus the more frequent
recipient of parasitic infestation. With the affirmed
influence of Tapinanthus bangwensis on its host
nonetheless, there is need to ecologically manage their
population for biodiversity conservation, ecosystem
stability and exploitation of their ethno-botanical value. Fabiana Alves Mourão, Claudia Maria Jacobi1, José Eugênio Côrtes
Figueira1 and Eugênia Kelly Luciano Batista, 2009. Effects of the
parasitism of Struthanthus flexicaulis (Mart.) Mart. (Loranthaceae)
on the fitness of Mimosa calodendron Mart. (Fabaceae), an
endemic shrub from rupestrian fields over ironstone outcrops,
Minas Gerais State, Brazil, Acta bot. bras. 23(3): 820-825. ( )
Harris D. J., 1996. A revision of the Irvingiacea in Africa, Bulletin du
Jardin Botanique National de Belgique, 65 (1-2): 55-64. Howell B. E. and Mathiasen R.L., 2004. Growth impacts of
Psittacanthus angustifolius Kuijt on Pinus oocarpa Schiede in
Honduras. Forest Ecology and Management, 198: 75–88. Judith L. N., Julius E. O., Samuel R. M., 2005. The effect of Irvingia
gabonensis seeds on body weight and blood lipid of obese
subject in Cameroon, Lipids Health Dis., 4: 12. Kwon-Ndung E.H. and Ismaila A., 2009. Prospects Of Host Resistance
In Improved And Domesticated Species of Parkia biglobosa To
African Mistletoes (Tapinanthus spp.) In Central Nigeria,
EJEAFChe, 8 (5): 382-388. Cite this Article: Edagbo DE, Ighere DA and Michael C (2013). The Influence of African Mistletoe (Tapinanthus
bangwensis) on the Conservation Status and Productivity of Irvingia gabonensis in Moor Plantation Area of Ibadan, Nigeria.
Greener Journal of Agricultural Sciences, 3(10): 743-747, http://doi.org/10.15580/GJAS.2013.10.101013896. DISCUSSION This
invariably implied that the presence of Tapinanthus
bangwensis constitute some measure of considerable
depleting force on the productive capacity of the Irvingia
host trees. The infested hosts could therefore be said to
manifest loss in productivity when compared to similar
uninfested hosts. And in this vein, the comparative total
cumulative output in fruit yield from the result of this
study was that of lower yield for the infested hosts and
hence reduced productivity. It therefore could be seen
that as had been noted in earlier research findings
(Tennakoon and Pate, 1996; Press et al., 1999; Aliero
and Ismaila, 2002; Howell and Mathiasen, 2004),
besides the lower fruit yield, susceptible hosts which are
in states of severe infestation usually display reduced
foliage, damaged allometry and architecture with even
deprived growth at branches with domineering parasitic
activities. All these features and many other attributes
associated
with
the
presence
of
Tapinanthus
bangwensis on its host, constitute a major means of loss
and value depreciation in the conservation status of
Irvingia gabonensis. A critical overview of the severity of parasitic
infestation on the Irvingia plantation was indicative of
gradual increase in the activities and population of
Tapinanthus
bangwensis. More
often
than
not,
assessment of the severity of infestation usually gives a
pointer to the phase of parasitic infestation in the
sampled host population. An infestation rated to be at
the level 4 (very high) effect on the crown of the host
tree(s) tells of advanced phase of infestation while
infestations at 1 (low) or 2 ( fairly high) effect could be
ascribed as being at the primary phase of infestation. With a conducive edapho-climatic condition, the parasitic
infestation as observed in the East Wing of the Irvingia
plantation
at
the
primary
phase
of
infestation
progressively degenerate to the advanced phase as
seen in the West Wing when external factors of limitation
or control are not introduced into the ecosystem. Oftentimes and in accordance with the assertion of
Mourão et al., 2009, it would be that for an interactive
nature like the type so described above; the negative
effects of the parasite may occur in cascade, because Edagbo et al / Greener Journal of Agricultural Sciences 747 REFERENCES Bako S.P, Onwuchekwa B. N, Bako L.S.P., Iortsuun D. N., 2001. Physiology of the African Mistletoe (Tapinanthus dodoneifolius
(DC) Danser) and its influence on the metabolism of two hosts
(Albizia lebbeck Benth and Citrus sinensis L.), Nig. J. Agric. Environ., 2: 1081-1092. Polhill Roger and Delbert Wiens, 1998. Mistletoes of Africa, The Royal
Botanic Gardens, Kew Ed. ISBN. pp. 370. pp
Press M. C., Scholes J. D. and Watling J. R., 1999. Parasitic plants:
physiological and ecological interactions with their hosts. In:
Press, MC, Scholes, JD, Barker, MG, eds. Physiological Plant
Ecology. Oxford, UK: Blackwell Science, 175–197. Benzing, D. H., 1990. Vascular Epiphytes. Cambridge Tropical Biology
Series. Cambridge University, Press. Press, M. C. and Phoenix, G. K., 2005. Impacts of parasitic plants on
natural communities. New Phytologist, 166: 737-751. Bright, E.O. and Okusanya, B. A., 1998. Infestation of economic plants
in Badeggi by Tapinanthus dodoneifolius (D.C) Danser and T. globiferus (A. Rich)Van Tiegh. Nigerian Journal of Weed Science,
11:51-56. Tennakoon K. U. and Pate J. S., 1996. Effects of parasitism by a
mistletoe on the structure and functioning of branches of its host. Plant, Cell & Environment, 19: 517–528. Devkota, Mohan Prasad, 2005. Biology of mistletoes and their status in
Nepal Himalaya. Himalayan Journal of Sciences 3(5): 85-88. Wahab, O. M., A. E. Ayodele and J. O. Moody, 2010. TLC
phytochemical screening in some Nigerian Loranthaceae, Journal
of
Pharmacognosy
and
Phytotherapy,
2(5):
64-70.
|
https://openalex.org/W992949557
|
https://teorievedy.flu.cas.cz/index.php/tv/article/download/447/445
|
Czech
| null |
Foucaultovo umění vidět
|
Teorie vědy
| 2,019
|
cc-by
| 13,977
|
* Kontakt na autora: John Rajchman, Th e Department of Art History and Arche-
ology, Columbia University, 913 Schermerhorn Hall, New York, NY 10027–6902,
USA (jr790@columbia.edu). Foucaultovo umění vidět John Rajchman TEORIE VĚDY XXX/2 2008 TEORIE VĚDY XXX/2 2008 Abstract Art of seeing, John Rajchman argues in his essay, was in the center of
Michel Foucault’s critical attention as well as practice. Foucault himself
was a visual thinker and writer. More importantly, however, the ways
in which historically changing vision determines not only what is seen,
but what can be seen, are one of his major concerns. Rupture with self-
evidences is then the fi rst step one must take to make the invisible – yet
not hidden – power visible. Th e invisibility of power, seen as the invis-
ible light that makes other things visible, is what makes it tolerable. Knowledge and the practice of knowing themselves are constructed
by the technology of the visual, such as the diff erent types of spaces
that bring about specifi c visibility. In Foucault’s histories, the prison
or the clinic are such spaces that have visualized criminality, sexuality
or madness in particular manner. However, problematization of these
things needs to go beyond new ways of looking at them and has to
question their entire fi eld of vision. Th is implies that Foucauldian eth-
ics is less concerned with what we do about things themselves, instead,
it rather asks how we see them in the fi rst place and how can they be
seen diff erently. It thus requires not to look within us, on the contrary,
we should look out, from outside of ourselves. Keywords: Foucault; visual; seeing; power; space; ethics Keywords: Foucault; visual; seeing; power; space; ethics 91 John Rajchman Gilles Deleuze ve své knize Foucault říká o Michelu Foucaultovi, že byl
velkým vidoucím, voyant. Tvrdí, že Foucaultovo vidění a jeho pojetí vidění
jsou neustálou a ústřední součástí nejen jeho dějin, ale rovněž myšlení. Říká, že ti, kdo tuto část jeho myšlení nevezmou v potaz, ho „překrucují“
do té míry, že se stává srovnatelným s analytickou fi losofi í, „s níž však má
jen velmi málo společného“.1 Deleuze přisuzuje vizuální části Foucaultova myšlení mnohé. Te-
ritorium viditelného zahrnuje vědění, umění, etiku a politiku, což pou-
kazuje na to, proč se Foucault nepotřeboval zabývat problémy týkajícími
se „vztahů mezi vědou a literaturou nebo mezi imaginací a vědeckostí či
mezi věděným a žitým“.2 Viditelné je také nejdůležitější ve způsobu, jakým
se Foucaultovo myšlení vyvíjelo. 1 Gilles DELEUZE, Foucault. Praha: Herrmann & synové 1996, s. 75. V Deleu-
zově čtení fi losofů lze vystopovat originální pohled na „vidění“. Tak kupříkladu
v 60. letech pro něj byl Spinoza, fi losof a brusič optických čoček, vivant-voyant.
Spinoza řekl, že geometrické demonstrace jeho Etiky jsou jako „oči duše“; Deleuze
tuto vitální optickou metodu vidí jako cosi, co napravuje smutné vášně, které boří
život, jako způsob broušení skla pro inspirované svobodné vidění. Pozdní Deleu-
zovo dílo se věnuje Leibnizovi a baroku. 2 Ibid, s. 76.
3 Ibid, s. 96.
4 V eseji nazvaném „In the Empire of the Gaze: Foucault and the Denigration
of Vision in Twentieth-Century French Th ought“ (in David Cousins HAY (ed.),
Foucault: A Critical Reader. London: Basil Blackwell 1986), Martin Jay předkládá
užitečný seznam některých míst, kde Foucault rozebírá záležitosti viditelného. Ale 2 Ibid, s. 76. 3 Ibid, s. 96. Abstract Je jeho další součástí, kterou společně
s „diskursem“ Deleuze chápe jako Foucaultovo „novokantovství“, tedy
propojené s tématem „transcendentální obrazotvornosti“ u Kanta a s po-
kusy na straně Merleau-Pontyho a Heideggera jít za intencionalitu k „ote-
vírání“ Bytí. Deleuze však na Foucaulta aplikuje také kategorie dánského
sémiologa Louise Hjelmsleva, které použil ve své práci o fi lmu. Říká, že
Foucault byl obrovským „audiovizuálním“ myslitelem, který měl „neoby-
čejně blízko k současnému fi lmu“.3 Myslím si, že Deleuze je prvním, kdo tuto stránku Foucaultova
myšlení „viděl“ a ukázal její obecnou důležitost v jeho díle.4 Nebudu zde 92 Foucaultovo umění vidět sledovat všechny spletité cesty, které Deleuze ve své analýze slučuje. Poku-
sím se ukázat jinými slovy, co si myslím, že měl Deleuze na mysli. Začnu
Foucaultovým uměním zobrazení dějin. sledovat všechny spletité cesty, které Deleuze ve své analýze slučuje. Poku-
sím se ukázat jinými slovy, co si myslím, že měl Deleuze na mysli. Začnu
Foucaultovým uměním zobrazení dějin. nejsem přesvědčen, že uspěl v pochopení toho, co je v tomto pojednání obsaženo.
Jeho základní myšlenkou je, že Foucault, jako mnozí jeho krajané, byl „proti
vidění“. Přesto není jasné, co tím mohl mít na mysli. Říci, že panoptický dozor
je „schématem“ formy moci nebo že přispívá k samozřejmosti této formy moci,
a tím ji činí přijatelnou, není proti vidění ani nečiní vidění ústřední záležitostí
(„okulo-centrismem“). Jay začíná hypotézou, že spousta francouzských myslitelů
byla sjednocena pod jakýmsi spiknutím, které mělo „zostuzovat“ vidění a že podél
Rýna, v německé sociologii, nacházíme mnohem „optimističtější“ pohled. Jestliže
se v této formulaci „vizuální“ nahradí „racionálním“, lze zde nalézt důvěrně známý
model vyloučení současného francouzského myšlení, které Apel vykládá ještě
ostřeji než Habermas. Aby se Jay skutečně k této polemice mohl připojit, musel
by ukázat, že příslušní francouzští myslitelé identifi kovali vizuální s racionálním
nebo byli v opozici k jednomu kvůli protikladu druhého. Myslím si, že by to značně
urovnalo obtíže či nesrovnalosti v původním napadání iracionalismu. A tím, že
zde neuspěl, dluží Jay výklad toho, co chápe jako „viditelné“ a co by mělo být tím,
co viditelné „zostuzuje“, či je proti tomu. Habermasovým pohledem se zabývám
v eseji „Habermas’s Complaint.“ New German Critique, 1988, č. 45, s. 163–191.
5 Mohlo by být zajímavé prozkoumat, jakým způsobem vlastně historici Annales 5 Mohlo by být zajímavé prozkoumat, jakým způsobem vlastně historici Annales
přispěli k dobovým fi lmům. V „Anti-Rétro“ (Cahiers du Cinéma, červenec 1974),
Foucault rozebírá takové fi lmy, jakými jsou Lacombe Lucien a Th e Night Porter ve
vztahu k „retro-stylu“ v odívání a výzdobě domovů. Jeho analýza návratu k dří-
vějším stylům není ani „simulací“ nebo prázdnou recyklací, ani anamnézou. Dějinné obrazy Foucault byl neobyčejně vizuálním historikem. Jeho dějiny jsou naplněné
živými obrazy, které se vrývají do mysli. Vizualizace událostí neboli
zobrazení dějin je bezpochyby uměním, které samotné má své dějiny. Události nebyly vždy nahlíženy stejným způsobem či popisovány stejně. Jedním z příkladů může být Michelet. Stejně tak celý aspekt „nových
dějin“, s nimiž Foucault spojuje své dílo v úvodu do Archeologie a v nichž
se pokusil „transformovat dokumenty v monumenty“ – noví historici byli
zaujati „prostorem“, v němž lidé žili, a rekonstrukcí tableaux de moeurs –
což je užitečná záležitost při vytváření „dobových fi lmů“.5 Ale Foucaultovy 5 Mohlo by být zajímavé prozkoumat, jakým způsobem vlastně historici Annales
přispěli k dobovým fi lmům. V „Anti-Rétro“ (Cahiers du Cinéma, červenec 1974),
Foucault rozebírá takové fi lmy, jakými jsou Lacombe Lucien a Th e Night Porter ve
vztahu k „retro-stylu“ v odívání a výzdobě domovů. Jeho analýza návratu k dří-
vějším stylům není ani „simulací“ nebo prázdnou recyklací, ani anamnézou. 93 John Rajchman obrazy jsou více než jen takovými tably. Jsou skládačkami, které volají po
analýze. Vytvářejí součást fi losofi ckého cvičení, v němž hraje roli vidění. obrazy jsou více než jen takovými tably. Jsou skládačkami, které volají po
analýze. Vytvářejí součást fi losofi ckého cvičení, v němž hraje roli vidění. Častým nástrojem Foucaultova psaní jsou „před-a-po“ obrazy. Ukáže
vám obraz z určitého období a poté další z jiného období. Tímto se otázka,
jak se přešlo z jednoho systému myšlení do jiného, stává viditelnou. Tento
nástroj se objevuje skrz celé dílo, ale nápadný je zejména ve dvou „počá-
tečních“ knihách – o zrodu vězení a zrodu kliniky. V knize Dohlížet a trestat poukazuje na obraz mučivé popravy Da-
miense, kralovraha, a poté na časový rozvrh sledovaných činností. Ve
Zrození kliniky ukazuje Pommeovu léčebnou koupel hysterika, při které
je „léčena“ „teplota“ nervového systému. Pak si všímá Bayleho důkladné
prohlídky mozkových lézí, té „rozplizle vypadající kaše“. V obou případech máme obrazy nejen toho, jak věci vypadaly, ale také
toho, jak byly zviditelňovány, jak byly podávány, aby mohly být viděny,
jak byly „ukazovány“ vědění a moci – dva způsoby, jakými se věci stávaly
nahlédnutelnými. V případě vězení jde o otázku dvou typů zviditelňování
zločinů v těle – skrz „podívanou“ a skrz „dohled“. V případě kliniky jde
o otázku dvou způsobů organizace „prostoru, v němž se setkávají těla
a oči“. Dějinné obrazy U Baylea oko získává na hloubce a tělo na objemu; při zkoumání
mozku nahlíží do hlubin jednotlivého těla, v němž je lokalizována nemoc. Pomme ještě stále hledal obecnější „portrét“ nemoci, který by umožnil
klasifi kaci horečných stavů. V obou případech dává Foucault do souvislosti techniky, jimiž byly
věci viditelné, v rámci širší koncepce vidění určité doby. Což se stává jed-
ním z témat u Deleuze – nazve jej visibilités. Máme dějiny nejen toho, co
bylo viděno, ale také toho, co mohlo být viděno, co bylo nahlédnutelné
či viditelné. „Vizualizace“, schéma, skrz které jsou věci vydány pohledu,
náleží k „pozitivitě“ vědění a moci své doby a místa. Máme zde však ještě druhý rys Foucaultových „před-a-po“ obrazů:
kde navrhuje fi losofi cké cvičení ve vidění. Proto nás na konci analýzy cesty
od „před“ k „poté“ vede k „vidění“ vylíčených událostí v novém světle 94 Foucaultovo umění vidět nebo jiným způsobem – ve světle jejich spodních, nespatřených koncepcí. Po přečtení díla Dohlížet a trestat je pak velmi těžké „nevidět“ konstrukci
kruhovitého vězení v novém světle, je obtížné nebýt překvapen, že se „vě-
zení podobá továrnám, školám, kasárnám, nemocnicím, když se všechny
podobají vězení“.6 Toto je hledisko Foucaultova dějinného zobrazení, které
Deleuze nazývá évidences. Foucault byl vidoucím právě způsobem, jakým
jsou „vidění“ a „evidence“ spojené v jeho dějinách a v jeho myšlení. 6 Michel FOUCAULT, Dohlížet a trestat. Praha: Dauphin 2000, s. 315. Viditelnost Foucault nachází v tom, co nazývá „klasickým věkem“, celou škálu
způsobů vidění a toho, jak se věci zviditelňují, což bylo nemyslitelné
v předcházejícím období, v němž bylo při dešifrování „podobností“ oko
spojeno s uchem: v klasifi kačních tabulkách forem vědění, v převaze
shody s vjemovou evidencí, v pojetí šílenství jako „oslepeného rozumu“,
v pojetí malířství, v utopické literatuře otevřené společnosti, v přírodních
historiích stejně jako ve způsobech „ukazování“ šílených lidí, které se
v mnohém liší od „lodí bláznů“. Foucault se pokusil vytvořit důkladné
pojmové uspořádání, které tyto způsoby vidění spojilo do formy „viditel-
nosti“, která byla odlišná od jiných. Foucaultův předpoklad byl takový, že existuje „pozitivní nevědomí“
vidění, které určuje nikoli to, co je viděno, ale to, co může být viděno. Předpokládal, že ne všechny způsoby vidění nebo zviditelňování jsou
možné najednou. Určitá doba umožňuje vidět jen některé věci, a nikoli
jiné. Určité věci „osvětluje“ a jiné tím vrhá do stínu. V tom, co můžeme
vidět, je mnohem více pravidelnosti, mnohem více donucení než předpo-
kládáme. Vidět vždy znamená myslet, neboť to, co je nahlédnutelné, je
součástí toho, co „strukturuje myšlení předem“. A naopak myslet znamená
vždy vidět. 95 John Rajchman To, co činí viditelné dostupným, nelze samotné vidět. Jde o anonymní
tělo praktik, které je rozptýlené na různých místech. Jak Deleuze říká,
„[visibilités] tedy nejsou ani akty vidoucího subjektu, ani danostmi vizu-
álního smyslu“.7 V Archeologii vědění Foucault pojednává o „výpovědních
modalitách“ jako o vlastnostech diskursu. Ale ve svých dějinách také
pojednává o „modalitách vidění“ jako o vlastnostech vizuální inteligence:
kdo vidí co nebo koho a kde jsou integrálními znaky vizuálního myšlení
určitého období. Nejde tedy o nezávislý fakt o jeho kontextu. Takovéto
vizuální myšlení je zakořeněno v určitém druhu „materiální existence“
– v prostorech, v nichž je vykonáváno (jako například v nemocnicích,
vězeních, muzeích nebo domácnostech), a v technikách, skrze které jsou
obrazy reprodukovány a skrze které kolují (například v tisku, na tržištích
atd.). V určitém smyslu jde o „subjekt“, který je dán ve formách „viditel-
ného“. Foucault zjistil, že stejné uspořádání, které určité období připisuje
vnitřním či psychologickým procesům, se opět vynořuje v procesech vněj-
ších, „veřejných“, jako například při vytváření map či ilustrací odborných
děl. Tak je v klasické době schéma renesančních podobností chápáno jako
zdroj omylu, jež má být objasněn patřičným pozorováním. Zrodem kli-
niky je tedy „vizionářský prostor“, v němž se o nemocech mluvilo, vložen
do hlavy pacienta. 8 Dávám do protikladu Freudův pohled na sny s Artemidórovým, jak o tom
pojednává Foucault v Péči o sebe (John RAJCHMAN „Ethics aft er Foucault.“
Socialtext, zima 1985. Ve svém prvotním úvodu k překladu Binswangerova díla
Dream and Existence (in Review of Existential Psychology and Psychiatry, roč. XIX,
1984–1985, č. 1). Foucault namítá Freudovi, že redukuje sny na záznamy o snech.
Ale v pozdějším díle chápe viditelnost snění historicky než spíše existenciálně. 7 DELEUZE, Foucault, s. 86. 9 Viz poznámky k Technologies of the Self: A Seminar with Michel Foucault.
Amherst: University of Massachusetts Press 1988, s. 14): „To, proti čemu se zde
stavím, je skutečnost, že mezi sociálními dějinami a dějinami myšlení je trhlina.
Sociální historici mají popisovat, jak lidé jednají bez myšlení, a historici myšlení
mají popisovat, jak lidé myslí bez jednání. Každý však jedná i myslí. A způsob,
jakým lidé jednají či reagují je propojen se způsobem myšlení, a to je bezpochyby
myšlení spojené s tradicí.“ Viditelnost Vizualizace náleží k důležitým internalizujícím a psy-
chologizujícím praktikám, které Foucault spojuje s modernitou. Freudova
myšlenka snění jako způsobu, jak sobě samému ukázat své nejniternější
touhy, je tedy v protikladu k Artemidórově pojetí, v němž jsou sny způso-
bem, jak činit osud člověka viditelným v hierarchické společnosti.8 96 Foucaultovo umění vidět Vizuální myšlení doby tak mělo pozitivní organizaci. Ale taková
organizace nespočívá v tom, že cosi drží v tajnosti. Foucault si postupně
uvědomil, že „klasický“ způsob zviditelňování šílenství nebyl založen
v potlačení nebo zatajení skutečného způsobu, jakým je šílenství vidět. Konceptuální schéma, které určuje to, co lze vidět, je ve fázi Archeologie
„neviditelné, nikoli však skryté“. Viditelnost doby se může zdát nevidi-
telnou, ale nikoli jako cosi skrytého či drženého stranou. Neviditelné je
pouze světlo, které osvětluje věci nebo je zviditelňuje. Zkrátka viditelnost je záležitost pozitivního, materiálního, anonym-
ního těla praxe. Její existence ukazuje, že jsme mnohem méně svobodní
v tom, co vidíme, než v tom, co si myslíme, neboť nevidíme omezení myš-
lenek v tom, co můžeme vidět. Ale také to ukazuje, že jsme mnohem více
svobodní, než si myslíme, neboť prvek viditelnosti je také cosi, co otevírá
vidění dějinné změně či transformaci.9 To je problém évidence. 9 Viz poznámky k Technologies of the Self: A Seminar with Michel Foucault. Amherst: University of Massachusetts Press 1988, s. 14): „To, proti čemu se zde
stavím, je skutečnost, že mezi sociálními dějinami a dějinami myšlení je trhlina. Sociální historici mají popisovat, jak lidé jednají bez myšlení, a historici myšlení
mají popisovat, jak lidé myslí bez jednání. Každý však jedná i myslí. A způsob,
jakým lidé jednají či reagují je propojen se způsobem myšlení, a to je bezpochyby
myšlení spojené s tradicí.“ y
j
Myšlenka visibilités se týká toho, jak lidé jednají a reagují, když vidí, že je něco
umožněno určitým způsobem myšlení vztaženým k tradici. V tomto světle lze
chápat projekt, který Foucault ohlásil na posledních stránkách Archeologie vědění,
aby byla malba studována jako „diskursivní praxe“ spíše než nahlížena jako „čistá
vize, která pak musela být přepsána do materiality prostoru“, nebo „nahé gesto“, či
„vždy jako způsob mluvení.“ (Michel FOUCAULT, Archeologie vědění, s. 289–290). Obraz byl „prostoupen pozitivitou“, samozřejmý charakter „viditelnosti“ byl od-
vozen od materiality zakořeněné v myšlení. Zdálo se, že myšlení činilo určitý druh
viditelnosti přirozeným či nezbytným pro obraz. 9 Viz poznámky k Technologies of the Self: A Seminar with Michel Foucault.
Amherst: University of Massachusetts Press 1988, s. 14): „To, proti čemu se zde
stavím, je skutečnost, že mezi sociálními dějinami a dějinami myšlení je trhlina.
Sociální historici mají popisovat, jak lidé jednají bez myšlení, a historici myšlení
mají popisovat, jak lidé myslí bez jednání. Každý však jedná i myslí. A způsob,
jakým lidé jednají či reagují je propojen se způsobem myšlení, a to je bezpochyby
myšlení spojené s tradicí.“
Myšlenka visibilités se týká toho, jak lidé jednají a reagují, když vidí, že je něco
umožněno určitým způsobem myšlení vztaženým k tradici. V tomto světle lze
chápat projekt, který Foucault ohlásil na posledních stránkách Archeologie vědění,
aby byla malba studována jako „diskursivní praxe“ spíše než nahlížena jako „čistá
vize, která pak musela být přepsána do materiality prostoru“, nebo „nahé gesto“, či
„vždy jako způsob mluvení.“ (Michel FOUCAULT, Archeologie vědění, s. 289–290).
Obraz byl „prostoupen pozitivitou“, samozřejmý charakter „viditelnosti“ byl od-
vozen od materiality zakořeněné v myšlení. Zdálo se, že myšlení činilo určitý druh
viditelnosti přirozeným či nezbytným pro obraz. Zkoumat takovouto „pozitivitu“
pak sestávalo z tázání, jak taková pojetí, podle kterých mohl být obraz viděn,
byla spojena, kde a jakým prostřednictvím, kým a tak dále. Skrze jaké „systémy
myšlení“ byly „objekty“ malby, třídy věcí, které mohly být malovány, určovány?
A jak bylo toto vymezení spojeno s prostory, v nichž mohly být viděny (kostely
či zámky, galerie či muzea), a s těmi, v nichž byly vytvářeny (ateliér, akademie
atd.), tedy s právními a ekonomickými pravidly, která ovládala jejich vlastnictví
a oběh? Jakým způsobem byl modus „bytí malířem“ chápán? Jak se technologické inovace staly součástí racionality či srozumitelnosti „technik“ otevřených ma-
lířství? Jak se „materialita“ obrazu stala více než „kontextem“, v němž byl obraz
viděn či vytvořen, součástí způsobu, jakým byl chápán (když kupříkladu Foucault
říká, že Manet byl prvním muzejním malířem)? A jakými způsoby je toto utvoření
konceptuálního prostoru obrazu spojeno s jinými či propojenými poli v myšlení
doby? Potom by zkoumání „událostí viditelného“ v dějinách malby předpoklá-
dalo, že lidé mají mnohem méně svobody v malbě než v myšlení, že v praktikách
malby je mnohem více konceptuálních „pravidelností“, než si lze představit. Ale
vzhledem k tomu, že tyto pravidelnosti jsou také tím, co otevírá malbu změně
a transformaci, lidé mají v malbě mnohem více svobody, než si představují.
10 Ian HACKING The Emergence of Probability Cambridge: Cambridge Univer Viditelnost Zkoumat takovouto „pozitivitu“
pak sestávalo z tázání, jak taková pojetí, podle kterých mohl být obraz viděn,
byla spojena, kde a jakým prostřednictvím, kým a tak dále. Skrze jaké „systémy
myšlení“ byly „objekty“ malby, třídy věcí, které mohly být malovány, určovány? A jak bylo toto vymezení spojeno s prostory, v nichž mohly být viděny (kostely
či zámky, galerie či muzea), a s těmi, v nichž byly vytvářeny (ateliér, akademie
atd.), tedy s právními a ekonomickými pravidly, která ovládala jejich vlastnictví
a oběh? Jakým způsobem byl modus „bytí malířem“ chápán? Jak se technologické 97 John Rajchman 11 Michelle PERROT, L’impossible prison. Paris: Seuil 1980, s. 44. 0 Ian HACKING, Th e Emergence of Probability. Cambridge: Cambridge Univer-
sity Press 1975. Evidence Slovo évidence jak v angličtině, tak ve francouzštině pochází ze slova
videre, vidět. V průběhu dějin získalo významy jako důkaz, svědectví
a jasnost či neklamnost mysli. Ian Hacking zkoumal jednu změnu pojmu
evidence v dějinách „vynoření se pravděpodobnosti“.10 Tu, jež umožnila
Huma. Ve francouzštině máme jeden význam slova évidence, který je pří-
značný pro Foucaultův vizuální styl – do angličtiny se překládá jako
„self-evidence“ (samozřejmost): tedy to, co se bere jako samozřejmost či je
přijímáno bez pochybností. Foucault tento pojem evidence představuje ve
svém novém zobrazení dějin. V rozpravě s historiky Foucault vysvětluje, že jeho způsob vidění
zrodu vězení byl pokusem vidět „události“ za samozřejmými entitami
a kontinuitami, a tak „zudálostnit“ dějiny. Člověk začíná pomocí rupture
d’évidence, odtržení od samozřejmosti, „od takových évidences, na kterých
spočívá naše vědění, dohody, praxe“.11 A pak se táže, jak takové „évidences“
vznikly a jak získaly formu. Tam, kde je samozřejmost, se člověk pokouší
odkrýt singulární formaci neviděné události. Evidence je v tomto smyslu použita v obou knihách zrození. Ve Zro-
zení kliniky Foucault říká, že „přesné vrstvení ‚těla‘ nemoci a těla nemoc- 98 Foucaultovo umění vidět ného člověka [...] je samozřejmé jen nám“12. A v Dohlížet a trestat odkazuje
k „samozřejmosti“ (le caractère d’évidence) „vězení, od které se tak těžko
odvracíme“.13 Ve formě vězeňství existuje jak právní, tak ekonomická sa-
mozřejmost. Společně vysvětlují, proč i přes skutečnost, že vězení nedělá
to, co mělo dělat, „není ‚vidět‘ nic, co by jej nahradilo“.14 „Vidění“ je v tomto smyslu součástí jednání. Nemůžeme vidět, co
máme dělat, protože jsme „vězni“ samozřejmosti jednoho způsobu vi-
dění toho, co máme dělat. Přidáváme svůj díl k praktikám, na kterých se
účastníme a které pro nás činí tento způsob vidění samozřejmým – jedná
se o účast či přijetí, které můžeme odmítnout. Pak je tedy u Foucaulta
évidence ve vztahu k přijatelnosti praxe. Představuje součást toho, co činí
„strategii moci“ tolerovatelnou i přes její obtíže. Tudíž nahlížení událostí,
skrze které se věci stávají důvěrně samozřejmé, umožňuje vidět, jakými
způsoby mohou být netolerovatelné či nepřijatelné. Jde o pokus vidět, jak
bychom mohli jednat v případě, kdy ještě nelze nahlédnout do toho, co
děláme. Zkrátka máme tu „kritické“ umění, a právě při jeho praktikování
byl Foucault podle Deleuze vidoucím neboli voyant. Pro Deleuze není v zásadě vidoucím – ani v první instanci – někdo,
kdo dokáže vylíčit budoucí události. 15 V „Eye of Power“, když odkazuje k dílu Jeana Starobinskiho, Foucault stručně
naráží na rousseauovský sen o otevřené společnosti (in Power/Knowledge. New
York: Pantheon 1980, s. 152–153). 12 Michel FOUCAULT, Th e Birth of the Clinic. New York: Vintage Books, 1975,
s. 3
13 FOUCAULT, Dohlížet a trestat, s. 320.
14 Ibid., s. 320. V Užívání slastí Foucault také říká: „Chtěl bych se nejprve zastavit
u onoho zcela běžného a nedávného pojmu ‚sexuality‘: zaujmout vůči němu od-
stup, vyhnout se jeho důvěrné samozřejmosti (contourner son évidence familière)
[...] celkově se jednalo o snahu sledovat, jak se v moderních západních společnos-
tech ustavila taková ‚zkušenost‘ [...] (Užívání slastí. Praha: Herrmann & synové
2003, s. 9–10).
15 V „Eye of Power“, když odkazuje k dílu Jeana Starobinskiho, Foucault stručně
naráží na rousseauovský sen o otevřené společnosti (in Power/Knowledge. New
York: Pantheon 1980, s. 152–153). 13 FOUCAULT, Dohlížet a trestat, s. 320.
14 Ibid., s. 320. V Užívání slastí Foucault také říká: „Chtěl bych se nejprve zastavit
u onoho zcela běžného a nedávného pojmu ‚sexuality‘: zaujmout vůči němu od-
stup, vyhnout se jeho důvěrné samozřejmosti (contourner son évidence familière)
[...] celkově se jednalo o snahu sledovat, jak se v moderních západních společnos-
tech ustavila taková ‚zkušenost‘ [...] (Užívání slastí. Praha: Herrmann & synové
2003, s. 9–10). 2 Michel FOUCAULT, Th e Birth of the Clinic. New York: Vintage Books, 1975
s. 3 16 „An Interview with Gilles Deleuze.“ History of the Present, jaro 1986, s. 1.
17 Ibid. Evidence Ani není nutně jakýmsi „vizionářem“
či „utopistou“, který nazírá budoucí rozložení tak, že vše, co by mělo být,
bude nakonec všemu otevřené.15 „Vidoucí“, říká Deleuze, „je někdo, kdo 99 John Rajchman vidí něco, co není viděno“.16 Foucaultovo umění vidět je uměním odkrý-
vání neviděných évidences, které pro nás činí věci, které v podstatě děláme,
přijatelnými či tolerovatelnými. Deleuze nachází tento druh vidění v práci, kterou Foucault dělal pro
Skupinu pro informaci o vězeních, jejímž byl Deleuze členem. Byl zde
zpřístupněn jakýsi „veřejný prostor“ diskuse s vězni. V takovémto prostoru
Foucault „viděl cosi, co v té době nikdo jiný neviděl“. Tento akt vidění
vyžadoval rupture d’évidence: trhlinu mezi samozřejmostí ekonomické
a právní koncepce vězení a tím, co se ve skutečnosti dělo. V této trhlině
bylo možné spatřit na praktikách cosi netolerovatelného, což otevřelo
tázání po dějinné analýze: analýze, která spustila nové způsoby vidění
a myšlení, nejen o francouzských trestních institucích, ale také o strategic-
kém uspořádání moci v moderních společnostech, jejím vztahu k formám
vědění a způsobům žití. Foucaultovo vidění spočívalo v tomto kritickém
otevření myšlení. Deleuze říká: Viděl věci a podobně jako všichni ostatní, kteří vědí, jak něco vi-
dět a jak to vidět do hloubky, shledával viděné netolerovatelným. Myslet pro něj znamenalo reagovat na netolerovatelné, na viděné
netolerovatelné. A netolerovatelné nebylo nikdy viditelné, bylo
čímsi více ...17 Jedním významem slova „evidence“ se v průběhu dějin stalo „očité svě-
dectví“ aktuálních událostí, které se liší od „oka“, jež nadcházející události
čte. Foucaultova myšlenka evidentních událostí má co do činění s okem
historiků. Ve fi ktivním vylíčení nachází Foucault podobný záměr, jak zvi-
ditelňovat neviditelné prostory vidění. V zobrazení „prostoru“ Mauricem
Blanchotem, kde schůzky vycházejí najevo a slova jsou vyměňována, viděl
ve fi kci pokus nikoli ukázat (faire voir) neviditelné, ale ukazovat míru, do 100 Foucaultovo umění vidět které je neviditelnost viditelného neviditelná. Tudíž [fi kce] s sebou nese
hlubokou spřízněnost s prostorem ...18 které je neviditelnost viditelného neviditelná. Tudíž [fi kce] s sebou nese
hlubokou spřízněnost s prostorem ...18 V mnohých rozhovorech Foucault také popisuje své vlastní dějiny
jako fi kci. Neznamená to, že tyto dějiny postrádají validitu, která by je
odlišila od fi kce. Spíše s fi kcí sdílejí záměr: záměr nikoli vysvětlovat nebo
ukazovat, jak jsou naše způsoby vidění a jednání dějinně podmíněné, ale
naopak ukazovat, jak věci mohou být jinak, mimo naši důvěrnou samo-
zřejmost. Evidence To je důvodem, proč dějiny „samozřejmostí“, způsobu, jakým
jsou věci viděny, zahrnují „samozřejmosti“ v myšlení historiků. „Vidět“
znamená otevírat dějiny novým doménám a novým otázkám, „dělat dě-
jiny ‚objektivizace‘ událostí, které historici berou jako objektivně dané“.19
Když Foucault říká, že píše historická díla, která jsou více než jen díla
historika,20 je to zčásti kvůli tomuto jinému záměru vidění, který fi losof
sdílí se spisovatelem. Vidění je v tomto smyslu v díle Foucaulta jako fi losofa a historika dů-
ležité jako umění pokusit se vidět, co je v našem vidění nemyšlené, a ote-
vřít to jako zatím neviděné způsoby vidění. Ve Foucaultově psaní se také
rozevírá nezvyklé spojení prostoru a pohledu, které se rozpohybovává do
mnohých směrů. Nyní bych chtěl poukázat na různé stránky tohoto pojetí,
podívat se na vidění ve vědění, vidění v jednání, vidění v myšlení a v žití. 18 Foucault/Blanchot. New York: Zone Books 1987, s. 24.
19 FOUCAULT, Užívání slastí, s. 9.
20 Richard RORTY, Philosophy and the Mirror of Nature. Princeton: Princeton
University Press 1979. 18 Foucault/Blanchot. New York: Zone Books 1987, s. 24. 19 FOUCAULT, Užívání slastí, s. 9.
20 Richard RORTY, Philosophy and the Mirror of Nature. Princeton: Princeton
University Press 1979. 20 Richard RORTY, Philosophy and the Mirror of Nature. Princeton: Princeton
University Press 1979. 19 FOUCAULT, Užívání slastí, s. 9. 21 Michel FOUCAULT, „Řád diskursu“ in: Diskurs, autor, genealogie. Praha: Svo-
boda 1994, s. 7–37.
22 Michel FOUCAULT, Slova a věci. Brno: Computer Press 2007, s. 43.
23 FOUCAULT, Slova a věci, s. 39. Mělo to být stejné „odloučení oka“, jež je ty-
pické pro klasickou převahu pozorování ve vědění, co mělo charakterizovat kla- OUCAULT, Slova a věci. Brno: Computer Press 2007, s. 43. 21 Michel FOUCAULT, „Řád diskursu“ in: Diskurs, autor, genealogie. Praha: Svo-
boda 1994, s. 7–37. 23 FOUCAULT, Slova a věci, s. 39. Mělo to být stejné „odloučení oka“, jež je ty-
pické pro klasickou převahu pozorování ve vědění, co mělo charakterizovat kla- Vidění ve vědění Foucault nechápal vědění jako jednoduše vystavěné z běžné samozřejmosti
vnímání skrze logiku důsledků, indukce či dedukce. Zajímaly ho způsoby,
jimiž bylo vidění ve vědění samo pojmově vystavěno. V tomto pojetí 101 John Rajchman savoir vyžaduje a vytváří způsob, jak se zprostornit, jakousi „technologii
viditelného“. Foucault chtěl opustit to, co lze nazývat moderní fi losofi ckou
posedlostí, „pozorování“ ve vědění – součást fi losofi cké „samozřejmosti“,
kterou objevil v různých formách jak ve fenomenologii, tak v pozitivismu. Naše fi losofi cké pojetí pozorování náleží do nedávné doby a zabraňuje
nám vidět, jak je ve skutečnosti vědění „zprostorněno“ či „viděno“. Ve vědě
je vidění více, než se ukazuje oku. „Zrakové metafory“, jak bylo často poznamenáno, zaujímají ústřední
místo v našem slovníku vědění: pravda je cosi, co říkáme, že vidíme. Ale
tyto metafory nefungovaly vždy stejným způsobem. Foucault si myslel, že
změny byly částečně dílem aktuálních způsobů, jež si lidé vymysleli, aby
„zprostornili“ své vědění, že byly aktuální rolí vidění ve vědění. Richard
Rorty přezkoumává literaturu, která ukazuje, jak se karteziánské chápání
vnímání odlišuje od „hylémorfi ckého“ vidění v myšlení scholastiky;
příroda je zrcadlena novým způsobem.21 A ve Foucaultově archeologii
viditelného je vynoření karteziánského upřednostňování vnímání, tedy
myšlenky evidence jako transparence pro mysl, čímsi spíše komplikova-
nějším. V „Řádu diskursu“ promlouvá o obecné změně ve vidění, která se
objevila v Británii šestnáctého a sedmnáctého století a je zachycena v pra-
vidle „dívej se, spíš než čti, prověřuj, spíš než komentuj“. Zahrnovalo to
celé „schéma možných, pozorovatelných, měřitelných, klasifi kovatelných
objektů“,22 schéma, jež předcházelo aktuálnímu souboru „obsahu“ vědění. Tato změna odpovídá tomu, co Foucault ve Slovech a věcech nazývá „reor-
ganizací kultury, ve které jsme neustále chyceni“, kde oko již nedešifruje
„prózu světa“, a kde tedy „oko bylo [...] určeno jen a pouze k vidění a ucho
jen a pouze k slyšení“.23 Zde vyvstává doktrína odtržení smyslů, která 102 Foucaultovo umění vidět je ústřední pro objevení se nové disciplíny „estetiky“ u Lessinga a Dide-
rota.24 je ústřední pro objevení se nové disciplíny „estetiky“ u Lessinga a Dide-
rota.24 Ve vědění vidíme změnu také v tom, jak se „zprostorňuje“ přírodní
historie klasické doby. V Linnaeově klasifi kaci byly zkoumány rostliny
bez mikroskopů z hlediska jejich vizuálního „charakteru“ jako bezbarvé,
nevonící „primární kvality“ dvojrozměrného prostoru. sickou převahu vnímání v malbě. Tradice ut pictura poesis měla být zpochybněna
tvrzením, že malba využívá jiné druhy znaků a jinou formu než poesie – měla
být předložena pouze samotnému oku, nikoli uchu. Zde se objevilo zkoumání
této formy, skrze kterou se malba samotná představuje vnímajícímu oku, jež mělo
připustit, aby byla „kritika“ malby odlišena od jejího „komentáře.“ Toto klasické
rozlišení mezi kritikou a komentářem nebo mezi formou a obsahem pak mělovést
k rozsáhlé rozpravě, v níž Foucault vidí Mallarméa jako změnu. (FOUCAULT,
Slova a věci, s. 65–67). sickou převahu vnímání v malbě. Tradice ut pictura poesis měla být zpochybněna
tvrzením, že malba využívá jiné druhy znaků a jinou formu než poesie – měla
být předložena pouze samotnému oku, nikoli uchu. Zde se objevilo zkoumání
této formy, skrze kterou se malba samotná představuje vnímajícímu oku, jež mělo
připustit, aby byla „kritika“ malby odlišena od jejího „komentáře.“ Toto klasické
rozlišení mezi kritikou a komentářem nebo mezi formou a obsahem pak mělovést
k rozsáhlé rozpravě, v níž Foucault vidí Mallarméa jako změnu. (FOUCAULT,
Slova a věci, s. 65–67). Vidění ve vědění Princip klasifi kace
prvků a jejich uspořádání v prostoru byl založen na určitém druhu „op-
tiky“ rostlinné morfologie, již bylo lze předvést na ilustracích umožněných
novými tiskařskými technikami, a jež proto zobrazovaly „reprodukce“ sickou převahu vnímání v malbě. Tradice ut pictura poesis měla být zpochybněna
tvrzením, že malba využívá jiné druhy znaků a jinou formu než poesie – měla
být předložena pouze samotnému oku, nikoli uchu. Zde se objevilo zkoumání
této formy, skrze kterou se malba samotná představuje vnímajícímu oku, jež mělo
připustit, aby byla „kritika“ malby odlišena od jejího „komentáře.“ Toto klasické
rozlišení mezi kritikou a komentářem nebo mezi formou a obsahem pak mělovést
k rozsáhlé rozpravě, v níž Foucault vidí Mallarméa jako změnu. (FOUCAULT,
Slova a věci, s. 65–67). 24 „Formalismus“ Clementa Greenberga je v tomto smyslu „klasický“. Dokonce
cituje Lessinga, když se pokouší najít princip obratu k abstrakci v moderní malbě
v procesu, v němž se každé z „klasických“ umění obrací ke specifi ckým problémům
svého „média“. To by pak bylo tajemstvím odtržení „formy“, která byla v centru
snah avantgardistů o zachování „hodnoty“ umění ve věku kýče a socialistického
realismu. Greenberg si myslel, že když tak učinil fyzické médium výsadním ob-
jektem vizuálního chápání, objevil tím podstatu viditelnosti či zrakovosti vizuál-
ních umění. Historicky byla tato podstata zakořeněná v klasické samozřejmosti
malby chápané jako vnímaný objekt – zde se nachází v Greenbergově příspěvku
o abstrakci v moderním umění jím prohlašovaný pozitivismus. Co se nyní zdá být
v Greenbergově koncepci podstaty vizuálního neviditelné, je právě ono známé
„oko“ formalistických kritiků, které se naučilo vidět jen formy a způsob, jakým
bylo oko přesunuto do malíře či sochaře jako povinnost „očistit“ jeho vizuální
chápání tím, že se bude na svůj objekt dívat „čistě“ formálně. To, co oko „vidět“
nemohlo, byla další koncepce viditelného založená Duchampem, dadaismem
a surrealismem. Zde je možné nahlédnout do díla Rosalindy Kraussové o optic-
kém nevědomí („In the Blink of an Eye“). Th ierry de Duve porovnává nezbytnost
Greenbergova pojetí avantgardy s „obrazovým nominalismem“ duchampovské
avantgardy, kde se otázka toho, jak se viditelné samotné, aby bylo pojmenováno či
pochopeno, stává ústředním uměleckým problémem (in Le nominalism pictural. Paris: Editions Minuit 1984). 103 John Rajchman rostlin samotných. 26 Ibid., s. 106. 27 Viz diskuse o vědění a ideologii v Archeologii vědění, s. 275–278. 25 FOUCAULT, Slova a věci, s. 106. 28 Ve svém díle Michel Foucault’s Archaeology of Scientifi c Reason (Cambridge:
Cambridge University Press 1989) Gary Gutting předkládá jasné a detailní vysvět-
lení Canguilhemova rozdělení mezi dějinami pojmů a dějinami teorií. Jeho kniha
je obstojnou korekcí pohledu, že Foucault byl proti objektivitě či racionalitě. Vidění ve vědění Tyto pojmové reorganizace či „zprostornění“ učinily
přírodní historii čímsi, co „není nic jiného než pojmenování viditelného“.25
„Přírodní historie“ klasického období, tvrdí Foucault, „nebyla umožněna
tím, že se lidé začali dívat lépe a z větší blízkosti,“ 26 ale když to, co viděli,
bylo uspořádáno novým způsobem. Ve Zrození kliniky zaznamenává další změnu ve vidění, která se ob-
jevuje ve francouzském lékařství na konci osmnáctého století. Foucault
znova usiluje o projednání pohledu, že se „člověk díval lépe a z větší blíz-
kosti“. Disputuje o důležitosti změny v lékařství, ve které se oko přesunulo
od neuvěřitelných představ k důkladnému pozorování věcí. To je součástí
jeho obecné pře o dichotomii mezi imaginárním a epistemickým či ideo-
logickým a vědeckým v dějinách vědění.27 V podstatě tvrdí, že „vizionářský prostor“, v němž lékaři, fyziologové
a pacienti pojednávali o nemoci, byl sám zcela řádnou a koherentní for-
mou „zprostornění“, které se zakládalo na rozpoznání „podoby“ nemoci
v těle. A právě ve „zrození kliniky“ byla obsažena změna celé myšlenky
toho, co má být „viděno“ lékařem – kde, jakým nástrojem a za pomoci
jakých pojmů. Foucault tvrdí, že změnu nelze vysvětlit „tematickým obsahem“ nebo
„logickými modalitami“ či použitím výlučně kvantitativních metod. Mění se „prostor“ nemoci samotné, místo konkrétního těla, kde je nemoc
lokalizována, a institucionální „prostor“, v němž se lokalizace objevuje. A navíc tato změna nebyla nevyhnutelná – muselo se čekat celá desetiletí,
než k léčení došlo. Změna je vysvětlena až na základě institucionálních
faktorů, které se objevily v nových programech francouzské revoluce. Tímto způsobem se Foucault pokouší ukázat, že na roli pozorování
v novém lékařství se podílelo souhrnné „zprostornění“ nemoci, a nikoli
nadřazení pozorování, jež se podílelo na nové koncepci nemoci. Procesy 104 Foucaultovo umění vidět „zprostornění“ však nejsou stejnou záležitostí jako „teorie závislosti“. Fou-
cault neříká, že lékařství začalo používat nový teoretický slovník, v němž
„záznamy pozorování“ byly „zatížené“. Jde spíše o záležitost vybudování
„prostoru“, v němž je umožněno nikoli pouhé pozorování, ale také teorie. V tomto ohledu je možné říci, že zde Foucault rozšiřuje rozlišení,
které ve svém zkoumání toho, jak se v sobě navzájem odráží „dějiny teorií“
a „dějiny pojmů“,28 rozvinul Georges Canguilhem. Dějiny pojmů, skrze
které se nám věci dávají, aby mohly být viděny, jsou oddělené od dějin
teorií, jichž se týkají. Foucaulta v dějinách obzvláště zajímalo, jak mohly
pojmy viditelného zakořenit v institucionálních praxích nebo v tom, čemu
říká „terciální zprostornění“. 29 FOUCAULT, Dohlížet a trestat, s. 263.
30 Ibid, s. 262. 30 Ibid, s. 262. Vidění ve vědění V Dohlížet a trestat se tak pouští do vysvět-
lení, že jednou ze základních podmínek epistemologického odblokování
medicíny na konci 18. století bylo organizování nemocnice jako
„zkoušejícího“ aparátu.29 Neboť skrze tento „aparát“ pak „konstruuje nad lidmi viditelnost, na
jejímž základě jsou rozdělováni a trestáni.“ 30 Prostorové „schéma“ formy vědění není pouze odlišné od teorií, které
se v něm objevují; často je předchází a umožňuje. Potom jednotlivé způ-
soby, v nichž nemocnice obecně šílence zviditelňovaly, předchází rozvinutí
klasické teorie šílenství a architektonická reorganizace vězení předchází
novou teorii zločinu. Vztah mezi teorií a viditelností ve vědění není vázán
nebo dán jako kantovská idea „schematismu“ uzamčeného v zákoutích
lidské duše. Je spíše záležitostí podmíněného dějinného uspořádání. 105 John Rajchman Když se Foucault táže po tom, jak byly entity jako „šílenství“, „ne-
moc“ nebo „zločin“ zviditelněny ve vědění různých období, zaměří se na
ucelenější praktiky „zprostornění“, praktiky, jež byly hlouběji zakořeněné
ve vnějších procesech než na pouhé zkoumání prostým okem a pomoc
teoretického slovníku. Ve své archeologii vidění ve vědění „odkryl“ to, jak
bylo pozorování okem na takové praxe zaměřeno, a to takovým způsobem,
který jednoduše nepocházel z teorie. Oživil tak starou fi losofi ckou debatu o vidění a skutečnosti. Vyplývá
potom z faktu, že když je nemoc „zprostorněna“, není „skutečná“? Vy-
plývá dále z toho, že když lékař vidí pacienta, nevidí cosi skutečného, ale
jenom přelud diskursu své doby? Jedním zdrojem takovýchto otázek je
dávná myšlenka, že skutečné je to, co je pozorovatelné. Ian Hacking se ujme právě této myšlenky, zda abstraktní či teoretické
entity jsou nebo nejsou ve fi losofi i a přírodních vědách skutečné.31 Hacking
prohlašuje, že právě posedlost „pozorováním“ ve fi losofi i a přírodních
vědách zastřela rozpoznání role složitých pokusných aparátů v tom, co
můžeme nazývat „vizualizací“ teoretických entit ve fyzice či genetice. Pří-
rodní věda má také své „mody zprostornění“, celou přírodní „technologii
viditelného“ – stejně jako tu lidskou: observatoře, mikroskopy, cyklotrony. A provádění pokusů je zde nejdůležitější. Pro Hackinga je „pozorování“ zavádějícím dědictvím logického pozi-
tivismu. Ve zřejmé narážce na Foucaulta říká, že fenomenologie i poziti-
vismus pocházejí ze „změny ve vidění“, která se objevuje okolo roku 1800. Poté byla vytvořena linie mezi tím, co je pozorovatelné a co je skutečné. V Hackingově příkladu se tedy jak fenomenologové, tak pozitivisté shod-
nou, že masové koule jsou skutečné, zatímco molekuly nikoli. 31 Ian HACKING, Representing and Intervening. Cambridge, England: Cambridge
University Press 1989. 32 FOUCAULT, Archeologie vědění, s. 76. Vidění ve vědění Hacking zvlášť uvádí, že velký zájem pozitivistů o pozorování vedl ke
dvěma fi losofi ckým tématům, jež společně zastřela roli pokusů v přírod-
ních vědách: myšlence sémantického zdvihu Willarda Quinea, myšlence 106 Foucaultovo umění vidět Normana Hansona o teorií zatíženém pozorování. Společně vedly k ome-
zené představě, že ve fyzikálních vědách je vidění říkáním. V podstatě
jsou však verbální „záznamy pozorování“, které prověřují teorie, ve fyzice
docela řídké. Mnohem více záleží na konstruování, jež odvozuje či vytváří
entity ve značně umělých podmínkách. Vztah mezi experimentálním kon-
struováním a teorií je složitý a proměnlivý. Je záležitostí dějin, není dán
v „logice“ teorie odvozované z percepce. Pro pochopení experimentu je
třeba chápat otázku, co činí moderní vědu moderní, neboť linie mezi teorií
a experimentem je součástí širších dějin linie mezi technologií a vědou,
která pomohla určit samotný smysl, který přisuzujeme „technologii“.i Hacking se obrací pochvalně k Francisi Baconovi, fi losofovi expe-
rimentu. Baconova myšlenka nikoli pozorování, ale „prerogativních in-
stancí“ nám dává lepší představu než Carnapova, tedy s ohledem na to,
jak jsou teoretické entity zviditelňovány. Hacking proto navrhuje nahradit
experimentální realismus realismem pozorovacím. Sám je přesvědčen, že
pokud je možné rozptýlit elektrony, pak jsou skutečné – právě tak skutečné
jako masové koule. Myslí si, že jde o hansonovsko-quineovskou myšlenku,
že vidění je mluvení. Ta vyvolala fi losofi cké pochyby o tom, jestli existují
nebo neexistují skutečné entity vně vědeckých formulací. Provádění po-
kusů je nápravou – nabízí lepší způsob chápání smyslu, v němž se o teore-
tických entitách může říci, že jsou skutečné. Přesto Foucaultova archeologie „zprostornění“ šílenství nebo nemoci,
i když zpochybňuje prostý pozorovací realismus, paralelně nevede k tako-
vému „pragmatickému realismu“, který říká: můžete-li vyléčit pacienta,
pak nemoc, kterou jste u něho viděl, je skutečná. Právě naopak, vede
k určitému druhu nominalismu – v Archeologii vědění Foucault například
mluví o „znepřítomnění“ samotných věcí, o nichž v „archeologii“ píše.32 Naše vědění chápe věci tak, že s nemocí či šílenstvím můžeme „za-
cházet“ stejně jako s elektrony nebo geny. Ale vidění a jednání spolu ne- 107 John Rajchman souvisejí stejným způsobem. Konstrukce vidění nemoci či šílenství a to,
jak vidění odpovídá institucím a je ve vztahu k ostatním oblastem, nikdy
neztrácí kontakt s tím, jak jsou viděny „skutečné“ společenské problémy. Vidění ve vědění V jednom významu je Foucaultova otázka týkající se toho, jak „vidíme“
psychotiky odlišným druhem otázky od té, která se týká našeho zacházení
s atomy a geny – dokonce i když by se naše nejlepší teorie o psychózách
nakonec měla ukázat jako otázka genetiky. Neptá se totiž na to, co dělat
s psychózami, jež nám naše vědění dovoluje vidět, nýbrž jestli můžeme
či chceme odmítnout „evidence“ tak, jak se naskýtají pohledu v celém
rozsahu praxe, a vymyslet další způsoby vidění/zacházení s nimi. Právě
k tomuto druhu vzájemných spojení mezi viděním, jednáním a praktic-
kou samozřejmostí se obrací v knize Dohlížet a trestat. Rozkresluje zde
rozdíl mezi disciplinárními a baconovskými způsoby vidění. Ústředním tématem je „normalizace“. „Normalita“ jako základní
kategorie našeho chování – a dokonce naší identity – se stává „viditelnou“
skrze rozšiřující se síť praktik v 19. století. Jednou ze základních věcí, již
naše vědění zviditelňuje, jsou anomálie jak osob, tak společností. Jedním zdrojem je právě lékařství, tedy změna v tom, co znamená
být „viděn“ lékařem. Kromě konstatování nedostatku zdraví v celém těle
neexistoval jiný nezávislý způsob, jak určit nemoc. „Normalita“ by mohla
být defi nována jako absence patologických symptomů v orgánech. Ab-
normalita začala být vztahována k úpadku. To vše bylo součástí změny
lékařského pohledu: když lékař „viděl“ pacienta, nezačal se ptát na to, „co
s vámi je“, ale „kde to bolí“. Tato nová racionalita normálního však začala být používána i na
jiných místech – na příklad v Durkheimově pokusu odlišit „normální“
stavy společnosti od „patologických“ nebo přesně určit „degenerativní“
podíl populace. Umění vidět „abnormalitu“ zapadá do sítě praxe. A právě
organizace této sítě byla vcelku odlišná od té, jež nám dovoluje rozptylovat
elektrony. 108 Foucaultovo umění vidět V Dohlížet a trestat vstupuje do Foucaultova metodologického slovníku
slovo „technologie“. Dohled zahrnuje novou „technologii viditelného“. Líčí
zde srovnání s pokusnými přístroji a s Baconem, fi losofem experimentu. Jakmile teleskopy, mikroskopy a hranoly pomohly transformovat nikoli
pouze to, co fyzika dokázala vidět, ale samotné místo „vidění“ v ní, pak
techniky dozoru a pokusu (jakýsi „mikroskop chování“) učinily nejen
takové věci, jež byly „abnormální“ či „zločinné“ povahy, viditelnými,
nýbrž také pomohly změnit místo viditelného ve vědění a moci. Tyto „po-
zorovatelny lidské mnohosti“, píše Foucault, zavedly „temné umění světla
a vidění“, jež „potají připravovalo nové vědění o člověku“.33 A přesto tu je základní rozdíl mezi dvěma typy vizuálních technologií
nebo místa vizuální „techniky“ ve vědění a moci. „Jiná moc, jiné vědění,“
říká Foucault. 33 FOUCAULT, Dohlížet a trestat, s. 245.
34 Ibid., s. 313.
35 Ibid., s. 313. 33 FOUCAULT, Dohlížet a trestat, s. 245.
34 Ibid s 313 35 Ibid., s. 313. 34 Ibid., s. 313. Vidění ve vědění Poté odkazuje k Baconovi: Na prahu klasické doby se Bacon, muž zákona a státník, pokusil
vypracovat pro empirické vědy metodologii. Který Velký Pozoro-
vatel vypracuje metodu zkoumání pro vědy o člověku?34 Ale vzápětí dodává: Ale vzápětí dodává: Ledaže by právě taková věc nebyla možná. Neboť je-li pravda, že se
vyšetřování, když se stalo technikou empirických věd, odpoutalo
od inkviziční procedur, v níž mělo své historické kořeny, pokud
jde o zkoušku, ta zůstala v těsné blízkosti disciplinární moci, jež ji
formovala. Je vždy a stále niternou součástí disciplín.35 Baconovo pojetí pokusu může mít kořeny v procedurách inkvizice –
dávat přírodu na skřipec, aby tak bylo možné dobýt její tajemství. Ale ona
technika byla již dávno předtím uvolněna z určitých problémů, se kterými
se inkvizice měla potýkat. V případě disciplíny a jejích technik vidění, 109 John Rajchman jejího „umění světla a viditelného“, naopak vidíme proces, skrze který se
zmnožovaly a komplikovaly souvislosti s problémy, kterými se měla zabý-
vat. Rozprostřely se v řadě institucí, kde si udržely racionalitu „technické
matrice“. Obtíže, jež vznikly se zavedením „těžké“ techniky, jako je například
parní motor, elektrárna nebo televize, vyvolaly „problematizaci“ nikoli
v „evidenci“ vidění páry, elektřiny nebo elektronů. Naopak, což Foucault
shledal důležitým v protestu proti psychiatrii či ve vězeňských revoltách,
byla to záležitost toho, jak byla zpochybňována samotná „technická mat-
rice“ disciplín, jež učinily šílence nebo zločince viditelnými, a jak protesty
odkryly samotnou „evidenci“, skrze niž jsou tyto praxe přijímány. „Filosofi cký“ problém „vidění elektronů“ tedy není tématem důležité
otázky, co s nimi dělat, zda válku či energii. Problém „vidění psychóz“
je zahrnut ve zpochybňování toho, co bychom s nimi měli dělat. Vidění
a jednání jsou ve vztahu různými způsoby. Jiné vědění, jiná moc. Proto
je možné, aby byl fi losofi cký postoj k jednomu postojem realistickým
a k druhému nominalistickým. Realisté pokusů a nominalisté disciplín se
mohou shodnout, že vidění je ve vědění spletitější záležitostí, než když je
vyvozované z vnímání. Neboť rozdíl spočívá ve způsobu, jakým je vědění
„zviditelňováno“ či „zprostorňováno“. 36 FOUCAULT, „Th e Eye of Power,“ s. 149–150. 37 HOY (ed.), Foucault Reader, s. 86. 36 FOUCAULT, „Th e Eye of Power,“ s. 149–150.
37 HOY (ed.), Foucault Reader, s. 86. Prostory vytvořené viditelnosti „Prostor“ je ve Foucaultových dějinách a v jeho myšlení ustavičným
tématem. Jak již bylo naznačeno, hraje význačnou roli v jeho práci
o lékařství, poté se toto téma objevuje v různém pojetí a je zobecněno
v práci o trestních praktikách. „Prostory“, které nazýváme „teritorii“
států se taktéž staly ústředním tématem v jeho práci o „policejní vědě“, jež
pomohla zavést nový druh správní racionality a „geo-politické“ orientace
a organizace války a diplomacie. 110 Foucaultovo umění vidět V dějinné práci o prostoru zaujala Foucaulta díla takových sociálních
historiků, jakými jsou Bloch, Braudel a Ariès. Myslel si, že by jejich práce
mohla sloužit jako náprava jedné tendence ve fi losofi i času u Bergsona,
Heideggera a Sartra – tendence dávat „prostor“ na stranu „prakticky-i-
nertního“, zatímco čas je ponechán velkým otázkám plánování a dějin.36 Značná část Foucaultova pojednání o „prostoru“ je věnována pro-
blému viditelnosti – jak byly prostory navrženy, aby věci zviditelňovaly,
a zviditelňovaly je také konkrétním způsobem. Ve svých dějinách viditel-
ného nemyšleného hraje prostor klíčovou roli. Během rozhovoru věnovanému prostoru Foucault říká: Myslím si, že je poněkud svévolné pokoušet se oddělovat účinné
praktikování svobody lidmi, praxi společenských vztahů a pro-
storové distribuce, v nichž se lidé sami nacházejí. Když se budou
oddělovat, nebude možné jim porozumět. Každý z nich lze chápat
pouze skrze ostatní.37 „Prostorové distribuce, v nichž se [lidé] sami nacházejí“ – stanovují
znovu se vynořující topos ve Foucaultově díle: nemocnice, chudobince,
muzea, veřejné lázně, školy, domovy, blázince a všechny prostory, v nichž
můžeme znovu ustavit racionalitu propracované výstavby toho, co lze
vidět. Jsou to prostory vytvořené viditelnosti. Jsme obklopeni prostory, které napomáhají vytvářet evidence způ-
sobů, jakými vidíme sebe samotné a sebe navzájem. Kde „přebýváme“, jak
jsme zabydleni; pomáhá tímto způsobem určit, kdo a co si myslíme, že
jsme – a tedy zahrnuje naši svobodu. Jsme bytosti, které jsou „zprostor-
něné“ různými způsoby; jsme sami dějinně zprostornění jako subjekty. Foucaultova analýza „prostorů konstruované viditelnosti“ odkrývá,
jak tyto prostory slouží k „vytváření subjektu“, jak slouží k vytváření
zprostornění subjektu nebo jeho „bytí v prostoru“. „Umění světla a vidi- 111 John Rajchman telného“, jež má takovéto prostory rozvinovat, nechává určité druhy našich
vlastností vystupovat jako samozřejmé. Foucault ukazuje, že právě tato linie mezi viditelností a vytvořeným
prostorem umožňuje „technologické“ dějiny umění architektury. Neboť
umění budov je mimo jiné uměním poskytování viditelného, a proto
odhaluje jednu ze svých ústředních propojení s mocí. Prostory vytvořené viditelnosti Architektura po-
máhá „zviditelňovat“ moc jinými způsoby, než pouze tím, že ji projevuje. Nejedná se zde pouze o záležitost toho, co budova ukazuje „symbolicky“
nebo „sémioticky“, ale také toho, co zviditelňuje o nás a uvnitř nás. Zámky a kostely to mohou činit skrze projevování božství, svrchovanosti
a mocnosti. Před vznikem muzea, jak zdůrazňuje Malraux, představovaly
ústřední „imaginární“ prostory, jež upevňovaly kategorie, skrze které bylo
umění zviditelňováno. Ale Foucault v Ledouxově solném mlýně nachází
další vztah mezi mocí, viditelností a vytvořeným prostorem, vztah propo-
jený s novými problémy chudoby a práce, který Bentham později nazval
„panoptismem“. Stavba budov souvisí s novým „uměním světla a viditel-
ného“, nevzhlíží ke slávě mocných či těch, co moc ztělesňují, ale shlíží
k opomíjené anonymní mase, jež jí uniká. Umění či technika vizuálního v „panoptické“ architektuře nejsou
vyčerpány úžasnými podivnými mechanismy, jež zneviditelňují neustálý
dohled nad vězni a toho, co je obklopuje. Panoptické ustavení také vpisuje
do kamene cel nové druhy klasifi kací, jež mají zacházet se svévolným
obyvatelstvem. Jsou vystavené tak, aby ulehčily zavedení „vyšetřovacích“
procedur, které třídí a posuzují lidi podle jejich „viditelných“ charakteris-
tik. Toto zprostornění činí nové klasifi kace narozdíl od těch současných
v botanice – „disciplinárními“. To, co činí osobu klasifi kovatelnou, ji
podřizuje „individualizující“ kontrole. Foucault tudíž říká, že tam, kde
přírodní taxonomie propojuje kategorie a charakter, tam disciplinární
taktiky propojují singulární a mnohé. Věnují pozornost každému jed-
notlivému a všem členům mnohosti jednotlivě. Disciplinární taktiky
vytvářejí kategorie, v nichž je „povaha“ lidí viditelná skrze „štěrbinu“; 112 Foucaultovo umění vidět vytvářejí v lidech „individualitu“, jež je obdařená určitými „nezbytnými“
či zřejmými druhy vlastností. Potom se jednoduše nejedná o to, jestli „oko
moci“ shlíží, nebo spíše vzhlíží. To, co vidí, již nejsou hrdinské činy, ale
dysfunkční osobnosti. Zaměřuje světlo nikoli na ilegální činy, ale na vady
v chování. „Zprostorňuje“ tuto novou věc, „osobnost“ jednotlivce. Benthamovo panoptické schéma je vztaženo k „samozřejmosti“ jeho
ušlechtilé morální zásady racionální vypočitatelnosti dobra v jednotliv-
cích. Proto, aby bylo možné sestavit do tabulky něčí dobro, jednotlivec
musí být „viděn“ určitým způsobem nebo podle určitých kategorií, a to
takových, které by architektura pomáhala zviditelňovat. Umění zpro-
storňování lidských mnohostí se stalo ústředním pro formulaci utilitární
etiky. Ludwig Wittgenstein se pokusil ukázat, že „pohled dovnitř“ či „intro-
spekce“ nebyly ničím více než pravidly řízené umění jazyka. Prostory vytvořené viditelnosti Foucaultova
analýza pravidel, jenž vládnou umění prostoru, ukazuje, že když se díváme
dovnitř, často nevidíme ani tak naše karteziánské mysli, jako spíše rušivé
zdroje našich vad v chování – ale tímto se neméně účastníme praktiky,
která pro nás činí to, co vidíme, samozřejmým. 38 Michel FOUCAULT, Dějiny sexuality. Praha: Herrmann & synové 1999, s. 102. Vidění v moci „Zprostornění“ je tedy jednou technikou uplatňování moci. Proto moc
nemůže být oddělena od „účinné praxe“ naší svobody nebo našich vzájem-
ných vztahů. Máme zde politické dějiny viditelného nemyslitelna: dějiny
toho, jak se moc sama „zviditelňuje“. Zásadou těchto dějin je „viditelnost“
jako jedna z velkých samozřejmostí“ chodu moci. Moc se stává přijatelnou
či tolerovatelnou skrze své zprostorňování nebo způsob, jakým se dává
pohledu. V Dějinách sexuality pak Foucault říká, že „[...] moc je totiž tolerova-
telná jen za podmínky, že významnou část sebe samé maskuje [...] byla 113 John Rajchman by přijatelná moc, jež by byla naprosto cynická?“38 Jedním ze způsobů,
jakým se maskuje, je, jak je lidem „zviditelňována“. Moc se skrývá tím, jak
se zviditelňuje. Její chod se stává přijatelným, protože člověk vidí jen to, co
mu dovoluje moc vidět, jenom to, co zviditelňuje. Jsme okouzleni honosností, „okázalými znaky“ moci. To přispívá
k „samozřejmosti“ naší představy, že ji máme, že se jí zmocňujeme, za-
tímco je anonymně vykonávána. Jeden z důvodů, proč nechápeme disci-
plínu jako formu moci, je to, že nevidíme, jak nás zviditelňuje. Dozorce
ve věži neovládá nebo neztělesňuje (nevidí) moc, kterou provádí. Druh
„viditelnosti“, kterou disciplíny zavádí, jednoduše neodkrývá jen jak fun-
gují, nýbrž čím jsou. „Nevidíme“ disciplínu jako moc, protože „nevidíme“
moc jako strategii. Konkrétně nám techniky vidění moci jako svrchovanosti, urozenosti
a práva zabránily, abychom je viděli jako anonymní techniky. V Dějinách
sexuality Foucault ukazuje, jak tato záležitost může být aplikována na
analýzu fašismu. Cosi jako neoklasické průčelí architektury panoptického
vězení odhalilo strategické vystavení vnitřní viditelnosti, takže velké fašis-
tické „předmoderní“ rituály okázalé svrchovanosti, práva a krve odhalily
samotný „moderní“ způsob, v němž její moc působila, tedy pomáhala je
činit tolerovatelnými. 39 Ibid., s. 84. 40 Ibid, s. 66–67.
41 Citováno z Jacques LAGRANGE, „Versions de la psychanalyse dans le texte de
Foucault.“ Psychanalyse à l’université, roč. 1987, č. duben, s. 263. In „Th e Ethics of
Care for the Self as a Practice of Freedom.“ (Philosophy and Social Criticism, roč. Vidění skrze touhu V Dějinách sexuality Foucault předkládá další téma: dějiny takového
druhu slasti, kterou nacházíme v tom, co vidíme. Myslel si, že se to tak
děje proto, že si ji představujeme jako součást naší „sexuality“, kterou jsme
fascinováni, že ji chceme odkrýt (nebo projevit), že jde o fascinaci a projev
propojený s tím, že o tom víme, nebo s takovou pravdou, která nám může
něco sdělit o nás samotných: „Vynalezli jsme,“ prohlašuje Foucault, 114 Foucaultovo umění vidět slast z pravdy o slasti, slast z vědění o slasti, z jejího vystavování
na odiv, z jejího odkrývání, z fascinace pohledem na ni, z mluvení
o ní.39 Nebylo to tak však vždy. To, co bylo považováno za nejskvělejší či nej-
problematičtější zkušenost v naší sexualitě, nebylo vždy záležitostí, která
nás fascinovala a kterou musíme odkrývat – „prostory“ a „techniky“, jež
ji tímto způsobem umožňují vidět, pro nás nebyly pokaždé takovými. Ne-
byli jsme odjakživa fascinováni naší sexuální touhou, onou nebezpečnou
věcí, na niž měl Freud jako první odvahu se podívat přímo. Foucault chtěl vymezit, jak se „samozřejmost“ této sexuality, kterou
musíme odkrývat a vidět, vynořila v našem vědění, našich ujednáních
a praktikách. Jedno místo, kterým se zabýval, lze nazvat „voyeurstvím“,
sexuální úchylkou v lékařství devatenáctého století: jeho zvláštnostmi,
chlípným zaujetím svým objektem – „fascinací“, která není strukturálně
příliš odlišná od „pornografi ckého“ hledání „tajného života“. Foucault
tento zájem chápe jako vlastnost dějin lékařského diskursu a praxe, nikoli
jako manýru jednotlivých lékařů. Ale jednalo se také o vlastnost „pro-
storů“, v nichž působili, o skutečnost lékařského umění, jež činilo sexualitu
viditelnou, o podněcování k vidění a ukazování její nebezpečné pravdy. Jedním příkladem je La Salpêtrière Jean-Marie Charcota, na němž
Freud učinil své „objevy“ nevědomí. Nejednalo se pouze o prostor vytvo-
řené viditelnosti, ale také o prostor podněcování k vidění. 40 Ibid, s. 66–67. Vidění skrze touhu „Byl to nesmírný
aparát k pozorování spolu se zkoušením, vyšetřováním a experimenty.“
Ale byla to také mašinérie podněcování se svými veřejnými demonstracemi,
s divadlem rituálních krizí pečlivě připravených éterem či amyl-
nitritem, s hrou dialogů, palpací, vkládání rukou a póz, jimiž
lékaři gestem nebo slovem něco vykouzlí či začarují, s osobní mašinérie podněcování se svými veřejnými demonstracemi,
s divadlem rituálních krizí pečlivě připravených éterem či amyl-
nitritem, s hrou dialogů, palpací, vkládání rukou a póz, jimiž
lékaři gestem nebo slovem něco vykouzlí či začarují, s osobní 115 John Rajchman hierarchií, která číhá, organizuje, provokuje, dělá si poznámky,
referuje a shromažďuje nesmírnou pyramidu pozorování a cho-
robopisů.40 A přesto odmítala pojmenovat to, co podněcovala vidět: sexualitu. O Charcotovi se říkalo – a sám si tak říkal – že byl un grand visuel. V nekrologu Freud o Charcotovi říká, že byl umělecky nadaným vidou-
cím, že chaos symptomů byl ustaven řádem oka jeho duše, že v oddělení
pro nemocné neustále hovořil o významu a obtížích vidění, v čemž, jak
říkal, nacházel největší uspokojení. Ale jak Freud dodává, přestože byl vi-
doucím, visuel, nebyl myslitelem či „hluboce přemýšlejícím“. To, co viděl,
nedokázal teoreticky pojmenovat. V tomto nekrologu lze vidět počátky
Freudova pozdějšího zájmu o „pozorování“ procesů nevědomé touhy. Charcot vynalezl odstupňované diagnostické schéma pro různé
„druhy“ le grande hystérie, vystavil ho podle scénografi ckých tabulek,
které zinscenoval. Tato typologie mu umožnila spojit hysterii s magií tak,
jak byla vyobrazena v malbách. Ale vysvětlující část jeho teorie spojovala
tělesné „pózy“ hysteriků pouze s tajemnými „orgánovými poškozeními“
mozkové kůry – sexualita nebyla kauzálním faktorem. Tímto způsobem překonal nejasnosti symptomů, jež hysterii přiřadily
k takovým mentálním poruchám, jejichž symptomy byly formou disi-
milace či lhaní. Ale inscenování hysterických postojů otevřelo možnost
jakéhosi protipohybu na straně hysteriků: možnost rozrušení jasného pro-
storu viditelného tím, že zavedla sexuální tělo. Takto se sexualita dostala
do obrazu. „Věřím,“ říká v roce 1974 Foucault na Collège de France, „že
to byla válka hysterie. [...] Hysterie byla řadou fenoménů zápasu, který
se odvíjel kolem tohoto nového lékařského aparát, tím byla neurologická
klinika.“41 116 Foucaultovo umění vidět S ohledem na své sexuální tělo byl Charcot nucen odvracet svůj „ob-
divný upřený pohled“. Babinski se vzdal divadelnosti nemoci a pro vysvět-
lení vymyslel „pithiatismus“ či schopnost dramatizovat se. Ale Freudovo
řešení spočívá v tom, že to teoreticky nazval tím, co prostor zviditelňoval. Původ hysterie, řekl, spočívá ve vztahu ženy k její sexualitě. Vidění skrze touhu A s tématem
sexuality začal Freud spojovat projev hysterika a to, co říkal, v novém typu
prostoru: v prostoru psychoanalýzy. Foucault doufal, že tímto druhem analýzy objasní jednu vlastnost, jež
odlišovala fascinaci od vystavování sexuality v naší společnosti. Vlastnost,
o níž se Foucault domníval, že ji zásadně rozšířil Freud– dal jí lékařský
status. V naší zkušenosti sexuality chceme neustále vidět a ukazovat její
abnormálnost, zvrácenost, nemoc. A tato fascinace je nedílnou součástí
slasti, kterou máme z toho, že o tom víme. Jednou technikou, jež byla použita v Charcotových projevech, bylo
nové umění fotografi e. A právě fotografi e hysterických póz byly publiko-
vány surrealisty, kteří, jak je dobře známo, se o vztah vidění a mentálních
poruch obzvláště zajímali. XII, 1987, s. 122), Foucault říká, že hysterie ho zasahuje jako „samotná ilustrace“
zápasu, kdy je člověk utvářen jako šílenec: „Nejedná se o pouhou náhodu, že hys-
terie byla zkoumána právě tam, kde byl jednotlivec donucen, aby se považoval za
šíleného.“ 42 Deleuze říká, že Foucaultova „koncepce viditelného je obrazová, blízká Del-
aunayovi, pro kterého světlo [...] vytvářelo své vlastní formy a své vlastní pohyby.
Delaunay říká: Cézanne rozlomil mísu s ovocem a my bychom ji neměli znova sle-
povat, jako to dělají kubisti.“ (Srv. také Obraz-čas, kde Deleuze říká podobné věci
o ne-expresionistickém použití světla ve fi lmech Rivetta (Obraz-čas. Praha: NFA 2006, s. 18 an.). To ovšem není jediný způsob, jak spojovat Foucaultovo umění
vidět s moderními obrazovými praktikami.
43 Foucault stručně pojednává o Platónově „známé metafoře oka“ in HOY (ed.),
Foucault Reader, str. 367–368. Oko myšlení Co to tedy znamená vidět události v tom, co je v naší mysli nemyšlené? Jedním vizuálním obrazem, který Foucault předkládá, je obklopení
události určitým „mnohostěnem srozumitelnosti“, jehož strany se roz-
prostírají do nekonečna v mnoha směrech.42 Má zmnožovat věci asocio- 117 John Rajchman vané se svojí „srozumitelností“ a způsoby, jakými jsou asociovány. Čím
větší a příznačnější vnitřní pojmová analýza události je, tím rozsáhlejší
jsou vnější procesy, se kterými je jejich „neviditelná“ či „evidentní“ sro-
zumitelnost spojena. Pak tedy Foucault ve své analýze události vězeňské
formy shledává srozumitelnost, která událost spojuje s pedagogickými
praktikami, profesionálními armádami, britským empirismem, novou
dělbou práce a vynálezem střelného prachu, a to skrze přenos technických
modelů do jiných oblastí, nové využití teorií nebo nové strategie, jež se
zabývají místními problémy. Foucault tedy začíná myšlenkou, že taková věc jako „esence“, jako
ono vizuální, nemusí vůbec být cosi, co může být popisováno jako „fe-
nomenologie percepce“ nebo „teorie upřeného pohledu“, cosi, proti čemu
by se Foucault podle Martina Jaye mohl postavit. Dějiny nám spíše uka-
zují mnohé odlišné singulární druhy viditelné srozumitelnosti, způsoby
vidění a zviditelňování, jednotu, kterou nelze najít v pohledu pouhým
okem, v empirickém či trancendentálním nebo „imaginárním řádu“. Tady
a tehdy, kdy „viditelné“ nabývá status esence, universality a nutnosti, musí
oko fi losofa hledat singulární a kontingentní procesy, které to činí samo-
zřejmým, tedy přijatelným.ii Podobně jako ve fi losofi ckém úkolu vystiženém známou Wittgenstei-
novou poučkou „nemysli, dívej se!“ není oko fi losofa, tedy oko myšlenky,
ani kontemplativní, ani introspektivní. Nevzhlíží, aby se dívalo na formy,
které zapomnělo, ani se nedívá dovnitř, aby vidělo bod, ze kterého by mělo
jednání vycházet, není sebejistotou, ze které by se mělo získávat vědění.43
Dívá se ven na ty události v mysli, skrze které se věci dávají vidění. Vyhlíží,
aby změnilo svůj způsob vidění. V roce 1981 potom Foucault tvrdil: 118 Foucaultovo umění vidět Vždy, když jsem se pokusil o teoretickou práci, bylo to na základě
prvků z mé zkušenosti – ve vztahu, v jakém jsem viděl, že zaujímají
místo kolem mě. Vlastně jsem se domníval, že jsem rozpoznal
jakousi prasklinu – netečně dráždivou či dysfunkční – ve věcech,
jež jsem viděl, v institucích, jimiž jsem se zabýval, v mém vztahu
k ostatním. 44 „Est-il donc important de penser?“ Libération, 30.–31. květen, 1981. Oko myšlení Pak jsem se ujal určité práce, mnohých fragmentů
autobiografi e.44 Když fi losof kolem sebe „vidí“ něco problematického nebo dysfunkč-
ního, neobrací své oko k ideálům, v jejichž světle se problémy jeví jako
nedokonalé či jako „protisituace“. Neobrací ho dovnitř, aby viděl pravdu
či autentické já, v jehož světle se problémy zobrazují jako zkreslené či jako
mystifi kace. Jeho vidění se pouští do teoretické práce, která analýzou toho,
jak problém vznikl a byl formulován, přetváří způsob, jakým problémy
vidí – a tedy jeho způsob žití. Ethos, krása, nebezpečí „Fragmenty autobiografi e“ – Foucaultovo umění vidět je také fi loso-
fi ckým uměním žít. Jeho teoretické dílo je „autobiografi cké“ nejen proto,
že je způsobem transkripce jeho zážitků, ale proto, že vytváří praktiky,
kterými se mají pravidelně zpochybňovat dané koncepce jeho zkušenosti,
tedy hledat nové. Autobiografi e v tomto smyslu není pokusem poskytnout
představu nebo obraz toho, čím kdo byl nebo jak by kdo měl být viděn,
ale vytvořit takové dílo, aby se mohl změnit přeměnou svého způsobu
vidění. Foucaultův způsob psaní se nerozvinuje jako jediná doktrína nebo
teorie věcí. V určitých aspektech měnil názor co do svých cílů, předmětů
a metod. Jako v případě Nietzscheho, Wittgensteina nebo Heideggera je
jeho myšlení přerušováno transformacemi, podle toho, jak pojímal svůj 119 John Rajchman vlastní fi losofi cký úkol. V úvodu ke svazkům, jež byly vydány těsně před
jeho smrtí, zobrazuje tento proces jako praktikování či askezi oproštění
se od sebe ve svém díle (se déprendre de soi-même), a to skrze „zkoušky“,
jimiž se snaží změnit způsob vidění věcí. Ale dodává, že tento proces je
také ironií. Právě takové snahy osvobodit se od sebe činí něčí dílo jeho
vlastním dílem – člověk nachází toho, kým vždy byl, neustálým unikáním
od sebe. Toto úsilí vynaložené na změnu způsobu vidění [façon de voir],
[...] není prosté určité ironie. Vedly skutečně tyto snahy k jinému
myšlení [penser autrement]? Možná, že nám umožnily jen myslet
jinak to, co už jsme mysleli, a spatřit to, co jsme dělali, z jiného
úhlu a v ostřejším světle.45 A ono nové světlo, v němž Foucault viděl, co dělal ve svém předešlém
díle, bylo světlem „problematizace“: Zdá se mi, že nyní lépe vidím, jak jsem se [...] zapletl do tohoto
podniku dějin pravdy: nikoli analýzy, [...] nýbrž problematizace,
skrze něž se bytí dává jako to, co může a má být myšleno, a prak-
tiky, na jejichž základě se tyto problematizace utvářejí.46 Zdá se mi, že nyní lépe vidím, jak jsem se [...] zapletl do tohoto
podniku dějin pravdy: nikoli analýzy, [...] nýbrž problematizace,
skrze něž se bytí dává jako to, co může a má být myšleno, a prak-
tiky, na jejichž základě se tyto problematizace utvářejí.46 Možná že žádné „osvícené“ řešení našich vztahů k bolesti, nemoci,
zločinu, šílenství a smrti není. 45 FOUCAULT, Užívání slastí, s. 19.
46 Ibid., s. 19. 45 FOUCAULT, Užívání slastí, s. 19.
46
b d 47 Ibid., s. 13.
48 FOUCAULT, Th e Birth of the Clinic, s. xiv. Ethos, krása, nebezpečí A přesto jde o dějinný fakt, že vyvstaly
různé formy srozumitelnosti či racionality v tom, jak lidé skutečně k ta-
kovým věcem přicházeli, a v tom, jak kolem sebe následně vztyčili formy
vědění a jednání, způsoby bytí. Tyto zážitky nebyly vždy viděny jako ty,
z nichž vyvstávají stejné druhy problémů. K analýze jejich dějin je třeba
vidět zvláštní druhy nebezpečí či problémů, jež vedly k evidenci určitého
způsobu jejich uchopení a zacházení s nimi. Potom podle nového způ-
sobu vidění zkoumal ve svém předešlém díle to, jak lidé viděli nebezpečí 120 Foucaultovo umění vidět v šílenství, nemoci či zločinu, jak tato nebezpečí zachycovali a činili je
„viditelnými“ či „zprostorněnými“ ve vědění a jednání. Foucault navrhoval, aby bylo na jeho dějiny šílenství, nemoci či zlo-
činu nahlíženo jako na dějiny, jakými byly ve zkušenosti šílenství, nemoci
či zločinu, a problematické bylo právě to, že obojí tak mohlo a muselo být
myšleno. A tyto problematizace zkušenosti začal asociovat s konkrétními
způsoby žití či bytí, způsoby bytí konkrétního druhu osoby. Pak se tedy
ptal, jaké jsou problematizace a následné praktiky, kdy „se člověk dostává
k myšlení svého vlastního bytí, když se sám pokládá za bytost, která mluví,
žije a pracuje, když sám sebe soudí a trestá jako zločince?“ 47 Ve Zrození kliniky Foucault hledal něco základnějšího než „bezmyš-
lenkovité fenomenologie“ v „setkání“ mezi lékařem a pacientem a „tak-
zvané ‚liberální‘“ pojetí smlouvy či paktu mezi dvěma jednotlivci. Pokusil
se vidět „hlavní událost ve vztahu člověka k sobě samotnému a k jazyku
věcí“.48 Nové „zprostornění“ nemoci v patologiích jednotlivých orgánů by
zásadně změnilo vztahy, jež měli lidé k sobě samotným a mezi sebou, když
„byli nemocní“. Zavedlo by to naprosto nový druh „etické“ srozumitel-
nosti problémů lékaře, pacienta a patologa. Podobně lze číst Foucaultovy dějiny šílenství jako podrobné zkoumání
toho, jak bylo „bytí šíleným“ nahlíženo jako zdroj nebezpečí pro společnost
a jednotlivce. Vyvstal tak nový způsob vidění problému šílenství. Skupina
chovanců ve všeobecné nemocnici se nám může zdát nesourodá. To odpo-
vídá na zcela srozumitelný způsob vidění problému – „sensibilitu“, u níž se
ústředním nebezpečím pro společnost a jednotlivce stala nečinnost. Tento
způsob vidění je částečně odvozen z teologického povyšování nečinnosti
nad marnivost, jež je smrtelným hříchem. Vyvstal také z nové koncepce
práce a chudoby, která se měla stát terčem nového správního druhu raci-
onality. Jednalo se tak o ústřední záležitost v myšlení, skrze které mohla
hysterie nečinné ženy vstoupit do lékařského diskursu. 47 Ibid., s. 13. 49 Ve svém L’Etat Providence (Paris: Grasset 1986) François Ewald tvrdí, že souhry
okolností ustavily nový druh nebezpečí či problému, jež například není znázor-
něn ve výčtu špatností 18. století, jak je tomu u Voltairova Candida. Se strategií,
jež měla být použita jako pojistka rizika náhod, se objevil nový druh „soudní
zkušenosti“ a nová třída práv. 50 HOY (ed.), Foucault Reader, s. 343. Ethos, krása, nebezpečí 121 121 John Rajchman Ale jak ukazují techniky morálních shromáždění v „osvícených“ ústa-
vech pro choromyslné v Tuke a Pinelu, vyvstaly otázky nejen nebezpečí
zvnějšku, nebo ve společenských vztazích, ale také zevnitř nebo ve vzta-
zích k sobě samotným, a tedy k ostatním. Vina, hanba, nezodpovědnost,
slabost nebo vůle jako formy vztahů k sobě samotnému vystupují v růz-
ných prostorech a pod různými koncepcemi – jsou pojímány ve vztahu ke
konkrétním nebezpečím či problémům.49 Nové světlo problematizace tak zavedlo pozornost na etické záležitosti
jeho předešlých dějin. Poskytlo Foucaultovi způsob myšlení o původu
a změnách v samotné koncepci etiky – její základní otázky, jak o nás
měla pravdivě vypovídat, druhy vztahů, jež měla mít k vědění, právu či
politice. Foucault se bezpochyby nesnažil ospravedlnit principy etického jed-
nání, ale zkoumal, jak etické myšlení a praxe viděly a jak reagovaly na
určité druhy problémů či nebezpečí: způsoby, jakými pojímaly překážky,
jež musíme překonat, abychom byli dobří nebo jednali správně; způsoby,
jakými racionalizovaly možnosti zacházení s tím, co bylo nahlíženo jako
špatné, hříšné či zhoubné. Možnost dějin etiky vznikla jako možnost zkoumání zvláštních
druhů nebezpečí či problémů, které měla překonávat. Ale ve zkoumání
dějin bychom neměli hledat „řešení nynějších problémů v řešení jiných
problémů, jež vyvstaly v jinou dobu u jiných lidí“50. Dějiny problematizací
„způsobů bytí“ v etice nejsou nostalgické. „Dějiny,“ říká Foucault, „nás
chrání před historicismem – před historicismem, který vyzývá minulost,
aby odpověděla na otázky přítomnosti.“ 51 122 Foucaultovo umění vidět Foucault říká, že „nebezpečná“ je spíš samotná analýza problematizací. Ve Slovech a věcech již Foucault vyslovil, že když už v moderním myšlení
nebyla „kosmologická“ forma morálního myšlení možná, byla chápana
jako nebezpečná – jako „nebezpečný akt“.52 A když se Foucault zmiňoval
o svém „pesimistickém aktivismu“ v pozdním rozhovoru, prohlásil: Rád bych udělal genealogii problémů, problématiques. Neříkám,
že všechno je špatné, ale že všechno je nebezpečné, což není to
samé jako špatné. Jestliže je všechno nebezpečné, pak vždy máme
co dělat.53 V poslední práci a v poslední koncepci své práce Foucault spojil své
umění vidět s „eticko-politickou volbou“, kterou člověk činí, když „určuje,
co je skutečným nebezpečím“. S volbou, jež spočívá v tom, pokusit se vidět,
co je to, proti čemuž musíme bojovat, abychom se osvobodili (a osvobodili
sebe od nás samotných). A právě tato svoboda je nebezpečná, neboť nikdy
nemůžeme předem znát, jaký je její rozhodující činitel či mít její úplný
obraz. 52 FOUCAULT, Slova a věci, s. 252.
53 HOY (ed.), Foucault Reader, s. 343. 53 HOY (ed.), Foucault Reader, s. 343. 52 FOUCAULT, Slova a věci, s. 252. Ethos, krása, nebezpečí Proto se Foucault jako myslitel a vidoucí zabýval situací, jež předchází
možnosti deduktivního normativního uvažování, kde to, co člověk vidí,
musí být uděláno bez toho, aniž by už věděl co. Prostor nikoli dedukce,
nýbrž tázání se a analýzy se tedy rozevírá mezi volbou, již člověk činí,
a tím, co dělá, v čemž se pokouší pochopit, co je oním nebezpečím, jež
zatím ještě plně nevidí, ale ve vztahu k němuž musí jednat. Jedná se o zod-
povědnost k věci, jež nás znepokojuje, ale již ještě nedokážeme popsat či
pojmenovat, jež vyžaduje naši práci na změně nás samotných. Tato práce
je pokusem změnit náš způsob vidění a žití ve vztahu ke konkrétním ne-
bezpečím, o nichž zatím ještě nevíme, co s nimi dělat. 123 John Rajchman Foucault si myslel, že tento druh vztahu mezi viděním, žitím a jedná-
ním, může být pojímán jako „estetika existence“, což byl opak toho, který
se pokusil rekonstruovat u starověkých etických škol savoir-vivre. Když Foucault rekonstruoval starověké etické myšlení, tedy to, co
bylo v sexu chápáno jako patřičně nebezpečné, aby se mohlo stát objektem
celé praktiky sebepřeměny, nebyly tam ještě hříchy těla či nezvyklé touhy,
které nás fascinují a skrývají se v patogenních zákoutích našich hlav. Jednalo se o nepřiměřenou činnost, která ohrožovala ztrátu sebeovládání
v tom, co mělo vládnout – nebezpečí pro ethos, náležitý modus bytí, pro
svobodného dospělého muže. Ale ethos „občanského člověka“, týkající
se jeho zdraví, domova, dvoření, kolem něhož se točily starověké etické
praxe, byl značně odlišný od ethosu křesťanského „niterného člověka“
a augustinského problému, že veškerá sexuální touha je protknuta Pádem
a je obecným modelem hříchu či „demokratického studu“, jenž spojoval
křesťanskou komunitu s problémem chudoby. Foucault usiloval o analýzu
těchto velkých změn v problémech „bytí sexuálním“ z hlediska etických
praxí, v nichž je po člověku požadováno, aby se měnil podle konkrétní
koncepce zkušenosti, cílů a závazků. V analýze starověkých umění ctnosti hrálo významnou roli vidění
a to, jak je člověk viděn. Jak již bylo mnohokrát poznamenáno, starověká
koncepce „krásy“ – krásy v etickém smyslu – byla obzvlášť vizuální. A pře-
sto takové aktivity jako „ukazování já“ byly „problematizovány“ a chá-
pány dosti odlišným způsobem narozdíl od otázek identity, autentičnosti
a zaujetí, jež Sartre dával do souvislosti s „upřeným pohledem“. Bylo-li
hrdé mužné tělo mistra „živoucím tělem“, bylo prožíváno jiným způsobem
než tím, jenž si Sartre představoval ve svém popisu našich „konkrétních
vztahů“ k nám samotným a mezi námi navzájem. 54 FOUCAULT, Užívání slastí, s. 213. In „Th e Ethics of Care for the Self as a Prac-
tice of Freedom,“ Foucault říká, že „Řekové ve skutečnosti považovali [...] svobodu
jednotlivce za etický problém. Ale etický ve smyslu, jakým mu rozuměli Řekové.
Ethos byl držením těla a způsobem chování. Byl to modus bytí subjektu a určité
způsoby jednání viditelné ostatním. To, jaký měl kdo ethos, bylo viditelné v jeho
oděvu, způsobech, chůzi, vyrovnanosti, se kterou reagoval na události atd. Pro
ně to byl konkrétní výraz svobody. To je způsob, jakým ‚problematizovali‘ svoji
svobodu,“ (s. 117). Ethos, krása, nebezpečí Vidění sebe a náš modus
žití měly v této etice odlišný druh srozumitelnosti. Sebeovládání bylo jako vizualizovaná evidence o způsobilosti člověka
vládnout. Bylo třeba ukázat – a ukázat pravdivě – stav kontroly a ukáz-
něnosti duše a těla jako čehosi ušlechtilého a krásného, aby mohlo být 124 Foucaultovo umění vidět velebeno příštími generacemi. Cíl či telos praxe sebeutváření byla krásná
shoda či harmonie mezi tím, čím kdo je, a tím, co dělá a říká. Krása byla
etickou kategorií – vidění a projevování já byly tedy součástí etho-poe-
tiky, estetiky existence. A linie mezi viditelnou krásou a způsobem bytí se
nacházela a byla vytvářena v určitých „prostorech“, oikos a agora. Oikos
byl „prostor vytvořené viditelnosti“. Rozprava o manželských povinnos-
tech byla rozpravou rozdělení rolí a přirozeností v „prostoru“ oikos, jehož
„strop“ odděloval to, co bylo uvnitř od toho, co bylo vně. Tak v rozpravě
o potěšeních a pravdě vznikl problém líčení a malování: líčení nemůže
zaujímat místo v krásném způsobu, jakým se má paní domu „prosazovat“,
v němž „vzpřímenost a přecházení dodají jejímu tělu onen postoj a způsob
chůze [allure], které v očích Řeků charakterizují postavu [la plastique]
svobodného jedince“.54 V této ideji krásy byla mimésis kategorií vztahu
člověka k sobě samotnému či své „postavě“ a vztahem „postavy“, jak se
pohybovala, k tomu, jak se jevila. Jestliže Hegel v této řecké zkušenosti krásy viděl první „moment“,
k němuž se duch navrací v průběhu dějin, jež procházejí od umění a ná-
boženství ke státu, pro Foucaulta je řecká zkušenost spíše „ztracenou
evidencí“, řešením nebezpečí, jež se nás již netýká. Umění vytváření já
v obrazu aktivní svobody již nadále nezaujímá v našem etickém myšlení
stejné ústřední a samozřejmé místo. Ztratili jsme „etho-poetiku“, která
z existence činila objekt estetiky. Vidíme další nebezpečí a zacházíme
s nimi jinými způsoby. Naše disciplinovaná a „poslušná a užitečná“ těla
nejsou „rozvážnými“ těly starověkého mistra. Benthamovské prostory, jež 125 John Rajchman napomohly dát vypočitatelnosti toho, co je pro nás dobré, její ústřední
etickou důležitost, jsou vlastně odlišné od prostorů, v nichž vyvstala sta-
rověká otázka moudrosti dobrého života. Aby mohla vzniknout moderní „estetika existence“, musel se změnit
samotný koncept krásy žití. Právě tuto změnu Foucault spojoval s Baude-
lairem a moderní zásadou, že „subjekt není dán“. Pro nás není nebezpečím
to, že se nám nepodaří stát se tím, čím se máme stát, nýbrž to, že můžeme
být pouze tím, čím sami sebe dokážeme vidět. 57 „Th e absence of the work of art“ u Richarda Howarda je překladem l’absence
de l’oeuvre a nikoli l’absence d’oeuvre. Alan Sheridan si myslí, že tento termín
znamená „neproduktivní nečinnost vně lidských úspěchů“ (viz Alan SHERIDAN,
Foucault: Th e Will to Truth. London: Tavistock 1980, s. 15). Ethos, krása, nebezpečí Ve starověkém myšlení byla
svoboda cosi krásného, viditelného jak v duši, tak v těle. A ztráta svobody
tak mohla být nahlížena jako cosi ošklivého. Ale když „není dán subjekt“,
naše svoboda přestává mít obraz. Je to cosi, co nikdy nedokážeme dopředu
vidět. Její krása spočívá v nebezpečí. Pro nás již krása nespočívá v dokona-
losti žité harmonie mezi námi samotnými a „prostory“, v nichž se můžeme
stát tím, co je pro nás přirozené, dané a možné, nýbrž v tom, co v nás zatím
nelze vidět či být pojmenováno v prostorech, jež obýváme. Krása žití spočívá v nesouladu či disharmonii mezi naší danou přiro-
zeností a možnostmi existence, mezi naší identitou a tím, co člověk vidí
v sobě a v procesech, jež se kolem něho dějí. Nebezpečí krásy tak dává
vystoupit oeuvre de soi, v němž vidění toho, co má člověk dělat, zahrnuje
změnu jeho způsobu vidění, façon de voir. Člověk se mění, když vidí, co
je v jeho existenci nebezpečným, a vidí, co je nebezpečné, tím, jak mění
sám sebe. Foucault se tak dostal k chápání umění vidět, jež praktikoval jako
„estetiku existence“, jako umění žít. V tomto umění pak události, jež člo-
věk kolem sebe vidí, narušují chápání sebe samotného a zapříčiňují to,
že o nich člověk přemýšlí a přehodnocuje je. Foucaultovo praktikování
vidění a myšlení by tedy bylo praktikováním moderního ethosu svobody
– svobody, pro niž zatím ještě nemáme obraz, a ethosu, v němž krása spo-
čívá v postupném vidění skutečných nebezpečí, jimž musíme čelit. 126 Foucaultovo umění vidět 56 Michel FOUCAULT, Madness and Civilization. New York: Vintage Books 1973
s. 229 a n.h 55 HOY (ed.), Foucault Reader, s. 42. 55 HOY (ed.), Foucault Reader, s. 42.
56 Michel FOUCAULT, Madness and Civilization. New York: Vintage Books 1973,
s. 229 a n.
57 „Th e absence of the work of art“ u Richarda Howarda je překladem l’absence
de l’oeuvre a nikoli l’absence d’oeuvre. Alan Sheridan si myslí, že tento termín
znamená „neproduktivní nečinnost vně lidských úspěchů“ (viz Alan SHERIDAN,
Foucault: Th e Will to Truth. London: Tavistock 1980, s. 15). 58 Deleuze má sklon spárovat „diskursivitu“ se slovy a pojmy a „viditelnost“
s věcmi a intuicí či vnímavostí. Ale podle jiného čtení Foucaultova způsobu psaní
lze „mluvit“ o věcech s obrazy a prostorem, stejně jako lze „ukázat“ věci ve slovech
a větách. Jazyk je jedním způsobem „zprostornění“ či „zviditelnění“ a máme
„evidence“ jak diskursů, tak „vnímavostí“. Prostory či obrazy mohou naopak vy-
tvářet výpovědi. Potom mohou být například písmena na klávesnici psacího stroje
považována za énoncé, podobně jako když v koloniálních dobách byla Británie na
mapách uprostřed. Dost možná vztah mezi způsoby vidění a způsoby mluvení
ve Foucaultovi spočívá v dějinách mlčenlivého myšlení, jež je utváří a nesnadno
odpovídá tradičním rozdílům mezi pojmem a intuicí nebo slovem a věcí.
59 Gilles DELEUZE, L’image-Temps. Paris: Editions Minuit 1985, s. 365. Vidění vnějšku Tento pokus o vystoupení ze sebe v „eseji“, jejž člověk píše, není tak odlišný
od „postoje modernity“, jejž Foucault nachází v Baudelairovi. V průběhu
pozdější rozpravy o Kantově práci o osvícenství říká, že Baudelairovo
„zkoušení na sobě“ jako spisovateli by se ukázalo v „jiném, odlišném
prostoru“, mimo sféru společnosti a politiky – v prostoru, jejž Baudelaire
nazýval „uměním“.55 Jak je potom Foucaultovo „umění vidět“ vztaženo ke
způsobu, jakým takovéto „umění“ viděl? A co konkrétně má toto „umění
vidět“ do činění s koncepcí „moderního“ umění a literatury, již sám pro-
sazoval v řadě esejů v 60. letech, ale od nichž se později chtěl distancovat? Při Deleuzově čtení je důležité vystavět kontinuitu skrze Foucaultovo dílo,
jež je založeno na jeho rané koncepci díla či oeuvre. Na posledních stránkách Šílenství a civilizace lze již v rozpravě o ab-
sence d’oeuvre najít zárodky této otázky, postromantickou ideu s kořeny
u Sada a Hölderlina, v níž je vztah k šílenství spojen s dílem nikoli jako
s expresivním obsahem, nýbrž jako s nevyslovitelným či nepopsatelným
zdrojem, z něhož se dílo objevuje a do něhož znovu mizí.56 „Absence d’o-
euvre“ neznamená, že dílo neexistuje nebo že je člověk vně díla, jak občas
navrhují anglické překlady.57 Spíše se člověk skrze dílo pokouší říct cosi
nevyslovitelného nebo vidět cosi, co je zatím neviditelné, a tak rozevírá
prostor jakéhosi rytmického „zmizení“ sebe v díle a skrze své dílo. „Bytí
jazyka“ (na rozdíl od jeho regulovaného používání v „diskursu“) nabízí
příležitost a podmínku tohoto moderního „postoje“ k sobě ve svém díle. V 60. letech Foucault tvrdil, že šlo o koncepci, jež byla základní v dílech
takových spisovatelů, jakými jsou Klossowski, Bataille a Blanchot. 127 John Rajchman Rozprava v anglickém jazyce toto téma ve Foucaultově díle opomíjela. A přesto ho Derrida ve své kritické práci o Foucaultovi obdivoval. Také
Deleuze se ho jiným způsobem dovolává svojí představou Foucaulta jako
vidoucího. Následně vidí Foucaulta jako toho, kdo uvedl praktikování
psaní jako désoeuvrement na pole dějin, politiky a epistemologie. Dele-
uze říká, že „bytí jazyka“, jež je podmínkou a příčinou literatury, je také
podmínkou a příčinou Foucaultovy archeologie diskursu. A říká také, že
otevření viditelnosti či „bytí světla“, jež je možností vizuálního umění, je
také podmínkou Foucaultova umění vidět.58 To je jedním z důvodů, proč Deleuze může říct, že Foucault má „vý-
jimečně blízko k fi lmu.“ Neboť ústředním pojmem v Deleuzově vlastních
analýzách role myšlení ve fi lmu je pojem désoeuvrement. Vidění vnějšku V odpovědi na
Godardovu předpověď konce fi lmové teorie říká, že „kinematografi cké
pojmy nejsou dány ve fi lmu.“59 Máme jistý druh „fi lmového nepředvída-
telna“, z něhož se fi lm neustále pokouší osvobodit, a tak se otevírá jiným
způsobům myšlení a hraní. Myšlení je ve fi lmu tout ouvert, konceptuálním
souborem otevřeným přeměnám. Jako takový může být analyzován jako
velké umění, jež určitým způsobem pojímá světlo, pohyb, čas a prostor
a vytváří pojmy vizuálna „prostorů“, skrze něž jsme vystavováni vidění. Deleuze napadá pokus Christiana Metze, jež činí příběh ústředním pro-
blémem, kolem něhož se má fi lmové myšlení točit, a následný výběr, jenž
člověk musí činit mezi dobrým shrnutím či teoretickým fi lmem a špatným 128 Foucaultovo umění vidět komerčním, ideologickým a výpravným fi lmem. Vizualita, již činí fi lm
srozumitelnou, se netýká fyzického média, jejž má teorie očišťovat ode
všech récit – jde o mlčenlivou konceptuální organizaci, jež spojuje fi lm se
způsobem, jakým jsou prostor, subjektivita a čas fi losofi cky chápány. A přesto po roce 1968, po jeho „politickém“ převratu, je ve Foucaul-
tově díle slyšet mnohem méně o „bytí jazyka“ (nebo světla) a postavy
Nietzscheho, Hölderlina a Bataille přestávají pronásledovat okraje jeho
institucionálních dějin. Jak již říkám ve své knize, Foucault na Sadeho
a Bataille změnil názor. A v posmrtně publikovaném rozhovoru z roku
1975 to Foucault říká sám.60 Tvrdí, že v esejích o takových postavách,
jakými byli Blanchot a Bataille v 60. letech, pro něj nebyla důležitá idea
literatury samotné. Odkaz k takovým postavám v jeho dějinách, jak
říká, byl záležitostí prostého constat, jako by si toho náhodně všiml při
procházce. U Blanchota pro něj byl důležitý pokus vymanit se z jistého
„hegelianismu“, jenž připisoval literatuře privilegovanou expresivní roli
v dějinách, a místo toho se ptát po singulárním místě, v němž by společ-
nost byla ve shodě se psaním, jež nazývá literárním. Blanchot se určitým
způsobem vymykal stylu fi losofi e, skýtal totiž možnost jiného způsobu
myšlení, v němž se člověk pokouší neustále vidět mimo hranice vidění
a myslet mimo hranice myšlení. Ale zároveň Foucault říká, že přijal „ne-
gativní postoj“ k „sakralizaci“ tohoto nového pojetí literatury, jenž se ujal
na univerzitách, sakralizaci, jež paradoxně zpočátku takovouto novou li-
teraturu chtěla odmítat. 60 „Foucault, passe-frontières de la philosophie.“ Le Monde, 6. září 1986. Vidění vnějšku Říká, že vyvstala nová „ultraracionální“ a „ultra-
lyrická“ idea literatury, jež radikálně odkazuje jen k sobě samotné, v níž
psaní získalo nedotknutelná práva k „rozvratu“, a čím spletitěji člověk
psal, tím více byl považován za „revolucionáře“. Foucault tuto ideu chápal
jako formu „politické překážky“ – a představuje své knihy o Rousselovi
a Rivièrovi jako vlastní způsob, jak se vymanit novému akademickému
posvěcování literatury. 129 John Rajchman Přesto můžeme tvrdit, že jeho rané myšlenky o dílech modernity
z jeho myšlení naprosto nevymizely. Jestliže absence d’oeuvre přestává být
předmětem jeho dějin, přichází, aby vyplnila cosi z ethosu jeho díla jako
historika. Spíš než aby byl Nietzsche temným hrdinou jeho dějin, je kýmsi,
koho může nově použít. A jiným způsobem by pokračoval s Baudelairo-
vým „postojem k modernitě“. Foucault tedy rozšiřuje místo, jež Baudelaire
nazýval „uměním“ o určitou etiku myšlení, vidění a žití. Ve svém eseji z roku 1966 o Blanchotovi Foucault říká, že „absence“
není uvnitř díla, nýbrž vně díla. Modernistická literatura není literaturou,
jež se obrací k sobě, ale literaturou, jež se otevírá vně sebe. Nenechává
krásné formy vynořovat uvnitř, ale vynáší je ven z toho, čím byly. Možná
se tento druh „absence“ stal součástí Foucaultova pohledu na jeho vlastní
dílo: to, co zatím nemůžeme vidět ve formách vědění, jednání a zkuše-
nosti, skrze níž se sobě dáváme. Foucaultova fi losofi e se týkala možnosti:
toho, co můžeme myslet a co můžeme změnit v tom, co si myslíme. Chtěl
vytvářet dějiny nikoli toho, co je pravdivé či nepravdivé, nýbrž toho, co
je možné; nikoli toho, co máme dělat, nýbrž toho, co může být uděláno;
nikoli toho, jak žít, nýbrž možností žití. Objevil tedy druh „nemožnosti“,
jež nebyla logická, nýbrž historická: nikoli nemožnost kvadratury kruhu
nebo neexistujícího boha, nýbrž toho, co již není či ještě není možné
myslet; nikoli toho, co je nesmyslné, nýbrž toho, co ještě není či už není
smysluplné. Taková „absence“ byla záležitostí dějinného omezení myšlení
– a fi losofi e to měla vynášet na světlo. V tomto smyslu bychom mohli o Foucaultově umění vidět říci, že
jde o umění vidět mimo nás samotné nebo vidět „absenci“ v našem díle. 61 HOY (ed.), Foucault Reader, s. 46. Vidění vnějšku Nejde o pohled dovnitř na naše pravé či autentické já, nejde o ovládnutí
našeho času tím, že ho budeme držet v našem myšlení, nejde o nalezení
místa pro sebe ve společnosti nebo státu, nýbrž máme se dívat ze sebe ven,
rozevřít náš čas tomu, co zatím nemohlo být viděno, přetvořit či přemístit
naši institucionalizovanou, přiřazenou identitu v čase a prostoru. V tomto
smyslu je Foucaultovo umění vidět uměním pohledu ven, který „dává nový, 130 Foucaultovo umění vidět co možná nejvíce dalekosáhlý a obšírný podnět – k neurčitému působení
svobody“.61 Z anglického originálu Foucault’s Art of Seeing (October, roč. 44, 1988,
s. 8–117) přeložila Hana Ondráčková. John Rajchman působí na Katedře dějin umění a archeologie Columbia
University. Věnuje se současné evropské fi losofi i a dějinám umění a ar-
chitektury. Je editorem časopisu Artforum a autorem řady monografi í
o předních současných myslitelích – např. Michel Foucault: Th e Freedom
of Philosophy (1985); Philosophical Events: Essays of the ‘80s (1991); Th e
Deleuze Connections (2000). 131 131
|
https://openalex.org/W4229368179
|
https://revistas.uned.ac.cr/index.php/cuadernos/article/download/v14i1.3786/5530
|
es
|
Efecto de las aves, las altas y bajas temperaturas sobre la germinación y viabilidad de las semillas de Rubus ulmifolius (Rosaceae)
|
Cuadernos de Investigación UNED/Cuadernos de investigación UNED
| 2,022
|
cc-by
| 4,595
|
Efecto de las aves y la temperatura sobre la germinación y viabilidad de las
semillas de Rubus ulmifolius (Rosaceae)
Claudia M. Dellafiore1
1.
2.
& Maximiliano Sainz2
Universidad Nacional de Río Cuarto, Departamento de Ciencias Naturales, Facultad de Ciencias Exactas, FísicoQuímicas y Naturales, Ruta 36 km 601, Río Cuarto, Córdoba, Argentina; cdellafiore@exa,unrc.edu.ar,
Universidad Nacional de Río Cuarto, Departamento de Ciencias Agrarias, Facultad de Agronomía y Veterinaria, Ruta
36 km 601, Río Cuarto, Córdoba, Argentina; pachisainz@hotmail.com
Recibido 21-IX-2021 Corregido 16-XI-2021 Aceptado 01-XII-2021
DOI: https://doi.org/10.22458/urj.v14i1.3786
ABSTRACT. “Effect of birds and temperature on the germination
and viability of Rubus ulmifolius (Rosaceae) seeds” Introduction:
Invasive species are causing serious modifications around the
world, affecting deserts and tropical forests. In Córdoba,
Argentina, the distribution and abundance of blackberry (Rubus
ulmifolius) has increased in rural and wild environments. Seed
dispersion by birds, and the high temperatures generated by
forest fires, have been mentioned as the main cause of this
increase. However, there are no studies about these hypotheses.
Objectives: To know if the birds disperse the blackberry seeds and
if they affect their germination, and to study the effect of high
and low temperatures on germination and viability. Methods:
Blackberry fruits and fresh feces from birds were collected in the
field and the seeds kept at constant temperature during 20
months to measure germination; 27 samples of seeds (50 fruits
per sdample) were kept at several temperatures and durations,
ranging from room temperature to -11ºC, and from a few minutes
to several days. Afterwards, they were germinated at constant
temperature for 20 months. Additionally, 33 similar samples were
treated for a viability test. Results: Neither seeds from the fruits,
nor seeds from feces, germinate after 20 months. Most (75%)
seeds from feces remained viable after 20 months. Viability was
affected by temperature (H: 21,50; p = 0,0054). Conclusion: These
blackberry seeds have a low germination power since they did not
germinate under any treatment after 20 months. On the other
hand, these seeds are highly resistant to high and low
temperatures, although surface fires could destroy them.
RESUMEN. Introducción: Las especies invasoras están causando
serias modificaciones por todo el mundo, afectando desiertos y
bosques tropicales. En Córdoba, Argentina, la distribución y
abundancia de la mora o zarzamora (Rubus ulmifolius) se ha
incrementado en ambientes rurales y silvestres. Se ha sugerido
que las causas principales del incremento son la dispersión de
semillas por aves, y las altas temperaturas generadas por los
incendios forestales. Sin embargo, no existen estudios sobre
estas hipótesis. Objetivos: Conocer si las aves dispersan las
semillas de zarzamora y si afectan a su germinación, y estudiar el
efecto de las altas y bajas temperaturas sobre la germinación y
viabilidad. Métodos: Se recolectaron en campo frutos de
zarzamora y heces frescas de aves; y las semillas se mantuvieron
a temperatura constante durante 20 meses para medir la
germinación. Se mantuvieron 27 muestras de semillas (50 frutos
por muestra) a varias temperaturas y duraciones, desde
temperatura ambiente hasta -11ºC, y desde unos pocos minutos
hasta varios días. Posteriormente, se germinaron a temperatura
constante durante 20 meses. Además, 33 muestras similares
fueron tratadas para una prueba de viabilidad. Resultados: Ni las
semillas de los frutos, ni las semillas de las heces, germinaron en
20 meses. La mayoría de las semillas de las heces (75%)
permanecían viables a los 20 meses. La viabilidad se vio afectada
por la temperatura (H: 21,50; p = 0,0054). Conclusión: Estas
semillas de mora tienen un bajo poder germinativo ya que no
germinaron en ningún tratamiento después de 20 meses. Por otro
lado, estas semillas son muy resistentes a las altas y bajas
temperaturas, aunque los incendios superficiales podrían
destruirlas.
Keywords: Germination power, fires, frosts, dispersal, blackberry.
Palabras clave: Poder
dispersión, zarzamora.
germinativo,
incendios,
heladas,
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
La introducción de especies invasoras está causando grandes modificaciones a nivel mundial
afectando tanto a los desiertos como a las selvas tropicales. Las especies invasoras son una de las
principales causas de pérdida de biodiversidad (Williamson, 1999; Walker & Steffen, 1997) y causan
serios daños ambientales con elevados costos sociales (Mack et al., 2000, Lodge & SgraderFrechette, 2003). Desde el punto de vista económico las especies invasoras cada vez adquieren
mayor importancia debido al alto impacto que generan sobre las cosechas y el rendimiento agrícolaganadero. En Estados Unidos dichas especies generan pérdidas de hasta 27millones de dólares por
año y en Reino Unido algunos planes de control, sin garantía de éxito, han generado costos de hasta
42 millones de libras esterlinas (Giorgis et al., 2006). Además, las especies invasoras tienen serios
efectos sobre la estructura, organización y composición de las comunidades vegetales y animales
debido a las múltiples interacciones mutualistas y antagonistas que establecen con las especies
nativas (Traveset & Richardson 2006; Alba-Lynn & Hen, 2010)
En la provincia de Córdoba, Argentina, la especie Rubus ulmifolius (NV: zarzamora), un
arbusto originario de Europa, norte de África y sur de Asia, ha incrementado notoriamente su
distribución y abundancia en los últimos años, tanto en ambientes naturales como en ambientes
rurales (Dellafiore, obs. pers.). Específicamente en las plantaciones forestales y en los bosques
nativos de las Sierra de Comechingones este incremento se ha hecho muy notable y ha obligado a
algunos productores a abandonar sectores de sus campos debido al alto grado de invasión por esta
especie la cual impide el acceso a los animales y a las maquinarias agrícolas (Dellafiore, obs. pers.).
Como ocurre con la mayoría de las especies invasoras, la zarzamora posee un crecimiento
rápido y puede multiplicarse vegetativamente, generando raíces desde sus ramas, además no
presenta una época de floración unitaria, sino que en un mismo ejemplar se encuentran flores y
frutos maduros e inmaduros al mismo tiempo. Esta especie posee, además, una elevada producción
de semillas, pero las mismas carecen de un mecanismo específico de dispersión por lo que caen y
permanecen en cercanías de la planta madre. La aparición de dichas plantas en áreas aisladas ha
sido atribuida a las aves las cuales han sido mencionadas como unas de las principales responsables
de su dispersión en su hábitat originario (Jordano, 1984). Sin embargo, en nuestro país no se han
realizado estudios acerca del rol de las aves sobre la dispersión de esta especie.
Por otro lado, los incendios y la proliferación de especies invasoras exóticas pueden
interactuar positivamente entre ellos incrementado el impacto de dichas especies sobre los
ecosistemas invadidos (D’Antonio & Vitousek, 1992; Mack & D’Antonio, 1998; D’Antonio, 2000). Así,
por ejemplo, las especies invasoras pueden acumular bancos de semillas cuya germinación es
estimulada por el fuego, (altas temperaturas, humo o combinación de ambos), lo que no ocurre con
las especies nativas las cuales, generalmente, pierden su viabilidad (Mack & D’Antonio, 1998;
D’Antonio, 2000; Alexander & D’Antonio, 2003). En el caso de la zarzamora, algunos productores
han mencionado que el fuego beneficia a la especie ya que luego de una quema observaron un
incremento en la cobertura y vigor de las plantas (Palacios, com pers.) Sin embargo, no existe
información científica a cerca del efecto del fuego sobre las semillas de zarzamora.
Otro efecto a tener en cuenta son las bajas temperaturas a las cuales suelen verse sometidas
las semillas durante la estación invernal. En los ecosistemas serranos de la provincia de Córdoba las
temperaturas suelen descender a -5°C (Servicio Meteorológico Nacional) durante el invierno. Estas
bajas temperaturas también pueden afectar a la viabilidad de las semillas de zarzamora las cuales
deben sobrevivir hasta encontrar las condiciones óptimas para su germinación.
Debido a lo expuesto anteriormente el presente trabajo tuvo por objetivos: a) estudiar si las
aves dispersan las semillas de zarzamora, b) conocer si las aves afectan la germinación de las semillas
de zarzamora y d) examinar el efecto de las altas y bajas temperaturas sobre la germinación y
viabilidad de las semillas de zarzamora.
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
MATERIALES Y MÉTODOS
El área de estudio comprende una superficie de 3,5km 2 y está ubicada en la ciudad de Alpa
Corral, Provincia de Córdoba, Argentina. Dicha área pertenece a la Región Fitogeográfica del Chaco
Serrano (Cabrera, 1976). Entre las especies vegetales nativas observadas en el área de estudio se
encuentran Lithraea ternifolia, Fagara coco, Celtis ehrenbergiana, Schinus areira, Schinus
fasciculatus, Prosopis torquata y varias especies de la familia Poaceae como Stipa sp. y Festuca sp.
Además, se encontraron especies exóticas como Ligustrum lucidum, Rubus ulmifolis, Pyracantha
atalantoides, Rosa eglanteria y Gleditsia triacanthos.
En el área de estudio se censaron veintiséis especies de aves nativas de América del Sur y al
menos dieciséis de ellas consumen frutos y semillas de forma regular (por ej: Saltator aurantiirostris,
Poospiza melanoleuca, Coryphospingus cucullatus, Zonotrichia capensis, Turdus rufiventris, entre
otras) (Dellafiore obs. pers).
Para conocer si las aves dispersan las semillas de zarzamora y si afectan su germinación, se
recolectaron fecas de aves sobre dos transectos lineales de 1 450 * 20m y 1 200 * 5m durante los
meses de verano de 2012 (momento en que aparecen las semillas de zarzamora en las fecas de las
aves, Dellafiore obs. pers.). Se recolectaron únicamente fecas frescas y debido a que las aves suelen
defecar varios pellets en un mismo lugar se consideró como una muestra a todos los pellets
recogidos en la misma posición geográfica (GPS).
En el laboratorio las fecas fueron pesadas y se realizó el análisis del contenido de las mismas.
Para ello fueron desmenuzadas mecánicamente mediante pinza diente de ratón y aguja de
disección. La búsqueda de las semillas de R. ulmifolius fue realizada bajo lupa estereoscópica y las
semillas observadas fueron contadas y analizadas en detalle para registrar posibles daños físicos
(semillas partidas, tegumento roto, exposición del embrión, deshidratación, etc.). En total se
obtuvieron 150 semillas de zarzamora las cuales fueron separadas al azar en 3 muestras de 50
semillas cada una y puestas a germinar en placas de petri con algodón y papel secante. Las placas
se regaron diariamente y se mantuvieron a temperatura constante de 20-25°C. El criterio de
germinación fue la emergencia de la radícula. La germinación de las semillas se registró diariamente
durante un período de 20 meses.
Al mismo tiempo en el área de estudio se recolectaron 100 frutos de zarzamora a las cuales
se les extrajeron las semillas (500 semillas). Posteriormente se seleccionaron al azar tres muestras
de 50 semillas cada una. Dichas semillas fueron puestas a germinar siguiendo el mismo
procedimiento mencionado para las semillas provenientes de las fecas de las aves y durante el
mismo período mencionado previamente.
Para conocer el efecto de las altas y bajas temperaturas sobre la germinación y viabilidad
de las semillas de zarzamora se recolectaron 1 000 frutos de 20 plantas diferentes. En el laboratorio
se extrajeron 2 500 semillas y se separaron al azar 60 muestras de 50 semillas cada una. Dichas
muestras fueron empleadas para llevar a cabo los siguientes tratamientos:
Tratamiento altas temperaturas
Las temperaturas que suele alcanzar el suelo durante una quema de baja, media y alta
intensidad es de 60°, 80° y 100°C respetivamente y el tiempo medio estimado de exposición es de
3 minutos (Carballas-Fernández, 2003). Teniendo en cuenta lo expuesto previamente, se realizaron
dos tests para conocer el efecto de dichas temperaturas sobre la germinación y viabilidad de las
semillas de zarzamora.
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
1. Prueba de germinación altas temperaturas
Se tomaron nueve muestras de 50 semillas cada una de las cuales tres se mantuvieron a
temperatura ambiente (control), 3 fueron sometidas a 80°C durante 3 minutos y tres se
mantuvieron a 100°C durante 2 minutos (Apéndice, Tabla 1). Tanto las muestras control como las
muestras sometidas a calor fueron puestas a germinar en placas de plástico con algodón y papel
secante. Las placas se regaron diariamente y se mantuvieron a temperatura constante de 20-25°C.
El criterio de germinación fue la emergencia de la radícula. La germinación de las semillas se registró
diariamente durante un período de 20 meses.
2. Prueba de viabilidad altas temperaturas
Se tomaron 12 muestras de 50 semillas cada una, de las cuales 3 se mantuvieron a
temperatura ambiente (control) y el resto fueron sometidas a 60°C, 80°C y 100°C durante 3 minutos
respectivamente (Apéndice, Tabla 2). Tanto las muestras control como las muestras sometidas a
calor fueron evaluadas mediante la prueba de tetrazolium (Cottrell, 1947; MacKay, 1972) para
determinar la viabilidad de las semillas.
Tratamiento frio
Teniendo en cuenta que la temperatura media del área de estudio es de 4,6°C durante el
invierno y que la temperatura mínima extrema, en el mes de julio, es de -11°C (datos de los últimos
20 años del Servicio Meteorológico Nacional), se realizaron dos tests para conocer el efecto del frío
sobre la germinación y vialidad de las semillas de zarzamora.
1. Prueba de germinación bajas temperaturas
Se tomaron 18 muestras de 50 semillas cada una de las cuales tres se mantuvieron a
temperatura ambiente (control) y el resto fueron sometidas a frío de 5°C durante 1,4,7 y 14 días y a
-11°C durante 7 días (Apéndice, Tabla 3). Tanto las muestras control como las muestras sometidas
a las temperaturas señaladas previamente fueron puestas a germinar durante un período de 20
meses siguiendo la misma metodología que la señalada previamente.
2. Prueba de viabilidad bajas temperaturas
Se tomaron 21 muestras de 50 semillas cada una de las cuales tres se mantuvieron a
temperatura ambiente (control) y el resto fueron sometidas a frío de 5°C durante 1,4,7 y 14 días y a
-11°C durante 1 y 7 días (Apéndice, Tabla 4). Tanto las muestras control como las muestras
sometidas a bajas temperaturas fueron evaluadas mediante la prueba de tetrazolium (Cottrell,
1947; MacKay, 1972) para determinar su viabilidad.
Las diferencias entre tratamientos fueron analizadas mediante la prueba de Kruskal-Wallis
y como prueba a posteriori se empleó la prueba de T para muestras independientes. En todos los
casos se consideró como significativo un p<0,05.
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
RESULTADOS
En total se recolectaron 21 muestras de fecas de aves durante el verano de 2012 en el área
de estudio de las cuales el 4,7% contenía semillas de zarzamora. En total se contabilizaron y
extrajeron 80 semillas y el 100% de las mismas no presentó ningún signo de daño físico. Al cabo de
20 meses de sembradas las semillas provenientes de los frutos y de las fecas de las aves no habían
germinado, pero el 75% de las mismas permanecía viable.
En cuanto a la germinación a altas temperaturas se observó que tanto las semillas
provenientes de los frutos como las sometidas a los distintos tratamientos no germinaron al cabo
de 20 meses. En relación al tratamiento de viabilidad a altas temperaturas se observó que el 82%
de las semillas control, el 72% de las semillas sometidas a 60°C y el 37% de las semillas sometidas a
80°C durante 3 minutos eran viables, mientras que ninguna de las semillas sometidas a 100°C
durante 2 minutos resulto viable (Fig. 1.).
En el tratamiento de las semillas al frío se observó que tanto las semillas provenientes de
los frutos como las sometidas a los distintos tratamientos no germinaron al cabo de 20 meses. La
viabilidad de las semillas sometidas a los diferentes tratamientos de bajas temperaturas mostró que
la misma oscilo entre el 54% para las semillas sometidas a -11°C durante 7 días y el 78% para las
semillas sometidas a 5°C durante 4 días (Fig. 1.).
Fig. 1. Porcentaje de semillas viables y no viables para los distintos tratamientos. C: control, F15: frío 1 día a 5°C, F45: frío
4 días a 5°C, F75: frío 7 días a 5°C, F145: frío 14 días a 5°C, F1-11: frío 1 día a -11°C, C360: calor 3 minutos a 60°C, C380:
calor 3 minutos a 80°C y C2100: calor 2 minutos a 100°C.
Al comparar los tratamientos mediante la prueba de Kruskal-Wallis se encontraron
diferencias significativas entre ellos (H: 21,50; p=0,0054). Mediante la prueba de la T para muestras
independientes pudimos observar diferencias significativas entre las semillas control y todos los
tratamientos excepto con los tratamientos de frío durante 4 y 7 días a 5°C respectivamente
(Apéndice, Tabla 5). Además, se observaron diferencias significativas entre todos los tratamientos y
las semillas sometidas durante 2 minutos a 100°C y las semillas sometidas durante 3 minutos a 80°C
(Apéndice, Tabla 5). También se observaron diferencias significativas entre la viabilidad de las
semillas sometidas durante 7 días a -11°C y el resto de los tratamientos, excepto con la viabilidad
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
de las semillas sometidas durante 3 minutos a 60°C y las sometidas a frío durante 1 día a -11 °C y el
resto de los tratamientos, excepto con frío 7 días a 5°C (Apéndice, Tabla 5).
DISCUSIÓN
De acuerdo con nuestros resultados las aves realizan una dispersión “legítima” de las
semillas de R. ulmifolius, dado que las mismas no sufren daño al pasar por el tracto digestivo de las
aves y al cabo de veinte meses un alto porcentaje de las mismas permanecía viable. Sin embrago,
no hemos podido comprobar que dicha dispersión sea “efectiva” ya que ninguna semilla germinó al
cabo de veinte meses. Estos resultados no coinciden con los observados por Jordano (1984) y
Traveset et al., (2001), quienes encontraron que las aves no afectan el porcentaje de germinación
de R. ulmifolius y que las semillas germinan al cabo de 70 días. Nuestros resultados coincidirían con
lo observado por Wada y Reed (2011), quienes manifiestan que la germinación de las semillas del
género Rubus está fuertemente limitada por una doble dormancia (física y fisiológica) y por lo
observado por Zasada y Tappeiner (2003) quienes mencionan que algunas especies de Rubus
poseen una lenta maduración del embrión. A pesar de que las semillas provenientes de las aves no
germinaron, el hecho de que sean llevadas a áreas alejadas de la planta madre y que permanezcan
viables por largos períodos de tiempo podría tener un impacto positivo para la especie. Es decir, que
las aves podrían tener un rol importante en la dispersión de las semillas a nuevas áreas abiertas a la
colonización.
Según Carballas-Fernández (2003), los incendios pueden ser de baja, moderada o alta
intensidad. Los incendios de baja intensidad son aquellos que alcanzan temperaturas en superficie
de 100 a 250°C y 100°C a los 2cm de profundidad. Los incendios de intensidad moderada alcanzan
los 300 a 400°C en superficie, 200 a 300°C a los 2cm de profundidad y 60 a 80°C a los 3cm de
profundidad. Los incendios de alta intensidad alcanzan los 500 a 700°C en superficie y 350 a 450°C
a 2cm de profundidad. De acuerdo con nuestros resultados, las semillas de zarzamora en superficie
perderán su viabilidad tanto en incendios de baja como de moderada o alta intensidad. Los
incendios de baja intensidad no afectarían la viabilidad de las semillas de zarzamora si forman
bancos a más de 3cm de profundidad. Mientras que los incendios de intensidad moderada podrían
llegar a disminuir el banco de semillas en un 63% si la temperatura alcanza los 80°C a los 3cm de
profundidad.
Las bajas temperaturas no parecen afectar considerablemente a las semillas de zarzamora.
Solo temperaturas menores a -11°C durante más de 7 días podrían llegar a afectar el banco de
semillas disminuyendo el mismo en un 46%. Teniendo en cuenta que R. ulmifolius posee una elevada
producción de semillas, esta disminución podría considerarse como significativa principalmente
para aquellas semillas que han llegado a nuevas áreas abiertas a la colonización.
De acuerdo con lo expuesto anteriormente, podemos concluir que R. ulmifolius posee
semillas altamente resistentes tanto a las bajas como a las altas temperaturas y a la endozoocoria
por aves. Sin embargo, dichas semillas poseen un escaso poder germinativo ya que no germinaron
bajo ningún tratamiento al cabo de 20 meses en condiciones favorables de luz, temperatura y
humedad. Por otro lado, podemos inferir que la invasión que se observa actualmente por zarzamora
en las sierras de Córdoba - Argentina se debería principalmente a su estrategia de reproducción
vegetativa y no a la alta producción de semillas. Sin embargo, la interacción mutualista que
establece zarzamora con las aves podría explicar su llegada a nuevas áreas abiertas a la colonización,
ya que las semillas permanecen viables en las fecas y podrían germinar cuando las condiciones les
sean favorables.
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
AGRADECIMIENTOS
Agradecemos a la Secretaría de Ciencia y Técnica de la UNRC por el apoyo financiero para la
realización del presente trabajo.
ÉTICA, CONFLICTO DE INTERESES Y DECLARACIÓN DE FINANCIAMIENTO
Declaramos haber cumplido con todos los requisitos éticos y legales pertinentes, tanto
durante el estudio como en la preparación de este documento; que no hay conflictos de interés de
ningún tipo, y que todas las fuentes financieras se detallan plena y claramente en la sección de
agradecimientos. Asimismo, estamos de acuerdo con la versión editada final de esta publicación. El
respectivo documento legal firmado se encuentra en los archivos de la revista.
Ambos autores hemos contribuido en cada paso de la preparación y aprobación final del
manuscrito.
REFERENCIAS
Alba-Lynn C., & Hen, S. (2010). Potential for ants and vertebrate predators to shape seed-dispersal dynamics of the
invasive thistles Cirsium arvense and Carduus nutans in their introduced range (North America). Plant Ecology,
210, 291-301. https://doi.org/10.1007/s11258-010-9757-2
Alexander J.M., & D’Antonio, C. (2003). Seed bank Dynamics of French Broom. In Coastal California grasslands: effects Of
Stand Age and prescribed burning on control and Restoration. Restoration Ecology, 11(2), 185-197.
https://doi.org/10.1046/j.1526-100X.2003.00169.x
Cabrera, A. L. (1976). Enciclopedia Argentina de agricultura y jardinería: Regiones fitogeográficas Argentinas. Editorial
Acme.
Carballas-Fernández, T. (2003). Los incendios forestales. En J. J. Casares (Ed.), Reflexiones sobre el medio ambiente en
Galicia (pp. 363-415). Consellería de Medio Ambiente Centro de Desenvolvemento Sostible.
Cottrell,
H. J. (1947). Tetrazolium salt
https://doi.org/10.1038/159748a0
as
a
seed
germination
indicator.
Nature,
159,
748.
D’Antonio, C.M., & Vitousek, P.M. (1992). Biological Invasions B Exotic Grasses, The Grass/Fire Cycle, And Global Change.
Annual Review of Ecology and Systematics, 23, 63-87.
D’Antonio, C.M. (2000). Fire, plant Invasions, And Global Changes. In H. A. Mooney & R.J. Hobbs (Eds.), Invasive Species in
a Changing World (65-95). Island Press.
Giorgis, M. A., Tecco, P. A., Marcora, P., Cingolani, M., Paiaro, V., & Renison, D. (2006). Las invasiones biológicas y su
problemática ambiental – Manual para docentes. CONICET.
Jordano. P. (1984). Seed weight variation and differential avian dispersal in blackberries. Oikos, 43(2), 149-153.
https://doi.org/10.2307/3544762
Lodge, D.M., & Sgrader-Frechette, K. (2002). Nonindigenous Species: Ecological Explanation, Environmental Ethics & Public
Policy. Conservation Biology, 17(1), 31-37. https://doi.org/10.1046/j.1523-1739.2003.02366.x
Mackay, D. B. (1972). The measurement of viability. In E. H. Roberts (Ed.) Viability of seeds (pp. 172-208). Chapman and
Hall.
Mack, M.C., & D’Antonio, C.M. (1998). Impacts of Biological Invasions on Disturbance Regimes. Trends in Ecology and
Evolution, 13(5), 195-198. https://doi.org/10.1016/S0169-5347(97)01286-X
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
Mack, R.N., Simberloff, D., Lonsdale, W.M., Evans, H., Clout, M., & Bazzaz, F.A. (2000). Biotic Invasions: Causes,
Epidemiology, Global Consequences and Control. Ecological Applications, 10(3), 689-710.
https://doi.org/10.1890/1051-0761(2000)010[0689:BICEGC]2.0.CO;2
Traveset, A., Riera, N., & Mas R.E. (2001). Passage through bird guts causes interspecific differences in seed germination
characteristics. Functional ecology, 15(5), 669-675. https://doi.org/10.1046/j.0269-8463.2001.00561.x
Traveset, A., & Richardson, D.M. (2006). Biological Invasions as disruptors of Plant-Animal Reproductive Mutualisms.
Trends in Ecology and Evolution, 21(4), 208-216. https://doi.org/10.1016/j.tree.2006.01.006
Wada S., & Reed B.M. (2011). Optimized scarification protocols improve germination of diverse Rubus germplasm. Scientia
Horticulturae, 130(3), 660-664.
Walker, B., & Steffen, W. (1997). An overview of the implications of global change for natural and managed terrestrial
ecosystems. Conservation Ecology, 1(2), 1-18. http://www.jstor.org/stable/26271662
Williamson, M. (1999). Invasions. Ecography, 22(1), 5-12. https://doi.org/10.1111/j.1600-0587.1999.tb00449.x
Zasada, J.C., & Tappeiner, J.C. (2003). Rubus L. The Woody Plant Seed Manual U.S.D.A. Forest Service, 162, 9-1638.
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
APÉNDICE
TABLA 1
Diseño experimental para evaluar la germinación de las semillas de zarzamora proveniente de los frutos y sometidas a
calor de 80 y 100ºC
Tratamientos
Control
Calor 3 minutos a 80º C
Calor 2 minutos a 100º C
Bandeja 1
50 semillas
50 semillas
50 semillas
Bandeja 2
50 semillas
50 semillas
50 semillas
Bandeja 3
50 semillas
50 semillas
50 semillas
TABLA 2
Diseño experimental para evaluar la viabilidad de las semillas de zarzamora proveniente de los frutos y las sometidas a
calor de 60º, 80º y 100ºC
Tratamientos
Control
Calor 3 minutos a 60ºC
Calor 3 minutos a 80ºC
Calor 2 minutos a 100ºC
Bandeja 1
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 2
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 3
50 semillas
50 semillas
50 semillas
50 semillas
TABLA 3
Diseño experimental para evaluar la germinación de las semillas de zarzamora proveniente de las de los frutos y
sometidas a temperaturas de 5ºC durante 1, 4, 7 y 14 días y a -11ºC durante 7 días.
Tratamientos
Control
Frio 1 día a 5º C
Frio 4 días a 5º C
Frio 7 días a 5º C
Frio 14 días a 5º C
Frio 7 días a -11ºC
Bandeja 1
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 2
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 3
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
TABLA 4
Diseño experimental para evaluar la viabilidad de las semillas de zarzamora proveniente de los frutos y las sometidas a
temperaturas de 5ºC y -11ºC durante 1, 4, 7 y 14 días y 1 y 7 días respectivamente
Tratamientos
Control
Frio 1 día a 5º C
Frio 4 días a 5º C
Frio 7 días a 5º C
Frio 14 días a 5º C
Frio 1 día a-11º C
Frio 7 días a -11º C
Bandeja 1
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 2
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
Bandeja 3
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
50 semillas
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
TABLA 5
Prueba de la T para muestras independientes (ns: diferencia no significativa).
Tratamientos
Control
Control
X
T: 3.5
P:0.025
*
Frio 1 día 5ºC
Frio 4 días
5ºC
Frio 7 días
5ºC
Frio 14 días
5ºC
Frio 1 día 1ºC
Frio 7 días
11ºC
Calor 2
minutos
100ºC
Calor 3
minutos
60ºC
Calor 3
minutos
80ºC
Frio 1
día 5ºC
Frio 4
días 5ºC
Frio 7
días 5ºC
Frio 1
día
11ºC
Frio 7
días
11ºC
Calor 2
minutos
100ºC
Calor 3
minutos
60ºC
Calor 3
minutos
80ºC
X
ns
ns
X
ns
ns
ns
X
ns
ns
ns
T: 3.2
P: 0.03
*
T: 5.3
P: 0.06
*
T: 41.2
P:0.0006
*
T:2.95
P:0.042
*
T: 4.7
P:0.0097
*
T: 18.7
P: 0.028
*
ns
T: 14.4
P:0.001
*
T: 3.5
P:0.025
*
T: 6.5
P:0.029
*
T: 7.7
P 0.016
*
T: 41
P:0.0006
*
T: 3.27
P: 0.03
*
T: 17.0
P:0.001
*
Frio 14
días
5ºC
X
T: 5.7
P: 0.046
*
T: 32.9
P:0.0009
*
T: 3.2
P:0.033
*
T: 5.3
P:0.006
*
T: 41.2
P:0.006
*
T: 3.0
P:0.039
*
T: 36.7
P:0.007
*
T: 17.7
P:0.032
*
ns
ns
ns
ns
ns
T: 9.1
P:0.008
*
T: 13.5
P:0.002
*
T: 14.4
P:0.001
*
T: 11.2
P:0.004
*
T: 4.9
P:0.008
*
ns
X
X
X
T: 31.2
P: 0.01
*
T: 12.2
P:0.003
*
X
T: 20.8
P: 0.03
*
X
UNED Research Journal (e-ISSN 1659-441X), Vol. 14(1): e3786, June, 2022
|
|
https://openalex.org/W2149231104
|
https://www.erudit.org/fr/revues/irrodl/2015-v16-n3-irrodl04980/1065975ar.pdf
|
English
| null |
Learners’ goal profiles and their learning patterns over an academic year
|
International review of research in open and distance learning
| 2,015
|
cc-by
| 10,313
|
Learners’ Goal Profiles and their Learning Patterns over an
Academic Year Clarence Ng Volume 16, numéro 3, juin 2015 Document généré le 24 oct. 2024 01:47 Document généré le 24 oct. 2024 01:47 Résumé de l'article Résumé de l'article The present study aimed to examine distance learners’ goal profiles and their
contrasting patterns of learning and achievements at three different points
during an academic year, i.e. in the beginning of the course in relation to
learners’ general orientations to learning, at the middle of the course in
relation to learners’ completion of an assignment, and towards the end of the
course in relation to learners’ preparation for course examination. Two
hundred seventy-six adult distance learners completed three survey
questionnaires that assessed their motivation and learning at these three
learning points. Using person-centred analytical procedures, this study located
four groups of learners based on different combinations of mastery and
performance-approach goals. MANOVA results have shown that multiple-goal
learners (High mastery/High performance, HH) who held strong mastery and
performance-approach goals used more deep and regulatory strategies and
showed a higher level of learning interest across three waves of surveys than
did those focusing solely on mastery (HL) or performance-approach goals (LH). However, the multiple-goal learners did not have better achievement levels
compared to those focusing solely on mastery goals (HL). Given that multiple
goal learners learnt with a more engaged pattern, it is less likely that these
motivated learners will drop out of distance learning courses and programs. Future studies should explore how these goals can be promoted
simultaneously in distance learning. Copyright (c) Clarence Ng, 2015
Ce document est protégé par la loi sur le droit d’auteur. L’utilisation des
services d’Érudit (y compris la reproduction) est assujettie à sa politique
d’utilisation que vous pouvez consulter en ligne. https://apropos.erudit.org/fr/usagers/politique-dutilisation/
Cet article est diffusé et préservé par Érudit. Érudit est un consortium interuniversitaire sans but lucratif composé de
l’Université de Montréal, l’Université Laval et l’Université du Québec à
Montréal. Il a pour mission la promotion et la valorisation de la recherche. https://www.erudit.org/fr/ Copyright (c) Clarence Ng, 2015 Ce document est protégé par la loi sur le droit d’auteur. L’utilisation des
services d’Érudit (y compris la reproduction) est assujettie à sa politique
d’utilisation que vous pouvez consulter en ligne. https://apropos.erudit.org/fr/usagers/politique-dutilisation/ Cet article est diffusé et préservé par Érudit. Érudit est un consortium interuniversitaire sans but lucratif composé de
l’Université de Montréal, l’Université Laval et l’Université du Québec à
Montréal. Il a pour mission la promotion et la valorisation de la recherche. International Review of Research in Open and Distributed Learning
Volume 16, Number 3 June – 2015 Learners’ Goal Profiles and their Learning Patterns
over an Academic Year Clarence Ng
Australian Catholic University, Australia Introduction The high dropout rate in many distance learning courses and programs (e.g. Simpson, 2013),
including the offerings of MOOCs (Liyanagunawardena, Adams, & Williams, 2013), begs the
question of what motivates distance learners to persist with their learning. This question is
particularly critical as distance learners often engage in an extended period of learning while
juggling commitments derived from personal, familial and employment concerns. Many studies
have investigated distance learners’ motivation; however, few have taken a longitudinal
perspective and utilised established motivational theories to inform their design (Simpson,
2008). This makes it difficult to pinpoint motivational variables that can be manipulated to
sustain distance learners’ learning motivation. In response, this study used a prospective
longitudinal design to examine distance learners’ motivation based on achievement goal theory,
which is currently a dominant theoretical model guiding the studies on learning motivation
(Hulleman, Schrager, Bodmann, & Harackiewicz, 2010). Achievement goals are students’
perceived cognitive purposes that define why and how students engage in learning or
achievement behaviours. Different goals are associated with a different pattern of cognition, affect
and behaviour (cf. Dweck, 1986; Hulleman et al., 2010). In the literature of distance education,
only a limited number of studies has taken an achievement goal perspective (e.g. Ng, 2009),
despite its dominance in motivational research and studies in campus-based samples. The current study aims to examine distance learners’ goal profiles and their contrasting patterns
of learning and achievement at three different points during an academic year, i.e. in the
beginning of the course in relation to learners’ general orientations to learning, at the middle of
the course in relation to learners’ completion of an assignment, and towards the end of the course
in relation to learners’ preparation for course examination. The results of the current study will
significantly contribute to our understanding of distance learners’ motivation and add to the
nascent literature base of achievement goal research in distance education. In this study, the
notion of learning was measured in terms of distance learners’ use of learning and regulatory
strategies, and their interest and self-efficacy beliefs in learning. The scores distance learners
received for their course assignment and examination were taken as measures for achievement
levels. Abstract The present study aimed to examine distance learners’ goal profiles and their contrasting patterns
of learning and achievements at three different points during an academic year, i.e. in the
beginning of the course in relation to learners’ general orientations to learning, at the middle of
the course in relation to learners’ completion of an assignment, and towards the end of the course
in relation to learners’ preparation for course examination. Two hundred seventy-six adult
distance learners completed three survey questionnaires that assessed their motivation and
learning at these three learning points. Using person-centred analytical procedures, this study
located four groups of learners based on different combinations of mastery and performance-
approach goals. MANOVA results have shown that multiple-goal learners (High mastery/High
performance, HH) who held strong mastery and performance-approach goals used more deep
and regulatory strategies and showed a higher level of learning interest across three waves of
surveys than did those focusing solely on mastery (HL) or performance-approach goals (LH). However, the multiple-goal learners did not have better achievement levels compared to those
focusing solely on mastery goals (HL). Given that multiple goal learners learnt with a more
engaged pattern, it is less likely that these motivated learners will drop out of distance learning
courses and programs. Future studies should explore how these goals can be promoted
simultaneously in distance learning. Keywords: Multiple goals; learning; achievement; distance learning; adult learners; prospective
d
i Keywords: Multiple goals; learning; achievement; distance learning; adult learners; prospective
design design 86 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng This work is licensed under a Creative Commons Attribution 4.0 International License. Alternative Achievement Goal Perspectives Two types of achievement goals have attracted the most research attention in the past three
decades. Mastery goals focus students on the learning task and relate to developing competence,
understanding and comprehension. In contrast, performance goals focus students on the self and
relate to demonstrating competence and eliciting favourable ability judgments from others
(Dweck, 1986; Hulleman et al., 2010; Senko, Hulleman, & Harackiewicz, 2011). Earlier studies 87 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng have confirmed the differences between these two types of achievement goals. Mastery goals are
usually associated with an adaptive learning pattern, characterised by positive cognition,
engaging learning behaviours and affect. In contrast, performance goals are often related to a
maladaptive pattern, which can be differentiated by a low level of learning engagement and a
relatively lower level of performance (e.g. Archer, 1994; Dweck, 1986; Meece & Holt, 1993). have confirmed the differences between these two types of achievement goals. Mastery goals are
usually associated with an adaptive learning pattern, characterised by positive cognition,
engaging learning behaviours and affect. In contrast, performance goals are often related to a
maladaptive pattern, which can be differentiated by a low level of learning engagement and a
relatively lower level of performance (e.g. Archer, 1994; Dweck, 1986; Meece & Holt, 1993). Based on these early studies, Midgley, Kaplan and Middleton (2001) argued that students should
be optimally motivated by mastery goals per se. This particular perspective was labelled as ‘the
mastery goal perspective’ and was formulated based on the findings derived from early studies on
achievement goals unanimously supporting the motivating effects of mastery goals on learning. The mastery goal perspective considers that all forms of performance goals are detrimental to
learning and the cost associated with performance demonstration outweighs its benefits. The
optimal goal profile should therefore include strong mastery goals but weak performance goals
(Meece & Holt, 1993). Achievement goal researchers such as Pintrich (2000) and Harackiewicz (Harackiewicz, Barron,
Carter, Lehto, & Elliot, 1997) disputed this mastery goal perspective and advocated a multiple
goal perspective based on a trichotomous framework of goals that classifies performance goals
into approaching and avoidance orientations. Alternative Achievement Goal Perspectives Research shows that maladaptive effects of
performance goals were confined to those with an avoidance orientation, such as avoiding looking
dumb whilst positive effects were found among performance goals with an approaching
orientation like getting a good grade (Elliot & Harackiewicz, 1996; Skaalvik, 1997). The multiple
goal perspective maintains that adopting performance-approach goals simultaneously with
mastery goals will enhance the positive effects on learning. In other words, the multiple goal
perspective suggests that having strong mastery and performance-approach goals in one's goal
profile will be most optimal for learning (e.g. Barron & Harackiewicz, 2001) and will facilitate
achieving different academic outcomes (Harackiewicz et al., 1997). However, the empirical support for adopting multiple goals over the mastery-only goals is far
from conclusive. First, the multiple goal perspective has built on the separation of performance
goals into approaching and avoidance orientations. While past research constantly showed that
performance-avoidance goals are detrimental to learning, empirical support on the positive
effects of performance-approach goals have not been consistent. Second, the current findings on
multiple goals cannot firmly establish the relative benefits of holding multiple goals over mastery-
only goals (Wolters, 2004). Some studies (e.g. Meece & Holt, 1993) found that having strong
mastery goals and weak performance-approach goals in one's goal profile are the most adaptive
form of motivation. Other studies found that students who endorsed both mastery and
performance-approach goals as compared to those who endorsed solely mastery goals were not
significantly different from each other in certain important aspects including their achievement
levels (e.g. Valle et al., 2003). To resolve the conceptual conflict between these two perspectives,
Pintrich (2000) argued that there are multiple pathways leading to similar learning outcomes and 88 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng achievement levels, albeit that students may have differential learning experiences due to the
particular goal mix they endorse. Certainly more studies are needed to clarify the effects of
multiple goals on learning and achievement despite that increasingly more research evidence (e.g. Darnon, Dompnier, Gilliéron, & Butera, 2010; Ng, 2009) has affirmed their positive effects on
learning. Alternative Achievement Goal Perspectives Researching multiple goals using a student group other than Euro-American samples of
on-campus students may provide new insights into the operation of multiple goals (Pintrich,
Conley, & Kempler, 2003). achievement levels, albeit that students may have differential learning experiences due to the
particular goal mix they endorse. Certainly more studies are needed to clarify the effects of
multiple goals on learning and achievement despite that increasingly more research evidence (e.g. Darnon, Dompnier, Gilliéron, & Butera, 2010; Ng, 2009) has affirmed their positive effects on
learning. Researching multiple goals using a student group other than Euro-American samples of
on-campus students may provide new insights into the operation of multiple goals (Pintrich,
Conley, & Kempler, 2003). This work is licensed under a Creative Commons Attribution 4.0 International License. The Current Study While accumulated evidence is supportive of the importance of performance-approach
goals to adult learners, some studies found contradictory evidence discrediting this (e.g. Eppler &
Harju, 1997). Sachs (2001) even argued that performance goals are not relevant to adult learners
because they have already demonstrated their academic abilities through their degree
qualifications. More research is warranted to examine the combined effects of performance-
approach goals and mastery goals on learning and achievement among adult distance learners. Taken together, this study argues that both mastery and performance-approach goals are
important motivational goals in the distance learning process and that they can contribute in
different ways to sustain an adaptive pattern of engagement for distance learners (cf. Barron &
Harackiewicz, 2001). Endorsing these two types of goals simultaneously allows distance learners
to vary their motivational focus on competence development or competence demonstration in
response to the specific demands of different learning situations. Most of the previous studies
(e.g. Ng, 2008, 2009), however, were limited by a cross-section design and failed to show the
effects of endorsing simultaneously mastery goals and performance-approach goals over time. More significantly, there is a lack of research that looks into distance learners’ motivation from a
longitudinally perspective covering learning at different points over an academic year. Addressing
this gap, the current study conducted three waves of surveys within an academic year to assess
distance learners’ motivated learning process in relation to 1) their general orientation to learning
at the beginning of the academic year (Time 1); 2) the completion of an assignment five months
later (Time 2); and 3) examination preparation at the end of the academic year (Time 3). This work is licensed under a Creative Commons Attribution 4.0 International License. The key research question is whether multiple-goal learners will hold a more engaged learning
pattern and have better achievement levels over the academic year when compared to other types
of goal users. To examine the effects of multiple goals at the individual level, four groups of
learners were examined: high-mastery/high-performance learners (HH), high-mastery/low-
performance learners (HL), low-mastery/high-performance learners (LH), and low-mastery/low-
performance learners (LL). The main issue was whether group membership moderated distance
learners’ engagement patterns in three data collection points. Engagement patterns were
measured by a host of dependent variables including learning strategies, regulatory strategies,
learning interest, self-efficacy and achievement levels. The Current Study In this specific study (Ng, 2008), it was found that distance learners who endorsed strong
performance-approach goals alongside other goals had a better achievement level than did those 89 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng focusing solely on mastery goals. In another study investigating the process of completing a
compulsory course assignment, Ng (2009) found that distance learners who held strong
performance-approach goals in their profiles had better achievement results compared to those
focusing on work avoidance goals. In addition, distance learners who endorsed both mastery and
performance-approach goals had the most adaptive pattern of engagement during the process of
completing the assignments. This adaptive engagement pattern was measured in terms of strategy
use, valuing of the task, self-efficacy, levels of anxiety and mastery focused responses to feedback
on their work. In a more recent study, Remedios and Richardson (2012) found that British
distance learners’ performance-approach goals predicted their course work and examination
marks. While accumulated evidence is supportive of the importance of performance-approach
goals to adult learners, some studies found contradictory evidence discrediting this (e.g. Eppler &
Harju, 1997). Sachs (2001) even argued that performance goals are not relevant to adult learners
because they have already demonstrated their academic abilities through their degree
qualifications. More research is warranted to examine the combined effects of performance-
approach goals and mastery goals on learning and achievement among adult distance learners. focusing solely on mastery goals. In another study investigating the process of completing a
compulsory course assignment, Ng (2009) found that distance learners who held strong
performance-approach goals in their profiles had better achievement results compared to those
focusing on work avoidance goals. In addition, distance learners who endorsed both mastery and
performance-approach goals had the most adaptive pattern of engagement during the process of
completing the assignments. This adaptive engagement pattern was measured in terms of strategy
use, valuing of the task, self-efficacy, levels of anxiety and mastery focused responses to feedback
on their work. In a more recent study, Remedios and Richardson (2012) found that British
distance learners’ performance-approach goals predicted their course work and examination
marks. The Current Study Adult distance learners are a unique student group who differ from campus-based students in
important ways. This student group is often populated by mature learners who often have
multiple roles derived from familial and employment responsibilities (Eppler & Harju, 1997;
Sachs, 2001). As a result, learning in a part-time mode and using a prolonged study period to
complete a degree programme are common characteristics among distance learners. This, of
course, calls into question distance learners’ sustained motivation; what motivates distance
learners to engage in such a prolonged process of learning? In the distance learning literature,
distance learners are generally described as learning-oriented. To learn for knowledge and
personal development are often distance learners’ main motivational foci when embarking on
distance learning (Eppler & Harju, 1997; Ng, 2006). In addition, Eppler and Harju (1997)
specifically found that adult learners, compared to young students, endorsed the use of mastery
goals over performance goals. Sachs (2001) found that mastery goals predicted adult learners’
enjoyment in completing course assignments but not their graded performance. More direct
support regarding the significant role of mastery goals for distance learners can be derived from
several recent studies (Ng, 2006, 2008, 2009; Remedios & Richardson, 2012) that produced
repeated empirical evidence supporting that learning for knowledge improvement, understanding
and competence development have been dominant forms of motivation for this group of learners. However, the extent to which mastery goals are the sole motivational source for distance learners
is questionable. Distance learners often have other learning goals that focus on career
advancement and different social considerations (e.g. Cochrane, 2000; Dearnley & Matthew,
2000; Cannon, Umble, Steckler, & Shay, 2001; Lyall & McNamara, 2000; Miller & Smith, 1998;
von Prummer, 1990; Wilson & Bagley, 1999). Adopting a multiple goal perspective, Ng (2008)
argued that different goals would provide different forms of motivational support that help
distance learners maintain their focus and cope with the demands of prolonged period of learning
while juggling work and family commitments simultaneously (cf. Barron & Harackiewicz, 2001). The Current Study Based on the multiple goal perspective, this
study hypothesised that the multiple-goal group (HH) would have a more engaged learning 90 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng pattern in Time 1, 2 and 3. In particular, compared to the other three groups, the HH group would
hold higher levels of interest and self-efficacy, use more deep and regulatory strategies but less
surface strategies during Time 1, 2 and 3. In terms of achievement levels, it was assumed that the
HH group would have higher levels of achievement in both Time 2 and 3 (cf. Ng, 2008, 2009;
Valle et al., 2003). pattern in Time 1, 2 and 3. In particular, compared to the other three groups, the HH group would
hold higher levels of interest and self-efficacy, use more deep and regulatory strategies but less
surface strategies during Time 1, 2 and 3. In terms of achievement levels, it was assumed that the
HH group would have higher levels of achievement in both Time 2 and 3 (cf. Ng, 2008, 2009;
Valle et al., 2003). Participants The participants were 272 adult learners enrolled in different degree programmes offered by the
four faculties (Education 43.5%, Science 9.4%, Arts 40.2%, and Business 6.9%) of a distance
learning university in Hong Kong. The majority of these participants were adult learners within
the age spans of 21-30 (30.8%), 31-40 (42%), and 41-50 (20.7%). The sample was gender biased
with 75% females and 19.5% males. Procedure A random sample of distance learners was generated from the university’s enrolment system, and
from which 400 potential participants were selected from major degree programmes offered by
the university’s four academic faculties in the beginning of an academic year. All the selected
learners had enrolled in a 10 credit-point year-long course that required learners to complete four
written assignments and an end-of-year examination. An invitation letter explaining the design of
the research was sent to these potential participants. In response, 308 distance learners agreed to
participate. The data were collected at the beginning of the academic year (Time 1), in the middle of the
academic year after the learners had completed their second written assignment (Time 2), and
finally, at the end of the academic year before the examination period (Time 3). In each round of
data collection, the participants were sent a cover letter together with a questionnaire and an
enclosed returning envelope. Follow-up strategies including sending a reminder letter and
contacting participants by phone were adopted to encourage them to complete and return the
questionnaires. Thirty-two learners failed to return at least one of the questionnaires and were
excluded from the study. The final sample was made up of 272 learners (a response rate of
88.31%) who had completed three questionnaires as required. This work is licensed under a Creative Commons Attribution 4.0 International License. Achievement goals. The measures of achievement goals for Time 1 and 3 were adapted from the Patterns of Adaptive
Learning Survey (PALS, Midgley et al., 2000). In the Time 2 survey, items were adapted from a
previous study on distance learners' completion of course assignments (Ng, 2009). Two scales of
achievement goals were included in all three surveys: mastery goals and performance-approach
goals. The mastery goals scale assessed a personal focus on learning and competence
development in the learning of the course, completion of an assignment and preparation for
examination. Five items were included in Time 1. A sample item in the Time 1 survey was "While
studying this course, I want to learn something new". Seven items assessed learners' mastery
goals for completing an academic assignment. A sample item was "When completing this essay, I
hoped to deepen my understanding of this course even though I might not get a high mark". Five
items assessed learners' mastery goals for examination preparation. A sample item was "While
preparing for the exam, I want to learn as much as possible in my revision". Performance-approach goals assessed learners' personal focus on gaining high achievement and
demonstrating competence. Five items were included in Time 1. A sample item in the Time 1
survey was "While studying this course, I want to get a good result". Four items assessed distance
learners' performance-approach goals for completing an assignment in Time 2. A sample item
was "While completing this essay, I intended to get a high mark". In Time 3, five items were
included in the scale of performance-approach goals for examination preparation but one of them
was dropped in order to improve the reliability score for this construct. Four items made up the
performance-approach goal construct in Time 3. A sample item was "While preparing for the
exam, I want to get a high mark". The original questionnaire in Time 1, 2 and 3 also included
items assessing performance-avoidance goals, which were not included in the subsequent
analyses due to low reliability. Measures The questionnaires of Time 1, 2 and 3 assessed learners’ achievement goals, use of strategies, self-
efficacy and learning interest in relation to their general learning orientation at the course level
(Time 1), the completion process of an academic essay (Time 2) and the preparation for final 91 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng examination (Time 3). All the items in Time 1 survey were preceded by a stem "While studying
this course …". Time 2 survey items were preceded by a stem, "When completing this essay…" and
were worded in such a way that learners were required to focus on how they had completed the
second written assignment. In Time 3, all the items were preceded by a stem, "While preparing
for the exam…". Items in Time 1 and 3 were set in a 5-point Likert scale (1=strongly disagree to 5
= strongly agree) while those in Time 2 followed a 7-point Likert-scale (1 = strongly disagree to 7
= strongly agree) in Ng (2009). Table 1 shows the Alpha values for each construct in three waves
of surveys. examination (Time 3). All the items in Time 1 survey were preceded by a stem "While studying
this course …". Time 2 survey items were preceded by a stem, "When completing this essay…" and
were worded in such a way that learners were required to focus on how they had completed the
second written assignment. In Time 3, all the items were preceded by a stem, "While preparing
for the exam…". Items in Time 1 and 3 were set in a 5-point Likert scale (1=strongly disagree to 5
= strongly agree) while those in Time 2 followed a 7-point Likert-scale (1 = strongly disagree to 7
= strongly agree) in Ng (2009). Table 1 shows the Alpha values for each construct in three waves
of surveys. Learning strategies. Learning strategies in the Time 1, 2 and 3 surveys included two contrasting scales: surface and
deep strategies. Surface strategies assessed learners' use of rehearsal-based strategies such as rote
learning and memorisation that lead to superficial understanding. In contrast, the deep strategies
construct included strategies that promote deep learning and encourage effort expenditure. Items This work is licensed under a Creative Commons Attribution 4.0 International License. 92 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng assessing these two contrasting types of strategies in Time 1 and 3 were adapted from the
Learning Approach Questionnaire (Biggs, 1987) while Time 2 items were taken from Ng (2009). Each surface and deep strategy scale in Time 1, 2, and 3 included five items. The sample items of
surface strategies in the Time 1, 2 and 3 respectively were, "I learn the course materials mainly by
rote, going over and over them until I know them by heart" (Time 1), "I just studied those
materials specified in the essay guidelines and thought it was unnecessary to study extra
materials" (Time 2), and "I go over and over the course materials to memorise the important
content that is likely to appear in the exam" (Time 3). The sample items for assessing deep
strategies in Time 1, 2, and 3 respectively were, "I think of real-life situations in which the
materials that I am learning would be useful" (Time 1), "I studied relevant materials related to
this essay and made notes carefully" (Time 2), and "I try to relate different parts of the course in
my revision" (Time 3). assessing these two contrasting types of strategies in Time 1 and 3 were adapted from the
Learning Approach Questionnaire (Biggs, 1987) while Time 2 items were taken from Ng (2009). Each surface and deep strategy scale in Time 1, 2, and 3 included five items. The sample items of
surface strategies in the Time 1, 2 and 3 respectively were, "I learn the course materials mainly by
rote, going over and over them until I know them by heart" (Time 1), "I just studied those
materials specified in the essay guidelines and thought it was unnecessary to study extra
materials" (Time 2), and "I go over and over the course materials to memorise the important
content that is likely to appear in the exam" (Time 3). Regulatory strategies. Regulatory strategies measured learners’ self-monitoring of their learning. Five items adapted
from the Motivated Strategies for Learning Questionnaire (MSLQ, Pintrich, Smith, & Garcia,
1993) assessed this construct in Time 1 and 3. Sample items in the Time 1 and 3 respectively were,
"I ask myself questions to help focus my understanding" (Time 1) and "I noticed that I often
missed important points and needed to study them again" (Time 3). Five items taken from Ng
(2009) were included in the construct of regulatory strategies for completing an assignment. A
sample item was "I have changed my usual way of study in order to understand the difficult
materials central to this essay". Learning strategies. The sample items for assessing deep
strategies in Time 1, 2, and 3 respectively were, "I think of real-life situations in which the
materials that I am learning would be useful" (Time 1), "I studied relevant materials related to
this essay and made notes carefully" (Time 2), and "I try to relate different parts of the course in
my revision" (Time 3). Efficacy beliefs. The efficacy beliefs scale used three items adapted from MSLQ (Pintrich et al., 1993) to assess
learners' perceptions of their abilities to complete the course and assignment, and prepare for the
examination. The sample items for Time 1, 2 and 3 respectively were, "I'm certain I can
understand the most difficult concepts and theories presented in the Course" (Time 1), "I believe I
am able to do well in this essay" (Time 2), and "I am confident that I am able to do well in the
exam" (Time 3). Outcome variables. Two achievement variables assessed the learning outcomes. Distance learners' scores of their
second assignment and examination results were collected from course convenors with the
permission from the participants. Results Table 1 shows the means, standard deviations and reliability scores for the variables in Time 1, 2
and 3. Table 2 shows the results of correlation analyses. As can be seen, distance learners' mastery
goals were related positively to self-efficacy, learning interest, and the use of deep and regulatory
strategies across Time 1, 2 and 3. These adaptive goals were negatively related to the use of
surface strategies across the three data collection points. These relational patterns were consistent
with those found among campus-based students (e.g. Barron & Harackiewicz, 2001). Performance-approach goals appeared to be adaptive in this study. These goals for performance
demonstration were related positively to self-efficacy, learning interest, and the use of deep and
regulatory strategies across Time 1, 2 and 3 but a negative relationship was found between these
goals and surface strategies. Both mastery and performance-approach goals were not related to
distance learners' assignment scores (Time 2) and examination results (Time 3). This work is licensed under a Creative Commons Attribution 4.0 International License. Interest. Distance learners' learning interest was assessed using 3 items adapted from MSLQ in Time 1, 2
and 3 (Pintrich et al., 1993). The sample items for Time 1, 2 and 3 respectively were, "I found this
course very interesting and enjoyed the time spent on it" (Time 1), "I enjoyed doing this
assignment" (Time 2), and "I enjoyed doing the revision for the exam" (Time 3). This work is licensed under a Creative Commons Attribution 4.0 International License. 93 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Table 1 Time 1 (Likert scale 1-5)
Time 2 (Likert scale 1-7)
Time 3 (Likert scale 1-5)
Mean
SD
Alpha
Mean
SD
Alpha
Mean
SD
Alpha
Mastery goals
3.78
.53
.72
5.23
.81
.86
3.72
.53
.73
Performance-
approach goals
3.50
.68
.63
4.95
.74
.64
3.38
.65
.66
Surface
strategies
2.82
.73
.63
3.75
.99
.69
2.84
.54
.54
Deep strategies
3.18
.54
.73
4.98
.84
.80
3.22
.54
.75
Regulatory
strategies
3.19
.46
.72
4.43
1.03
.63
3.16
.49
.79
Efficacy belief
3.07
.63
.78
4.57
.81
.70
3.09
.61
.76
Learning
interest
3.55
.67
.84
5.02
.92
.78
3.63
.70
.83 This work is licensed under a Creative Commons Attribution 4.0 International License. 94 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng rners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng Table 2 Table 2 Correlation Analyses
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
1. T1 Mastery goals
--
2. T1 Performance-
approach goals
.18
--
3. T1 Surface strategies -.19
-.12
--
4. T1 Deep strategies
.45
.21 -.32
--
5. T1 Regulatory
strategies
.30
.24 -.24
.67
--
6. T1 Efficacy
.31
.41 -.24
.37
.40
--
7. T1 Interest
.58
.13 -.30
.46
.34
.48
--
8. T2 Surface strategies
.21
-.14
-.15
-.15
--
9. T2 Deep strategies
.23
.26
.42
.46
.21
.18
--
10. T2 Regulatory
strategies
.19
.21
.24
.35
.48
--
11. T2 Efficacy
.18
.15
.15
.30
.32
.36
.24 -.38
.28
--
12. T2 Interest
.37
.17
.38
.24
.17
.42 -.19
.33
.13
.48
--
13. T3 Surface
strategies
-.20
.52 -.29 -.25 -.19 -.32
.28
.13 -.17 -.22
--
14. T3 Deep strategies
.39
.20 -.34
.65
.48
.34
.46 -.13
.45
.24
.36
.41 -.30
--
15. T3 Regulatory
strategies
.23
.24 -.32
.57
.62
.30
.27 -.13
.50
.27
.29
.25 -.24
.65
--
16. T3 Efficacy
.19
.26 -.19
.38
.40
.53
.31 -.35
.27
.49
.27 -.25
.46
.43
--
17. T3 Interest
.47
-.30
.39
.30
.30
.65 -.25
.24
.37
.49 -.37
.54
.35
.50
--
18. T2 Assignment
score
-.14
-.30
-.20
.31
.20
.13
--
19. Learners’ Goal Profiles and their Learning Patterns over an Academic Year
N Person-Centred Analyses Following Pintrich (2000), this study used the median split technique to classify learners into
different groups of goal users. To address the concern that median split may impose an artificial
structure without considering the underlying structure of the data, the current study used cluster
analysis and discriminant function analyses to verify the membership of the learner groups
produced by the median split technique. Using the median scores (3.80 for mastery goal and 3.33 for performance-approach goals in Time
1), four groups of distance learners were produced: high mastery / high performance learners
(HH; N=65), high mastery / low performance learners (HL; N=53), low mastery / high
performance learners (LH; N=68), and low mastery / low performance learners (LL; N=86). A
non-hierarchical cluster analysis specifying a 4-cluster solution was conducted to compare the
learners’ membership in different goal-user groups derived from both classifying techniques. The
resulting cluster solution located four clusters of learners with similar goal properties to the
groups derived from the median split technique. Comparing the membership of corresponding
groups produced by these two classification methods, 97.43% of learners were classified into
identical groups using both methods. Only seven learners had been classified differently. Further,
a discriminant function analysis significantly predicted the membership in these four groups
derived from the median split technique using mastery and performance-approach goals as
predictors. According to this significant discriminant function, 96.7% were classified correctly
into HH, HL, LH and LL groups. It can be concluded that the groupings of learners produced by
the median split technique were valid. To check the stability of these groupings of learners over time, the median split procedures were
repeated based on learners' scores on achievement goals in Time 2 and 3, respectively. The
membership in corresponding groups was then compared across three waves. It was found that
the classification of learners based on Time 2 and 3 data was 98% and 99% identical with the
Time 1 grouping described above. It can then be concluded that the groupings based on Time 1
data were stable over the whole academic year. These four groups did not differ in gender composition. However, a significant Chi square test
(χ2=30.86, d.f.= 6, p<.0001) found that HH and HL groups were populated by older learners
while LH and LL groups had more younger adults. The age factor was taken as a covariate in the
subsequent analyses. Table 1 T3 Exam score
-.14
-.12
-.23
-.21
.2
Note 1: * p<.05; **p<.01 (only significant correlation coefficients were shown) Correlation Analyses .29 This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. 95 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. Person-Centred Analyses Mixed ANOVAs were conducted using strategies, self-efficacy and interest
as within factors, groups as between-group factor, and the age factor was entered as a covariate. Standardised scores were used in these analyses. The main effects (time and group) and two-way
interactions were examined. Table 3 shows the descriptive statistics for these four goal user
groups in Time 1, 2 and 3. This work is licensed under a Creative Commons Attribution 4.0 International License. 96 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng Table 3 Means and Standard Deviations for Strategies, Efficacy, Interest, Goals and Achievement Levels
by Four Goal Groups by Four Goal Groups
High-
mastery/High
performance
(n=65)
High mastery /
low performance
(n=53)
Low mastery /
High performance
(n=68)
Low mastery /
low performance
(n=86)
Dependent
variables
M
SD
M
SD
M
SD
M
SD
Surface
strategies-T1
2.69 a
.71
2.68 a
.63
2.87 a
.78
2.93 a
.74
Surface
strategies-T2
3.47 a
.89
3.49 a
1.11
3.56 a
1.02
3.61 a
.92
Surface
strategies-T3
2.73 a,b
.53
2.67 b
.56
2.89 a, b
.56
2.98 a
.52
Deep
strategies-T1
3.52a
.52
3.37b
.46
3.05 c
.58
2.98 c
.39
Deep
strategies-T2
5.42a
.79
4.94b
.79
4.89 c
.98
4.78 c
.67
Deep
strategies-T3
3.53a
.51
3.35b
.56
3.07 c
.50
3.06 c
.43
Regulatory
strategies-T1
3.43 a
.48
3.22 b
.44
3.16 b
.50
3.04 b
.36
Regulatory
strategies-T2
4.81 a
1.04
4.30 b
1.06
4.49 b
.95
4.29 b
.92
Regulatory
strategies-T3
3.40 a
.41
3.24 b
.47
3.10 b
.52
3.07 b
.31
Efficacy
beliefs-T1
3.51 a
.62
3.00 b
.55
3.10 b
.56
2.81 b
.50
Efficacy
beliefs-T2
4.75 a
.71
4.61 b
.91
4.58 b
.85
4.45 b
.68
Efficacy
beliefs-T3
3.36 a
.52
3.09 b
.61
3.13 b
.59
2.98 b
.49
Interest-T1
3.95 a
.53
3.84 a
.54
3.34 b
.61
3.30 b
.59
Interest-T2
5.16 a
.96
5.07 a
.75
4.78 a
.98
4.52 b
.90
Interest-T3
3.91 a
.56
3.94 a
.63
3.44 b
.72
3.51 b
.43
Assignment
score
(0-100)
72.27 a
14.00
72.72 a
13.24
73.58 a
11.55
73.53 a
11.98
Examination
score
(0-100)
59.78 a
12.27
59.74 a
11.79
62.92 a
10.09
63.50 a
12.10
Note 1. T1=Time 1; T2=Time 2; T3=Time 3; Note 2. T1 & T3 scores ranged between 1 and 5; T2
scores ranged between 1 and 7; Note 3. Means within a row with different superscripts are
significantly different from one another; Note 4. Standardised scores were used in mixed ANOVA
analyses. Note 1. T1=Time 1; T2=Time 2; T3=Time 3; Note 2. T1 & T3 scores ranged between 1 and 5; T2
scores ranged between 1 and 7; Note 3. Means within a row with different superscripts are
significantly different from one another; Note 4. Standardised scores were used in mixed ANOVA
analyses. Note 1. T1=Time 1; T2=Time 2; T3=Time 3; Note 2. Surface strategies. The four groups differed in the use of surface strategies, F(1, 245)=2.65, p<.05, η2=.03. In
particular, the group differences were found in Time 3 where the LL group had the highest scores
in surface strategies and HL had the lowest scores; the HH and LH groups were not different from
each other in the use of surface strategies. However, both time and time x group interactive effects
were not significant. This work is licensed under a Creative Commons Attribution 4.0 International License. Table 3 T1 & T3 scores ranged between 1 and 5; T2
scores ranged between 1 and 7; Note 3. Means within a row with different superscripts are
significantly different from one another; Note 4. Standardised scores were used in mixed ANOVA
analyses. This work is licensed under a Creative Commons Attribution 4.0 International License. 97 Deep strategies. The four groups differed in the use of deep strategies, F(1, 245)=15.94, p<.0001, η2=.16. The HH
group had the highest scores in the use of deep strategies followed by the HL group across three
surveys. Both LH and LL groups had a similar low level in the use of deep strategies. However,
this significant group effect was qualified by a significant time x group interaction, Huynh-Feldt
F(5.85, 489.63)=2.61, p<.05, η2=.03, indicating that the inter-group difference in the use of deep
strategies was a function of Time. As shown in Figure 1, the HH and HL groups had higher levels
of deep strategies than did LH and LL groups in Time 1. An identical pattern was found again by
the end of the year. When completing the assignment, the HH group had the highest level of deep
strategies. The HL group, however, experienced a significant drop in the use of deep strategies
when completing the assignment in Time 2. This work is licensed under a Creative Commons Attribution 4.0 International License. 98 rners’ Goal Profiles and their Learning Patterns over an Academic Yea
Ng Figure 1. Time x Group Interaction: Deep strategies. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination
3
2
1
Time
0.75000
0.50000
0.25000
0.00000
-0.25000
-0.50000
Deep strategies
HH
HL
LH
LL 2
1
0.75000
0.50000
0.25000
0.00000
-0.25000
-0.50000
Deep strategies Figure 1. Time x Group Interaction: Deep strategies. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination Learners’ Goal Profiles and their Learning Patterns over an Academic Year Interest. A significant main effect, F(1, 245)=21.07, p<.0001, η2=.21, indicates that the four groups differed in
their levels of learning interest. A significant time x group interaction qualified this group
differential pattern, Huynh-Feldt F(5.72, 471.23)=2.17, p<.05, η2=.03. Figure 3 shows that both HH
and HL groups had higher interest levels than did LH and LL groups in Time 1 and 3. In Time 2,
the LH group had greater interest in completing the assignment. The HH, HL, and LH groups had
similar levels of interest. Self-efficacy. A significant main effect on group, F(1, 245)=11.01, p<.0001, η2=.12, indicates that these four groups
of learners differed in their levels of efficacy beliefs. HH and HL groups had a stronger sense of
self-efficacy than did the LH and LL groups. A significant time x group effect indicates that such a
differential pattern varied with time, Huynh-Feldt F(5.99, 509.04)=3.31, p<.005, η2=.04. As shown in
Figure 2, the HH group had higher levels of efficacy beliefs than did the other three groups in
Time 1, 2 and 3. Regulatory strategies. The four groups differed with each other in the use of regulatory strategies, F(1, 245)=9.89,
p<.0001, η2=.11. The HH group used more regulatory strategies than did the other three groups in
Time 1, 2, and 3. The main effect on time and time x group interactive effect were nonsignificant. This work is licensed under a Creative Commons Attribution 4.0 International License. 99 This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. Achievement scores. The four groups did not differ from one another on achievement scores. In other words, they had
similar scores in their assignments in Time 2 and examination in Time 3. This intriguing result
will be discussed in the next section. This work is licensed under a Creative Commons Attribution 4.0 International License. This work is licensed under a Creative Commons Attribution 4.0 International License. 100 100 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Figure 2. Time x group interaction: Efficacy beliefs
Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. 3
2
1
Time
0.80000
0.60000
0.40000
0.20000
0.00000
-0.20000
-0.40000
Efficacy beliefs
HH
HL
LH
LL 2
1
Tim
0.80000
0.60000
0.40000
0.20000
0.00000
-0.20000
-0.40000
Efficacy beliefs Efficacy beliefs Figure 2. Time x group interaction: Efficacy beliefs Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. This work is licensed under a Creative Commons Attribution 4.0 International License. 101 101 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Figure 3. Time x group interaction: Interest. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. 3
2
1
Time
0.60000
0.40000
0.20000
0.00000
-0.20000
-0.40000
-0.60000
Interest
HH
HL
LH
LL 3 Figure 3. Time x group interaction: Interest. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. This work is licensed under a Creative Commons Attribution 4.0 International License. Discussion The research question in this study focused on the differential patterns of learning and
achievement associated with distance learners holding different types of goals at three different
points of learning during an academic year. A key comparison can be made between distance
learners who were high in both mastery and performance-approach goals (HH) and those high in
mastery goals but low in performance-approach goals (HL). According to the multiple goal
perspective, the HH group should show a more adaptive pattern in the use of strategies, levels of
efficacy and learning interest in Time 1, 2 and 3. Aligning with previous studies (Ng, 2008, 2009),
the current results favoured the HH group over the HL group. While the two groups showed
similar levels in their interest in learning and endorsed a similar level of deep strategies at the
beginning and end of the academic year, the HH learners were more efficacious and used more
regulatory strategies. Unlike the HL group, HH learners did not show a significant decline in the
use of deep strategies when completing their assignments in Time 2. Overall these findings were
in line with previous research, such as Valle et al. (2003), that supports the benefits of endorsing
multiple goals at the individual level. In terms of achievement levels, the median split analyses failed to find any significant result
supporting the claim that endorsing multiple goals would be associated with a higher level of
achievement. Contrary to the prediction, the HH group did not have better scores in their
assignments and examination results. Though the four groups did not differ in their achievement
scores, the mean scores for the HH group were unexpectedly the lowest among the four groups. This intriguing finding was inconsistent with previous studies using similar student groups (Ng,
2008, 2009). More research is needed to examine the relationship between achievement and
goals. The inconsistency result calls into question the benefits of holding multiple goals and
whether distance educators should promote them if they will not necessarily lead to higher
achievement. In the discussion below, the concept of ‘multiple pathways’ (Pintrich, 2000) was
used to interpret the nonsignificant relationship between achievement levels and different goal
profiles. Following Pintrich (2000), it can be argued that different distance learner groups learnt through
different pathways but arrived at a similar level of achievement. Achievement scores. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. Note: HH=High Mastery, High Performance; HL=High Mastery, Low Performance; LH=Low
Mastery, High Performance; LL=Low Mastery, Low Performance; Time1=At the beginning of the
academic year; Time 2=After completing the second written assignment; Time 3=During the
preparation for the final examination. This work is licensed under a Creative Commons Attribution 4.0 International License. 102 102 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng This work is licensed under a Creative Commons Attribution 4.0 International License. Discussion The HH group showed a highly
engaged pattern characterised by strong regulation, deep learning, and high levels of interest and
confidence. The HL group had a similar learning trajectory over the year except that they used
less deep and regulatory strategies and had a lower level of interest. In contrast, the LH and LL
groups showed a rather disengaged pattern of learning over the academic year characterised by
surface learning and low levels of regulation, interest and confidence. This multiple pathway
argument alerts distance educators to different patterns of learning associated with different goal
profiles. The important question is which goal profile is associated with a more engaged pattern
that is likely to sustain distance learners’ motivation to learn. It is in this specific context that the 103 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Ng current findings support the promotion of multiple goals. Distance learners who learn with deep
and regulatory strategies and consider the learning interesting and maintain a higher level of
confidence are more likely to persist. It can be said that multiple goal learners are more
motivated, though this may not necessarily lead to better results. current findings support the promotion of multiple goals. Distance learners who learn with deep
and regulatory strategies and consider the learning interesting and maintain a higher level of
confidence are more likely to persist. It can be said that multiple goal learners are more
motivated, though this may not necessarily lead to better results. While this ‘multiple pathways’ argument may be sufficient to explain why multiple-goal learners
(HH) and mastery-only learners (HL) had similar levels of achievement in their course
assignments and examination, it does not help explain why learners in the LH and LL groups
could also achieve similar scores in these assessment tasks to those in the HH and HL groups. One plausible explanation is that the LH and LL learners might have engaged in "periods of
achievement spark" in order to deal with immediate demands for completing the written
assignments and passing the examination. During such highly motivated periods, these learners
might have engaged intensively and strategically in order to complete the required academic
tasks. This work is licensed under a Creative Commons Attribution 4.0 International License. Discussion However, for most of the time during the academic year, they might be settled with a
minimum level of motivation and engagement sufficient to get them through their courses. Another plausible explanation is that these two groups of learners might have relied on other
forms of motivation to drive their learning. For example, Ng (2008) found that distance learners
who held strong work-related goals had better performance than did those focusing on mastery
and performance-approach goals, combined or separately. Certainly, additional research is
required to look into the “achievement spark” hypothesis and explore distance learners’
alternative motivation in addition to mastery and performance considerations. The nonsignificant result between the HH and HL groups of distance learners in achievement
levels prevents us from making a strong claim about endorsing multiple goals over the use of
mastery goals. However, the current prospective study showed that multiple goals learners (HH)
maintained a more engaged pattern of learning than did the mastery-only learners across three
points of learning over an academic year. Given the challenge of sustaining engagement over the
prolonged period of distance learning, endorsing multiple goals in one's goal profile should be
optimal for distance learners. Of course, the current findings warn us that holding a multiple goal
profile does not necessarily lead to a better achievement level. Further research on the effects of
multiple goals on achievement among distance learners is still warranted. Finally, aligning with
previous studies (e.g. Meece & Holt, 1993), the current research and its findings highlighted the
importance of a person-centred analytical lens to examine the effects of multiple goals on learning
and achievement. This work is licensed under a Creative Commons Attribution 4.0 International License. The current results need to be interpreted with the following considerations. This study did not
assess learners' prior achievement levels, which is an important factor that should be examined or
controlled for in the future research. Further, Barron and Harackiewicz (2001) located significant
effects of multiple goals on achievement using a sample of students enrolled in a single course. The current study, however, involved distance learners in diverse courses and therefore variation
in assignment and examination grading processes in different courses may be another important 104 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng
factor that might have contributed to the nonsignificant difference in achievement levels between
the four groups of goal users. Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Discussion Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng
factor that might have contributed to the nonsignificant difference in achievement levels between
the four groups of goal users. d their Learning Patt
Ng ers’ Goal Profiles and their Learning Patterns ove
Ng Ng factor that might have contributed to the nonsignificant difference in achievement levels between
the four groups of goal users. factor that might have contributed to the nonsignificant difference in achievement levels between
the four groups of goal users. In conclusion, the current findings indicate that distance learners learn with different goal
profiles that are associated with different learning patterns. An important finding in this study
was that distance learners who endorsed both mastery and performance-approach goals engaged
in deep learning using adaptive strategies consistently throughout an academic year. These
multiple goal learners remained interested in learning and had confidence in their learning
abilities across three different survey points over the year. This finding is consistent with previous
research using distance learners (e.g. Ng, 2009) and campus-based students (e.g. Barron &
Harackiewicz, 2001). Given the associated engaged learning pattern, it is less likely for this type of
motivated learners who persistently find learning interesting and remain confident about their
abilities to drop out of their distance education courses and programs. While endorsing both goals
may not necessarily lead to higher achievement levels, it is important to recognise that distance
learners who learn with these goals are more engaged in their learning. Future research needs to
examine how these goals can be promoted in distance education courses and programs in order
that distance learners remain motivated and persist with their learning. This work is licensed under a Creative Commons Attribution 4.0 International License. 105 105 References Archer, J. (1994). Achievement goals as measure of motivation in university students. Contemporary Educational Psychology, 19, 430-446. doi:10.1006/ceps.1994.1031 Barron, K. E., & Harackiewicz, J. M. (2001). Achievement goals and optimal motivation: Testing
multiple goal models. Journal of Educational Psychology, 80(5), 706-722. doi:10.1016/B978-012619070-0/50031-3 Biggs, J. (1987). Student approaches to learning and studying. Melbourne: Australian Council
for Educational Research. Cannon, M. M., Umble, K. E., Steckler, A., & Shay, S. (2001). "We're living what we're learning":
Student perspectives in distance learning degree and certificate programs in public
health. Journal of Public Management Practice, 7(1), 49-59. Cochrane, C. (2000). The reflections of a distance learner 1977-1997. Open Learning, 15(1), 17-34. doi:10.1080/026805100115443 Darnon, C., Dompnier, B., Gilliéron, O., & Butera, F. (2010). The interplay of mastery and
performance goals in social comparison: A multiple-goal perspective. Journal of
Educational Psychology, 102(1), 212-222. doi:10.1037/a0018161 Dearnley, C., & Matthew, B. (2000). A group of nurses' experience open learning: Exploring the
impact. Open Learning, 15(2), 191-206. doi:10.1080/713688397 Dweck, C. S. (1986). Motivational processes affecting learning. American Psychologist, 41(10),
1040-1048. doi:10.1037//0003-066X.41.10.1040 Elliot, M. A., & Harackiewicz, J. (1996). Approach and avoidance achievement goals and intrinsic
motivation: A mediational analysis. Journal of Personality and Social Psychology, 70,
968-980. doi:10.1037//0022-3514.70.3.461 Eppler, M. A., & Harju, B. L. (1997). Achievement motivation goals in relation to academic
performance in traditional and non-traditional college students. Research in Higher
Education, 38, 557-573. doi:10.1023/A:1024944429347 Harackiewicz, J. M., Barron, E., Carter, S. M., Lehto, A. T., & Elliot, A. J. (1997). Predictors and
consequences of achievement goals in the college classroom: Maintaining interest and
making the grade. Journal of Personality and Social Psychology, 73, 1284-1295. doi:10.1037//0022-3514.73.6.1284 This work is licensed under a Creative Commons Attribution 4.0 International License. 106 Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Hulleman, C. S., Schrager, S. M., Bodmann, S. M., & Harackiewicz, J. M. (2010). A meta-analytic
review of achievement goal measures: Different labels for the same constructs or different
constructs with similar labels? Psychological bulletin, 136(3), 422. Liyanagunawardena, T. R., Adams, A. A., & Williams, S. A. (2013). MOOCs: A systematic study of
the published literature 2008-2012. International Review of Research in Open and
Distance Learning, 14(3), 202-227. Lyall, R., & McNamara, S. (2000). Influences on the orientations to learning of distance
education students in Australia. Open Learning, 15(2), 107-121. doi:10.1080/713688396 Meece, J. L., & Holt, K. (1993). A pattern analysis of students' achievement goals. Journal of
Educational Psychology, 85(4), 582-590. doi:10.1037//0022-0663.85.4.582 Midgley, C., Kaplan, A., & Middleton, M. (2001). Performance-approach goals: What, for whom,
under what circumstances, and at what cost? Journal of Educational Psychology, 93(1),
77-86. doi:10.1037//0022-0663.93.1.77 Midgley, C., Maehr, M. L., Hruda, L. Z., Anderman, E., Anderman, L., Freeman, K. E., Gheen, M.,
Kaplan, A., Kumar, R., Middleton, M.J., Nelson, J., Roeser, R., & Urdan, T. (2000). The
patterns of adaptive learning scales. Ann Arbor: University of Michigan. Miller, C., & Smith, C. (1998). Professional development by distance education: Does distance
lend enhancement? Cambridge Journal of Education, 28(2), 221-230. doi:10.1080/0305764980280207 Ng, C. H. (2006). The role of achievement goals in the completion of a course assignment:
Examining the effects of performance-approach goals and combined goals. Open
Learning, 21(1), 33-48. doi:10.1080/02680510500472189 Ng, C. H. (2008). Multiple-goal learners and their differential patterns of learning. Educational
Psychology, 28(4), 439-456. doi:10.1080/01443410701739470 Ng, C. H. (2009). Profiling learners' achievement goals when completing academic essays. Educational Psychology, 29(3), 279-296. doi:10.1080/01443410902797988 Pintrich, P. R. (2000). Multiple goals, multiple pathways: The role of goal orientation in learning
and achievement. Journal of Educational Psychology, 92(3), 544-555. doi:10.1037//0022-0663.92.3.544 Pintrich, P. R., Conley, A. M., & Kempler, T. M. (2003). Current issues in achievement goal theory
and research. International Journal of Educational Research, 39, 319-337. doi:10.1016/j.ijer.2004.06.002 This work is licensed under a Creative Commons Attribution 4.0 International License. 107 This work is licensed under a Creative Commons Attribution 4.0 International License. Learners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng Pintrich, P. R., Smith, D. A. F., & Garcia, T. (1993). Reliability and predictive validity of the
motivated strategies for learning questionnaire (MSLQ). Educational and Psychological
Measurement, 53(3), 801-813. doi:10.1177/0013164493053003024 Remedios, R., & Richardson, J. T. E. (2012). Achievement goals in adult learners: Evidence from
distance education. British Journal of Educational Psychology, 83(4), 664-685. doi:10.1111/bjep.12001 Sachs, J. (2001). A path model for adult learner feedback. Educational Psychologist, 21, 267-275. doi:10.1080/01443410123859 Senko, C., Hulleman, C. S., & Harackiewicz, J. (2011). Achievement goal theory at the crossroads:
Old controversies, current challenges, and new directions. Educational Psychologist,
46(1), 26-27. doi:10.1080/00461520.2011.538646 Simpson, O. (2008). Motivating learners in open and distance learning: do we need a new theory
of learner support? Open Learning, 23(3), 159-170. Simpson, O. (2013). Student retention in distance education: are we failing our students? Open
Learning, 28(2), 105-119. Skaalvik, E. M. (1997). Self-enhancing and self-defeating ego orientation: Relations with task and
avoidance orientation, achievement, self-perception, and anxiety. Journal of Educational
Psychology, 89, 71-81. doi:10.1037/0022-0663.89.1.71 Valle, A., Cabanach, R. G., Núñez, J. C., González-Pienda, J., Rodríguez, S., & Piñeiro, I. (2003). Multiple goals, motivation and academic learning. British Journal of Educational
Psychology, 73, 71-87. doi:10.1348/000709903762869923 von Prummer, C. (1990). Study motivation of distance students: A report on some results from a
survey done at the FernUniverstat in 1987/88. Research in Distance Education, 2(2), 2-6. Wilson, V., & Bagley, L. (1999). Learning at a distance: The case of the community pharmacist. International Journal of Lifelong Education, 18(5), 355-369. doi:10.1080/026013799293603 Wolters, C. A. (2004). Advancing achievement goal theory: Using goal structures and goal
orientations to predict students' motivation, cognition, and achievement. Journal of
Educational Psychology, 96(2), 236-250. doi:10.1037/0022-0663.96.2.236 This work is licensed under a Creative Commons Attribution 4.0 International License. 108 Learners’ Goal Profiles and their Learning Patterns over an Academic Year earners’ Goal Profiles and their Learning Patterns over an Academic Year
Ng © Ng This work is licensed under a Creative Commons Attribution 4.0 International License. 109 109
|
https://openalex.org/W1989109651
|
https://periodicos.uem.br/ojs/index.php/ActaSciBiolSci/article/download/5876/5876
|
Portuguese
| null |
Análise de processos alternativos na preparação de esqueletos para uso didático
|
Acta Scientiarum. Biological Sciences
| 2,008
|
cc-by
| 5,919
|
Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
uso didático
uso didático
uso didático
uso didático Márcio José da Silveira1,2*, Gustavo Monteiro Teixeira3 e Edson Fontes de Oliveira1,2
1Faculdade de Apucarana, Apucarana, Paraná, Brasil. 2Núcleo de Pesquisas em Limnologia, Ictiologia e Aquicultura, Universidade
Estadual de Maringá, Av. Colombo, 5790, 87020-900, Maringá, Paraná, Brasil. 3Universidade Estadual Paulista, Botucatu, São Paulo,
Brasil. *Autor para correspondência. E-mail: s.marciojs@gmail.com RESUMO. Laboratórios de Zoologia em Instituições de Pesquisa e Ensino têm
demonstrado grande demanda por peças anatômicas para utilização em aulas práticas. No
entanto, embora a bibliografia voltada para este tema não seja escassa, são raras as análises
das relações custo/benefício de tais processos levando em consideração aspectos técnicos,
como tempo de preparo, qualidade das peças e recursos materiais e humanos. Além de
técnicas usualmente utilizadas, novos produtos de baixo custo, não-citados na literatura e
encontrados facilmente no mercado, também foram testados. O principal objetivo deste
trabalho foi a elaboração e organização de informações sobre técnicas alternativas de
maceração de peças ósseas para coleções didáticas, realizando análises comparativas sobre as
relações custo/benefício de cada técnica. Foram testados 12 produtos, dos quais foram
avaliados os resultados a diferentes condições experimentais, tais como: concentração e
combinação dos reagentes, temperatura, pH e tempo de exposição das peças aos reagentes. Os resultados indicaram que os produtos mais recomendados para a utilização nos processos
de maceração foram: a água, o peróxido de hidrogênio e o suco de mamão (Carica papaya). Palavras-chave: maceração, clarificação de esqueletos, peróxido de hidrogênio, suco de mamão. ABSTRACT. Analysis of alternative processes of skeleton preparation for
educational uses. Zoology laboratories at institutions of research and education have
shown great demand for anatomical parts for practical lessons. However, although the
bibliography is not necessarily scarce, analyses of the cost/benefit relations of such processes
are rare, considering aspects such as preparation time, quality of the anatomical parts, and
material and human resources. In addition to the common techniques already used, new
low-cost products, not cited in literature and easily found in the market, were also tested. The main objective of this work was to elaborate and organize information on techniques of
bone parts maceration for study collections, making comparative analyses on the
cost/benefit relations of each technique. Twelve products were tested, evaluating
experimental conditions such as: concentration and combination of reagents, temperature,
pH and time of exposure of the parts to the reagents. Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
Análise de processos alternativos na preparação de esqueletos para
uso didático
uso didático
uso didático
uso didático The results indicated that the products
recommended for the use in the maceration processes were: water, hydrogen peroxide and
papaya juice (Carica papaya). Key words: maceration, skeleton clarification, hydrogen peroxide, papaya juice. Acta Sci. Biol. Sci. DOI: 10.4025/actascibiolsci.v30i4.5876 DOI: 10.4025/actascibiolsci.v30i4.5876 Introdução
Introdução
Introdução
Introdução estudos anatômicos e uso didático. Este preparo
deve seguir algumas etapas. Inicialmente, deve-se
evitar o uso de ossos de animais que tenham tido
como causa da morte enfermidades ósseas, pois as
mesmas podem descaracterizar as estruturas
morfológicas originais. O próximo passo seria o
descarnamento, o qual consiste na retirada da tela
subcutânea e músculos, evitando danificar as
superfícies ósseas. Esta etapa é realizada com o
auxílio de instrumentos cirúrgicos de dissecção
(Auricchio e Salomão, 2002). A última etapa
refere-se à maceração propriamente dita, ou seja,
manter as estruturas anatômicas em substâncias Laboratórios de Zoologia de Instituições de
Pesquisa e Ensino têm demonstrado grande demanda
por peças anatômicas para utilização em aulas práticas. O uso de esqueletos auxilia nas atividades científicas e
didáticas, pois fornece informações seguras sobre as
adaptações específicas dos vertebrados como, por
exemplo, sustentação, postura e modo de locomoção
(Hildebrand e Goslow, 2006). O preparo adequado de esqueletos tem grande
importância, pois por meio dele pode-se adquirir
peças ósseas de qualidade, mais propícias para Maringá, v. 30, n. 4, p. 465-472, 2008 466 Silveira et al. específicas
com
capacidade
para
dissolver
elementos não-ósseos. Nesta técnica, podem ser
utilizados
diferentes
processos
químicos,
biológicos ou mecânicos, aplicados isoladamente
ou combinados. Os processos químicos são,
geralmente,
os
mais
agressivos,
porém
possibilitam a obtenção mais rápida de resultados. específicas
com
capacidade
para
dissolver
elementos não-ósseos. Nesta técnica, podem ser
utilizados
diferentes
processos
químicos,
biológicos ou mecânicos, aplicados isoladamente
ou combinados. Os processos químicos são,
geralmente,
os
mais
agressivos,
porém
possibilitam a obtenção mais rápida de resultados. químico. Há vários fatores referentes às peças ósseas que
devem ser levados em consideração na preparação de
esqueletos, como, por exemplo, a necessidade de
desarticulação completa ou de manutenção de
cartilagens e articulações. Dessa forma, o objetivo
didático do uso das peças deve ser definido antes da
escolha da técnica a ser utilizada. Alguns métodos de
preparação tornam as peças inúteis para estudos
paleontológicos, ecomorfológicos e de morfologia
funcional que necessitem de comparação de
determinadas características entre indivíduos ou
espécies. Diversos produtos químicos são utilizados para a
maceração de esqueletos, por isso é preciso escolher
o produto que melhor se adapte às condições de cada
laboratório, ao custo, ao tempo de preparo e à
facilidade de clareamento. Kawamoto et al. Acta Sci. Biol. Sci. Introdução
Introdução
Introdução
Introdução (2006),
por exemplo, utilizaram, em seus experimentos,
peróxido de hidrogênio em diversas concentrações,
pois necessitavam de esqueletos desarticulados e
livres
de
todo
tecido
para
estudos
em
zooarqueologia. Embora tenha grande importância para o Ensino
e a Pesquisa, a manutenção de coleções zoológicas é
negligenciada em muitas Instituições, e mesmo as
mais criteriosas possuem acervos consideravelmente
incompletos. Por exemplo, é alarmante que quase
um terço dos espécimes de aves não esteja
representado em nenhuma coleção do mundo
(Matthiesen, 1993), e é possível que isso ocorra em
relação a outros grupos zoológicos. g
Muitas plantas acumulam substâncias orgânicas que
podem ser extraídas em quantidades suficientes para
serem utilizadas na maceração, como, por exemplo, o
suco do mamão (Balandrin et al., 1985; Baker et al.,
2003; Davis e Payne, 2003). Outros processos
biológicos bem conhecidos são a maceração por
Dermestes (Hildebrand, 1968; Maiorana e Valen, 1985;
Alvarenga, 1992; Ladeira e Höfling, 2007; Mendéz e
Höfling, 2007) e por Cirolana (Packard, 1959). Os
Dermestes, coleópteros com ampla distribuição
geográfica que se alimentam da carne seca residual da
carcaça de animais mortos, são indicados para a limpeza
de esqueletos delicados ou quando a estrutura óssea
está relativamente intacta. O tempo de permanência das
peças anatômicas sob a ação dos Dermestes depende da
abundância de larvas na colônia (Franco et al., 2001). Ainda que a literatura relacionada aos processos
de maceração não seja escassa, são raras as análises
das relações custo/benefício da aplicação desses
processos levando em consideração aspectos como
tempo de preparo, qualidade das peças e a
necessidade de recursos materiais e humanos. Com
o intuito de analisar processos alternativos de
preparação de esqueletos para uso didático, neste
trabalho foram realizados testes com diferentes
técnicas de maceração, variando as condições
experimentais, como qualidade e concentração dos
reagentes, temperatura, pH e tempo de exposição
das peças aos reagentes. Os Cirolana são isópodes carnívoros marinhos
com hábitos noturnos, também utilizados na
preparação de esqueletos. Os peixes aprisionados em
redes de espera são, frequentemente, atacados por
esses crustáceos, os quais entram pela boca e
brânquias e se alimentam do tecido abaixo da pele,
da região anterior em direção à base da nadadeira
caudal, reduzindo-os a pele e esqueleto (Packard,
1959). Nesses processos biológicos, os organismos
promovem a retirada de tecidos com eficiência e sem
risco de danificação das peças, no entanto, são
processos relativamente demorados. Preparo de esqueletos para uso didático Figura 1. Peças ósseas de Cachorro-do-mato (Cerdocyon thous)
após processo de descarne. As peças foram lavadas em água corrente por
alguns minutos para que o excesso de sangue
acumulado durante o descarne fosse removido,
minimizando a chance de ocorrência de manchas
que pudessem comprometer os processos de
clareamento. Todas as soluções utilizadas para a maceração,
testadas neste trabalho, foram preparadas em solução
aquosa com três concentrações iniciais de pH: (i)
ácida: 3,2-3,5; (ii) neutra: 7,0-7,3; (iii) básica: 9,7-
10,0. Após o preparo das soluções, os valores finais
de pH foram aferidos em Peagâmetro de bancada
(Digimed APA 200), para comparações posteriores. As diferentes soluções utilizadas para maceração
foram submetidas às três distintas concentrações de
pH, permitindo testar a hipótese de que as peças
expostas às soluções com valores mais baixos de pH
(ácida) apresentariam maior facilidade na retirada
dos tecidos. Figura 1. Peças ósseas de Cachorro-do-mato (Cerdocyon thous)
após processo de descarne. Tabela
1. Substâncias
utilizadas
nas
macerações,
suas
concentrações, intervalo de tempo de preparo, animais e
estruturas anatômicas utilizadas. Tabela
1. Substâncias
utilizadas
nas
macerações,
suas
concentrações, intervalo de tempo de preparo, animais e
estruturas anatômicas utilizadas. Substâncias
Concentração Condições e
tempo de
preparo
Animal utilizado
Estruturas
anatômicas
usadas
Água amoniacal
5%
Repouso
por três
dias e
fervura
por 25 min. Cachorro-do-mato
(Cerdocyon thous)
e avestruz
(Struthio camelus)
Tíbiatarso
e fêmur
Água amoniacal
1%
Repouso
por dois
dias e
fervura
por 15 min. Cachorro-do-mato
(Cerdocyon thous)
e avestruz
(Struthio camelus)
Tíbiatarso
e fêmur
Álcool
40%
Repouso por
sete dias
Passeriforme
Esqueleto
completo
Água
Potável
Repouso por
20 dias
Jabuti piranga
(Gerochelone
carbonaria)
Cintura
pélvica
Suco de mamão
(Carica papaya)
15%
Repouso
e fervura
por 30 min. Cachorro-do-mato
(Cerdocyon thous)
e avestruz
(Struthio camelus)
Vértebras
e fêmur
Ácido muriático
50%
Repouso
a 20 min.,
40 min. e 60 min. Cachorro-do-mato
(Cerdocyon thous)
Vértebras
Peróxido de
hidrogênio
9%
Repouso
por dois
dias e
fervura
por 15 min. Cachorro-do-mato
(Cerdocyon thous),
Avestruz
(Struthio camelus)
e Tatu galinha
(Dasypus
novemcinctus)
Costelas,
escápula
e fêmur
Hipoclorito
de sódio
23%
Repouso
por seis
dias e
fervura
por 25 min. Veado mateiro
(Mazama americana)
e avestruz
(Struthio camelus)
Cintura
pélvica
e fêmur
Intercapt
25%
Repouso
por dois
dias e
fervura
por 20 min. Cachorro-do-mato
(Cerdocyon thous),
e avestruz
(Struthio camelus)
Vértebras
e fêmur
Solupan
25%
Repouso
por dois
dias e
fervura
por 20 min. Material e métodos
Material e métodos
Material e métodos
Material e métodos Antes da realização dos testes com as diferentes
técnicas de maceração, todas as peças anatômicas
utilizadas foram submetidas a processos manuais de
descarne para retirada do excesso de músculos,
gordura, nervos e ligamentos (Figura 1), utilizando-
se pinças, bisturis e tesouras no laboratório de
Zoologia da Faculdade de Apucarana – FAP. Essas
peças foram obtidas de diversos cadáveres de
vertebrados silvestres de classes diferentes, bem
como de diversos animais domésticos (um exemplar
de cada espécie) (Tabela 1). Os cadáveres de animais
silvestres foram doados por uma empresa privada
produtora
de
papel
(Inpacel),
enquanto
os
domésticos foram doados pela comunidade do
entorno da FAP. Os processos mecânicos são aqueles que
eliminam
gordura,
músculos
e
cartilagens
manualmente, com auxílio de instrumentos como
pinças e bisturis. Trata-se do primeiro tratamento a
que
as
peças
anatômicas
são
submetidas,
constituindo na retirada da maioria dos tecidos
presentes nos esqueletos; em muitos casos, no
entanto, ainda é necessário passar por um processo
de maceração com a utilização de algum produto Maringá, v. 30, n. 4, p. 465-472, 2008 467 Acta Sci. Biol. Sci. Preparo de esqueletos para uso didático Cachorro-do-mato
(Cerdocyon thous)
e avestruz
(Struthio camelus)
Vértebras,
costelas
e tíbiatarso
Suco de abacaxi
(Ananas comosus)
Pedaços
do fruto
Repouso
por sete
dias e um
dia,
e fervura
por 30 min. Avestruz
(Struthio camelus)
e Galinha
(Gallus domesticus)
Tíbiatarso
e úmero
Amaciante
de carne
5%
Repouso
por dois
dias e
fervura
por 30 min. Cachorro-do-mato
(Cerdocyon thous)
e galinha
(Gallus domesticus)
Úmero
e fêmur Neste experimento, foram testadas 12 soluções
para maceração: (i) água amoniacal 5 e 1% e álcool
40% (Estado de São Paulo, 1967); (ii) água corrente
(Campos et al., 2002; Hildebrand e Goslow, 2006;
Cornélio, 2007); (iii) papaína, ácido muriático
(Rodrigues, 1973); (iv) peróxido de hidrogênio
(Rodrigues, 1973; Brandão et al., 2002; Nunes e
Perôncio, 2003; Bemvenuti, 2005; Pacheco et al.,
2005; Kawamoto et al., 2006; Ladeira e Höfling,
2007); (v) hipoclorito de sódio (Rodrigues, 1973;
Cornélio, 2007); e quatro soluções não citadas na
literatura corrente: Solupan (mistura tensoativa
aniônica com ácido fluorídrico, ácido muriático,
ácido dodecil benzeno sulfônico e corante; pH = 1,0
± 0,5) e Intercapt (mistura tensoativa aniônica com
hidróxido de sódio e corante; pH = 12,0 ± 0,5),
marcas comerciais de produtos utilizados para
limpeza de automóveis com grande eficácia na
remoção de determinados resíduos e, às vezes,
empregados para a limpeza de muros e calçadas, e
suco de abacaxi e amaciante de carne à base da
papaína. Esses produtos podem ser facilmente
encontrados em lojas especializadas em produtos
químicos,
feiras
de
produtos
agrícolas
ou
supermercados e, nos casos específicos do Solupan e
Intercapt, em postos de combustíveis. Todos
os
produtos
foram
utilizados
em
diferentes concentrações, algumas recomendadas
pelos autores acima citados. No entanto, as
concentrações
de
alguns
produtos
foram
modificadas, como nos casos do Hipoclorito de
Sódio, que foi utilizado a 23% ao invés de 2%, como
recomendado por Rodrigues (1973), concentração
esta considerada muito baixa, e da Papaína, que, em
razão do seu alto custo, foi substituída pelo Suco de Acta Sci. Biol. Sci. Maringá, v. 30, n. 4, p. 465-472, 2008 468 Silveira et al. Mamão; este último foi utilizado por Baker et al. (2003) e Davis e Payne (2003). Mamão; este último foi utilizado por Baker et al. (2003) e Davis e Payne (2003). muito baixo após o preparo da solução, como é o
caso do ácido muriático e do Solopan, com valores
de 1,0 e 2,21, respectivamente. Preparo de esqueletos para uso didático Por outro lado,
alguns produtos apresentaram altos valores de pH no
final, como é o caso do Hipoclorito de Sódio e do
Intercapt,
com
valores
de
11,50
e
11,73,
respectivamente (Figura 2). Cabe ressaltar que as
peças tratadas em solução com pH mais ácido
apresentaram maior facilidade para a retirada dos
tecidos. As peças anatômicas
foram colocadas
nas
diferentes soluções, acondicionadas em recipientes
específicos e, posteriormente, submetidas a diferentes
processos: repouso e fervura em distintos períodos de
tempo apresentados na Tabela 1. Os processos de
repouso e fervura foram sugeridos por Rodrigues
(1973) e Cornélio (2007), entretanto, o tempo de
permanência em fervura foi baseado nos trabalhos de
Baker et al. (2003) e Davis e Payne (2003). Figura 2. Valores do pH inicial da água para a elaboração das
soluções (A.I.: solução ácida; N.I.: solução neutra; B.I.: solução
básica) e valores do pH final das soluções utilizadas para a
maceração (A.F. = pH final da solução ácida; N.F. = pH final da
solução neutra; B.F. = pH final da solução básica). A Tabela 1 apresenta informações sobre as
substâncias
utilizadas
nas
macerações,
suas
respectivas concentrações e intervalo de tempo do
preparo, assim como as espécies de animais e
estruturas anatômicas utilizadas. Após a exposição das peças anatômicas aos
produtos
especificados,
elas
foram
lavadas
cuidadosamente em água corrente e limpas com
auxílio de bisturi e tesoura. Esta limpeza consiste na
retirada das sobras de músculos e ligamentos que
ainda estavam aderidas à estrutura óssea. Cabe
ressaltar que as peças que passaram pelo processo de
fervura foram deixadas em repouso para redução da
temperatura, a fim de evitar o risco da ocorrência de
trincas durante a lavagem pelas oscilações bruscas de
temperatura. Após todas essas etapas, as peças foram
analisadas e os resultados da utilização das diferentes
substâncias foram classificados como satisfatório ou
insatisfatório
para
utilização
no
preparo
de
esqueletos para uso didático. Figura 2. Valores do pH inicial da água para a elaboração das
soluções (A.I.: solução ácida; N.I.: solução neutra; B.I.: solução
básica) e valores do pH final das soluções utilizadas para a
maceração (A.F. = pH final da solução ácida; N.F. = pH final da
solução neutra; B.F. = pH final da solução básica). Acta Sci. Biol. Sci. Preparo de esqueletos para uso didático Critérios de avaliação: RT: remoção dos tecidos; DO: desgaste ósseo; FB: facilidade de
clareamento; TP: tempo de preparo; CP: custo do produto; DC: desarticulação
completa; RF: resultado final (soma das pontuações das avaliações de todos os critérios). Pontuação de avaliação para cada critério: ruim (-1); bom (0); e excelente (1). A utilização da água para a maceração de peças
ósseas apresentou um resultado satisfatório, sendo a
melhor entre todas as soluções aqui testadas. Após o
tempo determinado, as peças foram retiradas e pode-
se observar que as mesmas não apresentaram mais
tecidos, músculos e gordura, porém esse tratamento
apresenta um inconveniente: a demora e o mau
cheiro pela decomposição dos tecidos. A utilização da solução de papaína comercial,
indicada por Rodrigues (1973), apresenta custo
relativamente alto; neste, no entanto, foi utilizado
um produto extraído dos próprios frutos de
mamoeiro
(Carica
papaya). Foram
observados
resultados distintos nas condições aqui testadas. As
peças que estavam em repouso apresentaram
resultado insatisfatório e, após terem ficado expostas
ao produto por dois dias, ainda apresentaram grande
quantidade de nervos, músculos, ligamento e
gordura, para todas as condições de pH testadas. As
peças tratadas com a solução em fervura revelaram
resultados satisfatórios, apresentando remoção total
dos nervos, ligamentos, músculos e gordura. O
tempo de preparo foi considerado bom, em relação à
maioria dos outros produtos testados. Mesmo aparecendo como segundo melhor
produto na utilização nos processos de maceração, o
uso de peróxido de hidrogênio no tratamento das
peças em repouso mostrou-se insatisfatório, em
todas as condições de pH testadas. No entanto,
como apresentado na Tabela 2, os resultados foram
obtidos a partir do tratamento em fervura; dessa
forma, é de suma importância a observação e o uso
da Tabela 2 para a utilização desse e dos demais
produtos aqui apresentados. Após a maceração, ainda
persistiu grande quantidade de tecidos, porém as
peças que passaram pelo processo de fervura
apresentaram a retirada total de nervos, músculos,
ligamentos e gordura, com resultado satisfatório na
qualidade das peças, inclusive no que se refere à
facilitação em relação aos posteriores processos de
clareamento. Embora esse produto tenha custo
elevado
em
relação
aos
demais,
as
peças
apresentaram resultado excelente para quase todos
os critérios avaliados. Com isso, pode-se recomendar
esse produto para uso em laboratórios que
demandem rapidez e para os quais recursos
financeiros não sejam fator limitante. Outro produto testado extraído de plantas foi o
suco de abacaxi (Ananas comosus). Acta Sci. Biol. Sci. Preparo de esqueletos para uso didático Preparo de esqueletos para uso didático 469 Preparo de esqueletos para uso didático
Tabela 2. Critério de avaliação e resultado final de cada solução. Critérios de avaliação das peças
Produto utilizado
RT
DO
FB
TP
CP
DC
RF
Água
1
1
1
-1
1
1
4
Peróxido de hidrogênio
1
1
1
1
-1
0
3
Suco de mamão
(Carica papaya)
0
1
0
0
1
1
3
Suco de abacaxi
(Ananas comosus)
0
1
0
0
1
1
3
Solupan
1
1
0
0
0
0
2
Hipoclorito de sódio
1
0
0
0
1
0
2
Água amoniacal 1%
0
1
0
1
-1
1
2
Amaciante de carne
0
1
0
0
0
-1
0
Álcool 40%
0
1
0
0
0
-1
0
Ácido muriático
1
-1
-1
-1
-1
1
-2
Água amoniacal 5%
0
-1
0
-1
-1
1
-2
Intercapt
-1
1
-1
-1
0
0
-2
Critérios de avaliação: RT: remoção dos tecidos; DO: desgaste ósseo; FB: facilidade de
clareamento; TP: tempo de preparo; CP: custo do produto; DC: desarticulação
completa; RF: resultado final (soma das pontuações das avaliações de todos os critérios). Pontuação de avaliação para cada critério: ruim (-1); bom (0); e excelente (1). Tabela 2. Critério de avaliação e resultado final de cada solução. e gordura. Neste procedimento, peças menores,
como costelas, sofreram desarticulação indicando a
necessidade de manipulação dos esqueletos durante
a fervura, mantendo submersas apenas as partes que
ainda não atingiram o estágio de remoção de tecidos
desejado. Com esses resultados, o uso dessa solução
foi classificado, de acordo com a Tabela 2, como a
segunda melhor solução a ser utilizada para
maceração. Muitas plantas acumulam substâncias orgânicas
que podem ser extraídas em quantidades suficientes
para serem utilizadas para as mais variadas aplicações
científicas, tecnológicas e comerciais (Balandrin
et al., 1985), como, por exemplo, o uso dos sucos de
abacaxi ou de casca de mamão para maceração. Além
da aplicabilidade, estes produtos naturais apresentam
a vantagem de serem completamente atóxicos e de
não produzirem resíduos que ofereçam riscos ao
meio ambiente. Critérios de avaliação: RT: remoção dos tecidos; DO: desgaste ósseo; FB: facilidade de
clareamento; TP: tempo de preparo; CP: custo do produto; DC: desarticulação
completa; RF: resultado final (soma das pontuações das avaliações de todos os critérios). Pontuação de avaliação para cada critério: ruim (-1); bom (0); e excelente (1). Maringá, v. 30, n. 4, p. 465-472, 2008 Resultados e discussão
Resultados e discussão
Resultados e discussão
Resultados e discussão Devido ao problema de escassez de recursos
enfrentado por muitas Instituições de Ensino e
Pesquisa, há a busca constante pelo uso de produtos
com baixo custo e que consigam manter boa
qualidade final na preparação de esqueletos. Com
este intuito, foi realizada uma pesquisa de campo em
duas cidades do norte do Paraná (Apucarana e
Maringá), e constatou-se que dentre os produtos
químicos industrializados mais caros estavam o
ácido muriático e o peróxido de hidrogênio; por
outro lado, a água amoniacal, Intercapt, Solupan e o
amaciante de carne apresentaram os menores
valores. Quanto aos produtos naturais, os sucos de
mamão e abacaxi apresentaram valores relativamente
similares, com preço mais acessível em relação a
todas as soluções. A Tabela 2 apresenta os resultados da avaliação
de
cada
produto
investigado
quanto
ao
custo/benefício de sua utilização na preparação de
esqueletos para uso didático e indica quais as
melhores e piores opções. Ressalta-se que os dados
dessa tabela foram analisados de acordo com os
resultados obtidos nas soluções testadas em fervura,
em razão da maioria das soluções testadas em
repouso
não
ter
apresentado
resultados
significativamente expressivos. Depois de avaliado
cada produto, a partir dos resultados apresentados na
Tabela 2, constatou-se que alguns são mais
vantajosos em relação aos demais. O ranking de
classificação
quanto
aos
produtos
mais
recomendados, em ordem decrescente, revelou: 1º)
água; 2º) peróxido de hidrogênio, suco de mamão e
abacaxi; 3º) Solupan, hipoclorito de sódio e água
amoniacal 1%; 4º) amaciante de carne e álcool 40%;
5º) ácido muriático, água amoniacal 5% e Intercapt. A Figura 2 revela que houve modificação do pH
inicial dos produtos após o preparo de cada solução;
para todas as soluções, os valores de pH foram os
mesmos tanto para repouso quanto para fervura. Alguns produtos apresentaram valores de pH final Maringá, v. 30, n. 4, p. 465-472, 2008 Preparo de esqueletos para uso didático As peças que foram
colocadas em suco puro e ficaram em repouso por
sete dias apresentaram excessiva descalcificação,
perderam rigidez e adquiriram aspecto cartilaginoso. No segundo teste, as peças ósseas ficaram expostas
ao produto por apenas um dia; após a retirada, pode-
se constatar que a peça ainda apresentava grande
quantidade de nervos, músculos, ligamentos e
gordura. Com isso, para esses dois testes, o resultado
foi insatisfatório para utilização desse produto na
maceração de esqueletos. No teste realizado com o
produto diluído em água, nas três condições de pH,
e levado para fervura, pode-se observar resultado
satisfatório, apresentando remoção total de tecidos
moles sem ocorrência de desgaste ósseo. O produto
apresentou bom tempo de preparo, pois alguns
minutos de fervura foram suficientes para que as
peças ficassem prontas para o clareamento. Com Outra vantagem considerável do uso de peróxido
de hidrogênio é a possibilidade de realizar a fervura
assistida, permitindo a retirada da peça em diferentes
estágios de remoção de tecidos. Um bom exemplo
da aplicação dessa técnica foi a obtenção de uma
coluna vertebral de Cachorro-do-mato (Cerdocyon
thous) completamente articulada e livre de músculos Maringá, v. 30, n. 4, p. 465-472, 2008 470 Silveira et al. classificação dos resultados; porém, se for utilizada a
solução na concentração recomendada pelo autor, a
mesma pode obter melhor classificação entre os
produtos testados. esses resultados, os produtos naturais aqui testados
foram classificados como segundo melhor produto
para maceração, e pode-se constatar a eficiência de
produtos naturais com baixo custo para maceração
de esqueletos, porém é recomendado observar as
formas de utilização dos mesmos para melhor
aplicá-los. Na solução de água amoniacal na concentração
de 1%, nas condições tanto de fervura como de
repouso, pode-se observar resultado satisfatório,
com boa remoção de tecidos moles e sem marcas de
desgaste ósseo. O tempo de preparo foi considerado
excelente e, com isso, a solução apresentou resultado
final satisfatório quando comparada aos outros
produtos. Dessa forma, aparece no ranking de
classificação como terceira melhor solução a ser
utilizada na maceração de esqueletos. A observação
dos
resultados
indicou
que
esta
técnica
é
recomendada especialmente para esqueletos de
médio porte. Os produtos Intercapt e Solupan apresentam
grande poder de reação química pelas suas
composições, por isso foram preparadas diluições
maiores em relação ao ácido muriático para
minimizar o risco de danos às peças, como ocorreu
com este último (ver resultados). Preparo de esqueletos para uso didático Para as peças
deixadas em repouso por dois dias, constatou-se que
ainda apresentavam grande quantidade de tecidos,
portanto, nessas condições de preparo, os produtos
mostraram-se
insatisfatórios
para
o
uso
em
maceração. No entanto, também foram testados em
fervura e, nesta condição, houve intensa destruição
de proteínas, mostrando-se eficientes para a remoção
da
musculatura;
apenas
o
Solupan,
porém,
apresentou a retirada total dos nervos, ligamentos e
gordura, enquanto o Intercapt revelou resultado
insatisfatório mesmo em fervura. Com esses
resultados, pela Tabela 2, observa-se que os dois
produtos se encontram em posições distintas de
classificação: o Intercapt apareceu como um dos
menos
recomendados
para
a
utilização
na
maceração, juntamente com o ácido muriático e a
água amoniacal, enquanto o Solopan aparece na
terceira posição e é considerado produto eficaz para
maceração. p
O amaciante de carne, facilmente encontrado em
mercados, apresenta a papaína em sua composição e
pode ser comparado com o uso do suco de mamão. Resultados distintos foram observados para as peças
que estavam em repouso e em fervura. Quanto às
primeiras, grande quantidade de nervos e músculos
foi mantida, tornando inviável a sua utilização. Neste
processo, a solução preparada com o produto foi
classificada como insatisfatória independentemente das
condições de pH que foram testadas. As peças que
passaram pelo processo de fervura apresentaram boa
remoção de tecidos sem ocorrência de desgaste
ósseo; para estas condições, o produto foi
classificado como satisfatório e como o quarto
melhor na classificação geral dos produtos. Assim,
mais uma vez pode ser observado que novos
produtos podem ser utilizados para maceração de
peças ósseas, enfatizando uma atenção maior nos
métodos de preparo para aquisições de boas peças. O hipoclorito de sódio foi utilizado em peças de
aves e de mamíferos, as quais foram fervidas por um
curto período de tempo e/ou expostas por um tempo
em repouso. Resultados distintos para ambas as
classes de vertebrados foram observados, com
avaliação satisfatória para as peças de mamíferos que
passaram tanto pelo processo de fervura quanto pelo
de repouso. Em contrapartida, as peças de aves
tratadas
nas
mesmas
condições
apresentaram
excelente retirada de tecidos moles, mas também
grande desgaste ósseo. Isso pode estar relacionado ao
fato de que ossos de aves apresentam um tecido
ósseo esponjoso, mais sensível que o compacto
(Didio, 1974). Acta Sci. Biol. Sci. Preparo de esqueletos para uso didático Pode-se constatar que a utilização
desta técnica não é recomendada para essa classe de
vertebrados, sendo classificado como insatisfatório o
uso do produto nestas condições para o preparo de
ossos de aves. No entanto, recomenda-se o uso do
produto na diluição proposta por Rodrigues (1973),
a 2%, evitando-se, assim, a danificação das peças,
principalmente de aves. Nestas condições, essa
solução
aparece
como
terceira
melhor
na Para pequenos animais, pela fragilidade dos ossos,
muitas vezes é necessário uso de técnicas específicas
de
maceração,
limpeza
e
clareamento,
recomendando-se a utilização de álcool diluído a 40%
(Estado de São Paulo, 1967). No experimento com
este
produto,
utilizaram-se
esqueletos
de
passeriformes e não foram avaliadas as variações de
pH. Após o tratamento, pode-se observar que as peças
apresentavam pequenas quantidades de nervos,
músculos e ligamentos. Elas permaneceram um dia
em água para a retirada do restante dos tecidos,
embora tenha sido necessário, ainda, o tempo
sobressalente de um dia para complementar o
tratamento para que o resultado fosse considerado
satisfatório. Entretanto, foi necessária maior atenção
por se tratarem de peças de pequeno porte, pois
algumas podem, ao final do tratamento, apresentar
resultado de articulação completa, se isso for desejado. Maringá, v. 30, n. 4, p. 465-472, 2008 Acta Sci. Biol. Sci. Referências
Referências
Referências
Referências ALVARENGA, H. Coleções osteológicas: perspectivas
para a ornitologia no Brasil. Bol. Mus. Para. Emilio Goeldi,
Zool., Belém, v. 8, n. 1, p. 247-257, 1992. AURICCHIO, P.; SALOMÃO, M.D.G. Técnicas de coleta e
preparação de vertebrados para fins científicos e didáticos. São
Paulo: Aruja: Instituto Pau-Brasil de História Natural,
2002. BALANDRIN, M.F. et al. Natural plant chemicals:
sources of industrial and medicinal materials. Science,
Washington, D.C., v. 228, n. 4704, p. 1154-1160, 1985. BAKER, P. et al. On preparing animal skeletons: a simple
and
effective
method. International
Council
for
Archaeozoology, México, v. 4, n. 1, p. 4-15, 2003. BEMVENUTI, M.A. Comparative osteology among the
species of silverside Odontesthes Evermann & Kendall
(Osteichthyes, Atherinopsidae) in the Patos-Mirim
lagoons systems, in the Southern of Brazil. Rev. Bras. Zool.,
São Paulo, v. 22, n. 2, p. 293-305, 2005. Conclusão
Conclusão
Conclusão
Conclusão Em síntese, alguns produtos foram equivalentes
no ranking de classificação, ficando a critério de cada
laboratório qual das soluções deva ser utilizada. Com
esses resultados, para laboratórios que mantêm
atividade constante de maceração, para os quais os
recursos financeiros sejam escassos e o tempo de
preparado não seja problema, é recomendada
utilização da água, que apresentou o melhor
resultado
entre
todos
os
produtos
testados. Entretanto, para laboratórios que necessitam da
aquisição de peças com uma maior rapidez e
apresentam melhor condição financeira, recomenda-
se a utilização do peróxido de hidrogênio, que
também apresentou excelente resultado para a
maceração de peças ósseas. Por outro lado, os
produtos menos viáveis foram o ácido muriático,
água amoniacal 5% e o Intercapt, nas concentrações
aqui utilizadas, pelo grande prejuízo que os mesmos
trouxeram às estruturas das peças. BRANDÃO, C.V.S. et al. Substituição do ligamento da
cabeça do fêmur com auto-enxerto de fáscia lata na
luxação coxofemoral em cães. Cienc. Rural, Santa Maria,
v. 32, n. 2, p. 275-280, 2002. CAMPOS, D.B. et al. Biometria do osso do pênis em
correlação com a da coluna vertebral em cães (Canis
familiaris) sem raça definida. Biosci. J., Uberlândia, v. 18,
n. 1, p. 85-92, 2002. CORNÉLIO, M.O.V. Tratamento de ossos de boi para
artesanato. Minas Gerais: Fundação Centro Tecnológico de
Minas Gerais, 2007. DAVIS, S.J.M.; PAYNE, S. 101 modos de tratar um erizo
muerto: notas sobre la preparación de esqueletos
desarticulados para uso zooarqueológico. Archacofauna,
México, v. 12, n. 618, p. 203-211, 2003. DIDIO, L.J.A. Sinopse de anatomia. Rio de Janeiro:
Guanabara Koogan, 1974. ESTADO DE SÃO PAULO. Departamento de Zoologia. Manual de coleta e preservação de animais terrestres e de água doce. São Paulo: Secretaria de Agricultura do Estado de São
Paulo, 1967. Acta Sci. Biol. Sci. Preparo de esqueletos para uso didático 471 Zoologia da FAP que ajudaram no descarne dos
cadáveres, ao Prof. M.Sc. Edson Scolin pela
avaliação das peças anatômicas, à Inpacel e aos
moradores do entorno da FAP pela doação dos
cadáveres utilizados, à mestranda Solana M. Boschilia pela revisão do abstract, ao mestrando
Roger P. Mormul pela ajuda na confecção e análise
crítica da Figura 1 e à doutoranda Priscilla Carvalho
pelas valiosas sugestões ao manuscrito, todos do
Programa de Pós-graduação em Ecologia de
Ambientes Aquáticos Continentais (PEA/Nupélia)
da Universidade Estadual de Maringá. Zoologia da FAP que ajudaram no descarne dos
cadáveres, ao Prof. M.Sc. Edson Scolin pela
avaliação das peças anatômicas, à Inpacel e aos
moradores do entorno da FAP pela doação dos
cadáveres utilizados, à mestranda Solana M. Boschilia pela revisão do abstract, ao mestrando
Roger P. Mormul pela ajuda na confecção e análise
crítica da Figura 1 e à doutoranda Priscilla Carvalho
pelas valiosas sugestões ao manuscrito, todos do
Programa de Pós-graduação em Ecologia de
Ambientes Aquáticos Continentais (PEA/Nupélia)
da Universidade Estadual de Maringá. Dentre os produtos químicos utilizados, a
solução amoniacal a 5%, testada em fervura nas três
condições de pH, apresentou boa retirada de
músculos, nervos, ligamentos e gordura; em
contrapartida,
apresentou
desgaste
ósseo
considerável, tanto em fervura como em repouso
por três dias. Isso aconteceu tanto para ossos de aves
como de mamíferos, sendo, assim, considerada
insatisfatória. De acordo com os critérios de
avaliação, apresenta-se como uma das piores
soluções para maceração, ficando na sexta posição. Embora tenha a desvantagem de destruir ossos
pequenos, principalmente ossos de crânio, o uso de
ácido muriático como alternativo de maceração
química apresenta a vantagem da rapidez no preparo
do esqueleto (Rodrigues, 1973). Este ácido não foi
testado em fervura pelo seu grande poder de reação
química. A solução resultante revelou resultado
insatisfatório, independentemente do tempo que as
peças ficaram em exposição à solução. As peças
apresentaram intensa descalcificação óssea em suas
extremidades, as quais se tornaram amolecidas, com
aspecto de cartilagem, provavelmente pela perda
excessiva de material mineral; com isso, foi umas das
piores soluções, a sexta a ser utilizada nos processos
de maceração, de acordo com o critério de avaliação
e as condições aqui apresentados. Received on October 02, 2007.
Accepted on July 04, 2008. Acta Sci. Biol. Sci. Maringá, v. 30, n. 4, p. 465-472, 2008 Agradecimentos
Agradecimentos
Agradecimentos
Agradecimentos Agradecemos à Faculdade de Apucarana (FAP)
pelo aporte logístico para o desenvolvimento do
experimento, aos estagiários do laboratório de FRANCO, T.C.B. et al. Utilização de larvas de
coleópteros (Dermestídeos) na preparação de material
osteológico. Arqueologia em Conexão, Rio de Janeiro, Maringá, v. 30, n. 4, p. 465-472, 2008 472 Silveira et al. Cerylinae (Coraciiformes: Alcedinidae). Bol. Mus. Para. Emilio Goeldi, Zool., Belém, v. 2, n. 1, p. 155-182, 2007. n. 7, set., 2001. HILDEBRAND, M. Anatomical preparations. Berkeley:
University of California Press, 1968. NUNES, P.V.; PERÔNCIO, C. Implantação e proposta
de informatização da coleção osteológica de referência do
laboratório de zoologia e anatomia comparada do Unileste, HILDEBRAND, M.; GOSLOW, J.R. Análise da estrutura
dos vertebrados. 2. ed. São Paulo: Atheneu, 2006. MG. 2003. Disponível
em:
<http://www.unileste
mg.br/revistaonline/volumes/02/downloads/artigo_19.pdf>. Acesso
em: 14 mar. 2008. KAWAMOTO, H.S. et al. Confecção da coleção
osteológica de referência (Mastofauna e Ictiofauna) para
aplicação de zooarqueológicos em Mato Grosso de Sul. In: PACHECO, M.L.A.F. et al. Confecção de coleção
osteológica de referência e sua aplicação em análises de
vestígios faunísticos resgatados no sítio arqueológico
Maracaju-1, Maracaju, MS. Canindé - Revista do Museu de
Arqueologia de Xingó, Xingó, v. 6, n. 6, p. 85-114, 2005. REUNIÃO ANUAL DA SBPC, 58., 2006, Florianópolis. Anais... São Paulo: SBPC/UFSC, 2006. Disponível em:
<http://www.sbpcnet.org.br/livro/58ra>. LADEIRA, L.M.C.E.B.; HÖFLING, E. Osteologia
craniana de Bucconidae. Bol. Mus. Para. Emilio Goeldi
Zool., Belém, v. 2, n. 1, p. 117-153, 2007. PACKARD, A. Preparation of skeletons by marine
animals. Tuatara: Journal of the Biological Society, Wellington,
v. 7, n. 3, p. 120, 1959. MAIORANA, V.C.; VALEN, V.M.L. Terrestrial isotops
for preparing delicate vertebrate skeletons. Syst. Zool.,
Washington, D.C., v. 34, n. 2, p. 242-245, 1985. RODRIGUES, H. Técnicas anatômicas. Juiz de Fora:
Unijui, 1973. MATTHIESEN, D.G. La curación de las colecciones
osteológicas de aves. In: ESCALANTE-PLIEGO, P. (Ed.)
Curación moderna de colecciones ornitológicas. Washington,
D.C.: American Ornitological Union, 1993. p. 41-68. MENDÉZ, A.C.; HÖFLING, E. Osteologia craniana de Maringá, v. 30, n. 4, p. 465-472, 2008
|
https://openalex.org/W3080856654
|
http://www.scielo.br/j/aib/a/B7Pj9CftKmfrYCYwcLQfYLz/?format=pdf&lang=en
|
English
| null |
Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil
|
Arquivos do instituto biológico/Arquivos do Instituto Biológico
| 2,020
|
cc-by
| 4,584
|
Isolation of entomopathogenic nematodes
in the west region of Santa Catarina, Brazil
Isolamento de nematoides entomopatogênicos
na região oeste de Santa Catarina, Brasil Isolamento de nematoides entomopatogênicos
na região oeste de Santa Catarina, Brasil Dannyelle Cristine Orsolin de Morais1 (orcid.org/0000-0003-4990-0571)
Marco Aurélio Tramontin1* (orcid.org/0000-0002-3888-8696)
Vanessa Andaló2
(orcid.org/0000-0002-6310-1680) ABSTRACT: Entomopathogenic nematodes (EPNs) are
potential candidate for integrated pest management programs. As little is known about the presence of these organisms in the
state of Santa Catarina, it was aimed to perform soil sampling
in the cities of Chapecó, Palmitos, Seara and Concordia for
the isolation of EPNs. In total, 200 samples (100 g soil) were
collected. In Chapecó, 40 samples from soil containing green
manure (Raphanus sativus), five samples from native forest area
and five samples from riparian forest were collected. In the city
of Palmitos, 40 soil samples were obtained in the areas of soybean
(Glycine max), corn (Zea mays), oats (Avena strigosa), and pasture
(Pennisetum purpureum), and in each location 10 samples were
taken. Sixty soil samples were collected in the city of Concordia,
in a pasture area (A. strigosa). In Seara, the 50 soil samples
were collected at a pasture consortium site between ryegrass
(Lolium multiflorum) and black oats (A. strigosa). For the isolation,
the collected soil samples were conditioned in 350 mL plastic
containers and sent to the laboratory of the university. Later,
four larvae of Tenebrio molitor of last instar were inserted, and the
sets were maintained at the temperature of 25°C for seven days. After this period, the presence of dead larvae was verified, and the
confirmation of the mortality by EPNs was evaluated using of
White’s trap. The positive samples for EPNs were obtained from
the cities of Chapecó and Concordia, which corresponded to 2%
of the total soil samples. RESUMO: Os nematoides entomopatogênicos (NEPs) apresen-
tam potencial para utilização em programas de manejo integrado
de pragas. Como pouco se conhece sobre a presença desses orga-
nismos no estado de Santa Catarina, objetivou-se realizar amostra-
gens de solo nas cidades de Chapecó, Palmitos, Seara e Concórdia
para o isolamento de NEPs. No total foram coletadas 200 amostras
(100 g solo). Em Chapecó, foram coletadas 40 amostras em solo
contendo adubo verde (Raphanus sativus), cinco amostras de área de
floresta nativa e cinco amostras de mata ciliar. Em Palmitos, foram
obtidas dez amostras em cada área, totalizando 40. Foram elas: soja
(Glycine max), milho (Zea mays), aveia (Avena strigosa) e pastagem
(Pennisetum purpureum). Realizaram-se 60 amostras de solo na
cidade de Concórdia, em área de pastagem (A. strigosa). PHYTOPATHOLOGY / SCIENTIFIC COMMUNICATION PHYTOPATHOLOGY / SCIENTIFIC COMMUNICATION DOI: 10.1590/1808‑1657000322019 1Universidade Federal da Fronteira Sul – Chapecó (SC), Brazil
2Universidade Federal de Uberlândia – Monte Carmelo (MG), Brazil
*Corresponding author: marco.silva@uffs.edu.br
Received on: 04/28/2019. Accepted on: 05/15/2020 PALAVRAS-CHAVE: Nematoda; manejo integrado de pragas;
controle biológico. Isolation of entomopathogenic nematodes
in the west region of Santa Catarina, Brazil
Isolamento de nematoides entomopatogênicos
na região oeste de Santa Catarina, Brasil Em Seara,
as 50 amostras de solo foram retiradas em um local de consór-
cio de pastagem entre azevém (Lolium multiflorum) e aveia preta
(A. strigosa). Para o isolamento, as amostras de solo coletadas foram
acondicionadas em recipientes plásticos de 350 mL e alocadas no
laboratório da universidade. Foram posteriormente inseridas qua-
tro larvas de Tenebrio molitor de último instar, e mantiveram-se os
conjuntos em temperatura de 25°C por sete dias. Após esse perí-
odo, verificou-se a presença de larvas mortas, e a confirmação da
mortalidade por NEP foi feita por meio de armadilha de White. As amostras positivas para NEPs foram obtidas da cidade de Chapecó
(População 7, 18, 26) e Concórdia (População Concórdia), o que
correspondeu a 2% do total de amostras de solo. PALAVRAS-CHAVE: Nematoda; manejo integrado de pragas;
controle biológico. PALAVRAS-CHAVE: Nematoda; manejo integrado de pragas;
controle biológico. KEYWORDS: Nematoda; integrated pest management;
biological control. 1 Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 D.C.O. Morais et al. amazonensis, Metarhabditis rainai, Oscheios tipulae and S. rarum (DE BRIDA et al., 2017). The entomopathogenic nematodes (EPNs) are associated
with symbiotic bacteria, and after penetration into the host
insect the release of these symbionts causes mortality of the
target within 72 hours after infection (FERRAZ et al., 2008;
DILLMAN et al., 2012). EPNs have a potential as biologi-
cal control agents since these organisms have a wide range of
geographic areas, soil types and are adapted to several hosts. They do not cause damage to the environment and may also
have synergistic effect with some phytosanitary products
(GREWAL, 2012; LACEY, 2015; KAPRANAS et al., 2017). In places where these microorganisms have not yet been
explored, studies for sampling and isolation of EPNs are
necessary (ACEVEDO et al., 2005). When EPNs are locally
adapted, they provide effective control compared to exotic spe-
cies (LU et al., 2016; RIVERA et al., 2016). Therefore, this
study aimed to isolate EPNs in different agricultural areas of
the west region of Santa Catarina, Brazil. For the isolation of EPNs, surveys were conducted obtain-
ing soil sampling from the cities of Chapecó, Palmitos, Seara
and Concórdia between April and June 2017. According
to Köppen’s classification, the climate of the region is Cfa,
with average annual temperature of 20°C and annual average
rainfall of 1,830 mm (UHLMANN et al., 2012). Isolation of entomopathogenic nematodes
in the west region of Santa Catarina, Brazil
Isolamento de nematoides entomopatogênicos
na região oeste de Santa Catarina, Brasil The red clay
latosol soil is predominant of western region of Santa Catarina
state (POTTER et al., 2004). In addition to the potential of these entomopathogens to
control insects, studies have been developed to isolate these
microorganisms from the soil and to perform pathogenicity
tests on insects of agricultural importance (LU et al., 2016). Soil samples and isolation of EPNs in Brazil were made in the
states of Minas Gerais (ACEVEDO et al., 2005; ANDALÓ et al.,
2018), Rondônia (DOLINSKI; MOINO, 2006), Rio Grande
do Sul (BARBOSA-NEGRISOLI et al., 2010; FOELKEL
et al., 2017), São Paulo and Paraná (DE BRIDA et al., 2017). Samples were collected from 10-cm depth from the soil
surface, with the aid of a garden shovel, and the difference
between each collection was at least 1 m apart. At each point
of sampling, about 100 g of soil was collected, packed in plas-
tic bags and transported to the laboratory in styrofoam box. In the agroecosystem, EPNs are affected by soil proper-
ties such as soil texture, moisture, temperature and organic
matter, which might be drastically altered by agricultural
management practices, such as crop rotation and cover crop
rotation (JAFFUEL et al., 2016). In Chapecó, 40 samples were collected in a revolved soil,
containing previously Raphanus sativus, five samples in native
forest area and five samples in riparian forest. In Palmitos,
there were 10 samples in each area with annual crops: soy-
bean (Glycine max), corn (Zea mays), oats (Avena strigosa) and
pasture (Pennisetum purpureum), generating 40 samples. Sixty
soil samples were collected in the city of Concórdia, in a pas-
ture area (A. strigosa). In Seara, the 50 soil samples consisted
of a pasture consortium between ryegrass (Lolium multiflo-
rum) and black oats (A. strigosa). These collections totaled
200 soil samples (Fig. 1). In the state of Minas Gerais, the natural populations of
Heterorhabditis amazonensis were found in cerrado and gal-
lery forest areas (ANDALÓ et al., 2018). In the state of Rio
Grande do Sul, the occurrence of the species Steinernema
feltiae, Steinernema rarum and Steinernema riobrave was
reported for the first time in Brazil (BARBOSA-NEGRISOLI
et al., 2010). Nematodes of the genera Oscheius are found
in apple orchard (FOELKEL et al., 2017). EPNs also iden-
tified in agricultural areas of São Paulo and Paraná were H. Figure 1. Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil than 20% and clay varying between 15 to 80%, and they are
strongly to well drained (EMBRAPA, 2013). The clay tex-
ture did not interfere in the survival of EPNs, as reported by
BARBERCHECK; KAYA (1991). Instead, each type of soil
has a variety of unique characteristics that may have different
effects on EPNs species (SHAPIRO et al., 2000). The insect-trap technique was used to obtain the EPNs. The soil samples were conditioned in plastic containers (9 × 12 cm)
containing 100 g of soil and moistened with distilled water
(when necessary). After that, four larvae of Tenebrio molitor L. (Coleoptera: Tenebrionidae) of last instar were added to each
container, and all of them were closed with voil (BEDDING;
AKHURST, 1975). The plastic containers were kept at the tem-
perature of 25 ± 20°C in the Laboratory of Botany, Ecology and
Entomology of Universidade Federal de Fronteira Sul. f
In this study, it was observed that the cambisoils have
medium or fine texture, while the argisoils can be sandy and
have medium to clayey texture, and the nitosols are character-
ized by clayey to very clayey texture. However, these three soils
have the common characteristic of good drainage. Anyway,
organosoils come from predominantly organic material and
are commonly associated with poorly drained environments,
in which case the availability of oxygen may be difficult
(EMBRAPA, 2013). Later, the dead larvae were transferred to a white trap at
25 ± 2°C for ten days. The infective juveniles (IJs) that left the
T. molitor carcasses were collected daily with distilled water
and stored at 18 ± 2°C. To purify and confirm the parasitism,
Koch’s postulates were applied by multiplications in larvae of
T. molitor. The larvae of T. molitor were raised according to
the methodology of POTRICH et al. (2007). The soil texture can also influence the efficacy of the nem-
atode. As the clay content increases, the nematode disper-
sion and the survival are influenced (SHAPIRO et al., 2000;
KOPPENHOFER; FUZY, 2006). The moisture is one of the
major factors affecting survival, virulence and persistence of IJs
in the soil (LU et al., 2016), and different ranges of soil mois-
ture affect the EPNs to find and infect a host (ACEVEDO;
NÚÑEZ, 2003; SALAME; GLAZER, 2015). The cities that presented positive samples for EPNs were
Chapecó and Concórdia. Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil From the soil samples from Chapecó,
three samples were positive to EPNs, obtaining three isolates —
Population 7, Population 18 and Population 26 — from a total
of 40 (7.5%) (Table 1). From the samples collected in the city of
Concórdia, only one sample presented EPN, which corresponded
to 1.6% of the total of 60 samples taken at the area (Table 1). The isolate captured at this site was called Population Concórdia. The cities that presented positive samples for EPNs were
Chapecó and Concórdia. From the soil samples from Chapecó,
three samples were positive to EPNs, obtaining three isolates —
Population 7, Population 18 and Population 26 — from a total
of 40 (7.5%) (Table 1). From the samples collected in the city of
Concórdia, only one sample presented EPN, which corresponded
to 1.6% of the total of 60 samples taken at the area (Table 1). The isolate captured at this site was called Population Concórdia. In relation to the positive samples obtained, there are sev-
eral favorable conditions that may have benefited the occur-
rence of EPNs in these areas, such as moisture, organic matter
and associated cultures that favor the establishment of EPNs
(LEWIS et al., 2015). Although these parameters were not
directly compared in this study, soil samples positive for EPNs
have clayey texture (POTTER et al., 2004). In addition, optimum moisture levels will vary by nem-
atode species and soil type, since excess moisture can cause
oxygen deprivation and restrict movement. Soil character-
istics must also be considered (SHAPIRO et al., 2006),
as already mentioned in the soil texture. Studies gener-
ally report that lighter soils and soils with higher clay con-
tent restrict nematode movement and have reduced aera-
tion potential, which may result in less nematode survival
(GEORGIS; POINAR, 1983; MOLYNEUX; BEDDING,
1984). However, exceptions have been observed (SHAPIRO
et al., 2000), and this study proved the occurrence of EPNs
in soils with clayey texture (latosoil). In relation to the positive samples obtained, there are sev-
eral favorable conditions that may have benefited the occur-
rence of EPNs in these areas, such as moisture, organic matter
and associated cultures that favor the establishment of EPNs
(LEWIS et al., 2015). Although these parameters were not
directly compared in this study, soil samples positive for EPNs
have clayey texture (POTTER et al., 2004). Isolation of entomopathogenic nematodes
in the west region of Santa Catarina, Brazil
Isolamento de nematoides entomopatogênicos
na região oeste de Santa Catarina, Brasil Cities of Santa Catarina State where soil samples were collected for the isolation of entomopathogenic nematodes. Source: QGIS DEVELOPMENT TEAM (2019). 10 points in corn (Zea
mays), soybean (Glycine
max), oats (Avena
strigosa) and pasture
(Pennisetum
purpureum) respectively. Georeferencing:
27o6’23.57”S
53o9.10’55”N
40 points in green
adubation
(Raphanus
sativus), 5 points
native forest and 5
points in riparian
forest. Georeferencing:
27o6’23.57”S
52o9.10’55”N
Consorted pasture
(Lolium
multiflorum and A. strigosa), 50 points
collected. Georeferencing:
27o11’10.44”S
52o18.22’50”N
60 points collections in
culture Avena strigosa. Georeferencing:
27o13’47.54”S
52o1.28’68”N 10 points in corn (Zea
mays), soybean (Glycine
max), oats (Avena
strigosa) and pasture
(Pennisetum
purpureum) respectively. Georeferencing:
27o6’23.57”S
53o9.10’55”N Source: QGIS DEVELOPMENT TEAM (2019). Figure 1. Cities of Santa Catarina State where soil samples were collected for the isolation of entomopathogenic nematodes. Source: QGIS DEVELOPMENT TEAM (2019). atarina State where soil samples were collected for the isolation of entomopathogenic nematodes. Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 2 Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil The soils from the cities of Chapecó and Concórdia
where EPNs were found are classified according to Brazilian
Soil Classification System (SIBCs) as latosoil (EMBRAPA,
2013). These soils have as characteristics the silt content less Collection Location
City
Total number
of collections
Number of positive
samples for EPNs
Percentage of positive
samples for EPNs (%)
Soil type
Green adubation (Raphanus sativus)
Chapecó
40
3
7,5
Latosoil
Native forestin
Chapecó
5
-
-
Organosoil
Riparian forest
Chapecó
5
-
-
Nitosoil
Corn (Zea mays)
Palmitos
10
-
-
Argisoil
Soybean (Glycine max)
Palmitos
10
-
-
Argisoil
Oats (Avena strigosa)
Palmitos
10
-
-
Argisoil
Pasture (Pennisetum purpureum)
Palmitos
10
-
-
Argisoil
Pasture (Avena strigosa)
Concórdia
60
1
1,6
Latosoil
Consorted pasture
(Lolium multiflorum and A. strigosa)
Seara
50
-
-
Cambisoil
Total
200
4
2
EPNs: entomopathogenic nematodes. Table 1. Relationship of collection sites and positive samples for entomopathogenic nematodes. Table 1. Relationship of collection sites and positive samples for entomopathogenic nematodes. Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 3 3 D.C.O. Morais et al. In a study investigating the presence of EPNs in the cit-
ies of Barretos, Botucatu, Garça, São Manuel, São Paulo, and
Palotina, Paraná, samples were taken of agricultural soils with
annual, fruit and forest crops, totaling 201 samples. From
this total, 16 samples presented EPNs, which corresponded
to 8%. Areas with positive samples were forest plantations
(seven samples), annual crops (three samples) and orchards
(six samples). The species identified were H. amazonensis, M. rainai, O. tipulae and S. rarum. EPNs samples were not found
in plowed soil, native forest nor pasture areas. This result may
also indicate the need for a higher number of samples collected
at different soil depths (DE BRIDA et al., 2017). It demon-
strates the difficulty in obtaining positive samples, even when
they are collected in different environments. Several studies have demonstrated negative effects of inten-
sive soil management (chemical fertilization, agrochemicals,
monoculture, harrowing, among others) on EPNs (CAMPOS-
HERRERA et al., 2012; CAMPOS-HERRERA et al., 2014;
JAFFUEL et al., 2016). Though, the EPN populations isolated
in the present study were found in agricultural areas. This fact
can be justified by the adoption of smaller quantities of plant
protection products, better vegetation cover on the soil and
less soil rotation in the isolation areas when compared to soy-
bean areas and corn. Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil FUNDING: This work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. CONFLICTS OF INTEREST: The authors certify that they have no commercial or associative interest that represents a
conflict of interest in connection with the manuscript. ETHICAL APPROVAL: Not applicable. AVAILABILITY OF DATA AND MATERIAL: All data generated or analyzed during this study are included in this published
article. AUTHORS’ CONTRIBUTIONS: Conceptualization: Tramontin, M.A; Ándalo, V. Visualization, Writing – original draft:
Orsolin, D.C.M. Writing – review & editing: Tramontin, M.A; Ándalo, V. REFERENCES
ACEVEDO, J.P.M.; NÚÑEZ, J.C.L. Supervivencia y parasitismo de
nematodos entomopatógenos para el control de Hypothenemus
hampei, (Coleoptera: Scolytidae) en frutos de café. Boletín Sanidad
Vegetal de Plagas, v.29, n.4, p.523-533, 2003. ACEVEDO, J.P.M.; MOINO, A.J.; CAVALCANTI, R.S.; DOLINSKI, C.;
CARVALHO, F.A. Amostragem e avaliação de técnicas para isolamento
de nematóides entomopatogênicos nativos obtidos em Lavras,
Minas Gerais. Nematologia Brasileira, v.29, n.1, p.17-23, 2005. his work did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil The cultures to be implemented, as well as the history
of the area (crop rotation), have an important effect on the
abundance and activity of EPNs (JAFFUEL et al., 2016). In this study, the consortium of pastures favored greater root
activity in the soil profile, such as nutrient recycling in the
soil provided by R. sativus. This study did not present positive samples for EPNs in native
forest nor in agricultural crops such as maize, which differs from
the positive samples for EPNs found in other studies in Brazil
(BARBOSA-NEGRISOLI et al., 2010; ANDALÓ et al., 2018;
DE BRIDA et al., 2017). However, this study presented posi-
tive samples in pasture and soil areas with green manure, and it
corroborates with BARBOSA-NEGRISOLI et al. (2010) and
differs from the results presented by DE BRIDA et al. (2017). In a study of native EPN, 15.70% of the samples from the
state of Rio Grande do Sul from the set of 121 soil samples col-
lected contained EPNs. For the EPN positive samples, 7.69 to
18.18% were observed in forests, native pastures, fruit trees and
corn, and between 21.42 and 25% were identified in soybean
and tobacco. The species identified were S. rarum, Heterorhabditis
bacteriophora, H. amazonensis, S. feltiae, Steinernema glaseri and
S. riobrave (BARBOSA-NEGRISOLI et al., 2010). The variation of sites with or without EPNs requires a great
deal of effort mainly in sampling (sample area, number of sam-
ples, sample size), as well as the combination of extraction tech-
niques of these soil microorganisms to avoid losses during the
isolation of these entomopathogens (BARBOSA-NEGRISOLI
et al., 2010; DE BRIDA et al., 2017). The isolation of the
three populations in the west of Santa Catarina demonstrates
the wide distribution of these organisms in Brazil, besides the
potential use in pest control in the region, because they are
adapted to the local conditions. In a verification survey of EPNs in the state of Minas
Gerais, a total of 216 soil samplings was performed, from which
three populations of EPNs were identified as H. amazonen-
sis — two populations found in the cerrado area and one in
the gallery forest (forest that forms corridor along rivers). Both
areas are characterized by the presence of high-density vege-
tation cover, due to the characteristics associated with these
biomes (ANDALÓ et al., 2018). 4
Arq. Inst. Biol., v.87, 1-6, e2019032, 2020
ACKNOWLEDGEMENTS: Not applicable. REFERENCES A simple technique for the detection
of insect parasitic rhabditid nematodes in soil. Nematologica,
v.21, n.1, p.109-110, 1975. 10.1163/187529275X00419
CAMPOS-HERRERA, R.; EL-BORAI, F.E.; DUNCAN, L.W. Wide
interguild relationships among entomopathogenic and free-living
nematodes in soil as measured by real time qPCR. Journal of
Invertebrate Pathology, v.111, n.2, p.126-135, 2012. https://
doi.org/10.1016/j.jip.2012.07.006 BEDDING, R.A.; AKHURST, R.J. A simple technique for the detection
of insect parasitic rhabditid nematodes in soil. Nematologica,
v.21, n.1, p.109-110, 1975. 10.1163/187529275X00419 KAPRANAS, A.; MALONE, B.; QUINN, S.; MCNAMARA, L.;
WILLIAMS, C.D.; O’TUAMA, P.; PETERS, A.; GRIFFIN, C.T. Efficacy
of entomopathogenic nematodes for control of large pine weevil
Hylobius abietis: effects of soil type, pest density and spatial
distribution. Journal of Pest Science, v.90, n.2, p.495-505, 2017. https://doi.org/10.1007/s10340-016-0823-y CAMPOS-HERRERA, R.; EL-BORAI, F.E.; DUNCAN, L.W. Wide
interguild relationships among entomopathogenic and free-living
nematodes in soil as measured by real time qPCR. Journal of
Invertebrate Pathology, v.111, n.2, p.126-135, 2012. https://
doi.org/10.1016/j.jip.2012.07.006 KOPPENHOFER, A.M.; FUZY, E.M. Effect of soil type on infectivity and
persistence of the EPNs Steinernema scarabaei, Steinernema glaseri,
Heterorhabditis zealandica, and Heterorhabditis bacteriophora. Journal of Invertebrate Pathology, v.92, n.1, p.11-22, 2006. https://doi.org/10.1016/j.jip.2006.02.003 CAMPOS-HERRERA, R.; EL-BORAI, F.E.; EBERT, T.E.; SCHUMANN,
A.; DUNCAN, L.W. Management to control citrus greening alters the
soil food web and severity of a pest-disease complex. Biological
Control, v.76, p.41-51, 2014. https://doi.org/10.1016/j. biocontrol.2014.04.012 LACEY, L.A.; GRZYWACZ, D.; SHAPIRO-ILAN, D.I.; FRUTOS, R.;
BROWNBRIDGE, M.; GOETTEL, M.S. Insect pathogens as biological
control agents: back to the future. Journal of Invertebrate
Pathology, v.132, p.1-41, 2015. https://doi.org/10.1016/j. jip.2015.07.009 DE BRIDA, A.L.; ROSA, J.M.O.; DE OLIVEIRA, C.M.G.; CASTRO,
B.M.D.C.; SERRÃO, J.E.; ZANUNCIO, J.C.; GARRIGÓS, L.L.;
WILCKEN, S.R.S. Entomopathogenic nematodes in agricultural
areas in Brazil. Scientific Reports, v.7, p.45254, 2017. https://
doi.org/10.1038/srep45254 LEWIS, E.E.; HAZIR, S.; HODSON, A.; GULCU, B. Trophic relationships of
entomopathogenic nematodes in agricultural habitats. In: CAMPOS-
HERRERA, R. (Ed.). Nematode pathogenesis of insects and other
pests. Sustainability in plant and crop protection. Cham: Springer,
2015. https://doi.org/10.1007/978-3-319-18266-7_5 DILLMAN, A.R.; CHASTON, J.M.; ADAMS, B.J.; CICHE, T.A.;
GOODRICH-BLAIR, H.; STOCK, S.P.; STERNBERG, P.W. An
entomopathogenic nematode by any other name. PLoS Pathogens,
v.8, n.3, p.e1002527, 2012. https://doi.org/10.1371/journal. ppat.1002527 LU, D.; BAIOCCHI, T.; DILLMAN, A.R. Genomics of entomopathogenic
nematodes and implications for pest control. Trends in Parasitology,
v.32, n.8, p.588-598, 2016. https://doi.org/10.1016/j. pt.2016.04.008 DOLINSKI, C.; MOINO J.R.A. Utilização de nematóides
entomopatogênicos nativos ou exóticos: o perigo das introduções. Nematologia Brasileira, v.30, n.2, p.139-149, 2006. EMPRESA BRASILEIRA DE PESQUISA AGROPECUÁRIA
(EMBRAPA). REFERENCES ACEVEDO, J.P.M.; NÚÑEZ, J.C.L. Supervivencia y parasitismo de
nematodos entomopatógenos para el control de Hypothenemus
hampei, (Coleoptera: Scolytidae) en frutos de café. Boletín Sanidad
Vegetal de Plagas, v.29, n.4, p.523-533, 2003. ACEVEDO, J.P.M.; NÚÑEZ, J.C.L. Supervivencia y parasitismo de
nematodos entomopatógenos para el control de Hypothenemus
hampei, (Coleoptera: Scolytidae) en frutos de café. Boletín Sanidad
Vegetal de Plagas, v.29, n.4, p.523-533, 2003. ACEVEDO, J.P.M.; MOINO, A.J.; CAVALCANTI, R.S.; DOLINSKI, C.;
CARVALHO, F.A. Amostragem e avaliação de técnicas para isolamento
de nematóides entomopatogênicos nativos obtidos em Lavras,
Minas Gerais. Nematologia Brasileira, v.29, n.1, p.17-23, 2005. ACEVEDO, J.P.M.; MOINO, A.J.; CAVALCANTI, R.S.; DOLINSKI, C.;
CARVALHO, F.A. Amostragem e avaliação de técnicas para isolamento
de nematóides entomopatogênicos nativos obtidos em Lavras,
Minas Gerais. Nematologia Brasileira, v.29, n.1, p.17-23, 2005. ACEVEDO, J.P.M.; MOINO, A.J.; CAVALCANTI, R.S.; DOLINSKI, C.;
CARVALHO, F.A. Amostragem e avaliação de técnicas para isolamento
de nematóides entomopatogênicos nativos obtidos em Lavras,
Minas Gerais. Nematologia Brasileira, v.29, n.1, p.17-23, 2005. Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 4 Isolation of entomopathogenic nematodes in the west region of Santa Catarina, Brazil Brazil and its virulence to Anastrepha fraterculus (Diptera:
Tephritidae) larvae, under laboratory conditions. Brazilian
Journal of Biology, v.77, n.1, p.22-28, 2017. https://doi. org/10.1590/1519-6984.08315 ANDALÓ, V.; MIEKO, J.; CARVALHO, F.J.; DE ASSIS, G.A.; DE
FARIA, L.S.; DE ASSIS, F.A.; ROSA, F. Entomopathogenic nematode
distribution and edaphoclimatic conditions in the Cerrado of Minas
Gerais, Brazil. Applied Entomology and Zoology, v.53, n.1, p.129-
136, 2018. https://doi.org/10.1007/s13355-017-0538-4 GEORGIS, G.O.; POINAR, J.R. Effect of soil texture on the
distribution and infectivity of Neoaplectana carpocapsae
(Nematoda: Steinernematidae). Journal of Nematology, v.15,
n.2, p.308-311, 1983. BARBERCHECK, M.E.; KAYA, H.K. Effect of host condition and
soil texture on host finding by the entomogenous nematodes
Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae)
and Steinernema carpocapsae (Rhabditida: Steinernematidae). Environmental Entomology, v.20, n.2, p.582-589, 1991. https://
doi.org/10.1093/ee/20.2.582 GREWAL, P.S. Entomopathogenic nematodes as tools in integrated
pest management. Integrated pest management: principles and
practice. Wallingford, UK: Cabi Publishing, 2012. p.162-236. BARBOSA-NEGRISOLI, C.R.B.; GARCIA, M.S.; DOLINSKI, C.;
NEGRISOLI, J.R.A.S.; BERNARDI, D.; DOS SANTOS, F.J. Survey of
entomopathogenic nematodes (Rhabditida: Heterorhabditidae,
Steinernematidae) in Rio Grande do Sul State, Brazil. Nematologia
Brasileira, v.34, n.4, p.189-197, 2010. JAFFUEL, G.; MÄDER, P.; BLANCO-PEREZ, R.; CHIRIBOGA, X.;
FLIESSBACH, A.; TURLINGS, T.C.; CAMPOS-HERRERA, R. Prevalence
and activity of entomopathogenic nematodes and their antagonists
in soils that are subject to different agricultural practices. Agriculture, Ecosystems & Environment, v.230, p.329-340,
2016. https://doi.org/10.1016/j.agee.2016.06.009 BEDDING, R.A.; AKHURST, R.J. REFERENCES Sistema Brasileiro de Classificação dos Solos
(SIBCS). Brasília: EMBRAPA-SPI, Centro Nacional de Pesquisa
de Solos, 2013. MOLYNEUX, A.S.; BEDDING, R.A. Influence of soil texture and
moisture on the infectivity of Heterorhabditis sp. D1 and
Steinernema glaseri for larvae of the sheep blowfly Lucilia
cuprina. Nematologica, v.30, n.3, p.358-365, 1984. https://
doi.org/10.1163/187529284X00266 MOLYNEUX, A.S.; BEDDING, R.A. Influence of soil texture and
moisture on the infectivity of Heterorhabditis sp. D1 and
Steinernema glaseri for larvae of the sheep blowfly Lucilia
cuprina. Nematologica, v.30, n.3, p.358-365, 1984. https://
doi.org/10.1163/187529284X00266 FERRAZ, L.C.C.B.; LEITE, L.G.; LOPES, R.B.; MOINO, J.R.A.; DOLINSKI,
C. Utilização de nematóides para o controle de pragas agrícolas
e urbanas. In: Alves, S.B.; Lopes, R.B. Controle microbiano de
pragas na América Latina: avanços e desafios. Piracicaba: FEALQ,
2008. p.171-202. POTTER, R.O.; CARVALHO, A.P.; FLORES, C.A.; BOGNOLA, I. Solos do Estado de Santa Catarina. Embrapa Solos-Boletim de
Pesquisa e Desenvolvimento (INFOTECA-E). 2004. Available
from: https://www.infoteca.cnptia.embrapa.br/bitstream/
doc/964417/1/BPD462004SantaCatarina.pdf. Access on:
Apr. 23 2019. FOELKEL, E.; VOSS, M.; MONTEIRO, L.B.; NISHIMURA, G. Isolation
of entomopathogenic nematodes in an apple orchard in Southern Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 5 5 D.C.O. Morais et al. POTRICH, T.D.; LORINI, I.; VOSS, M.; STEFFENS, M.C.S.; PAVANI,
D.P. Methodology to rear Tenebrio molitor in laboratory to obtain
larvae. Passo Fundo: Embrapa Trigo, 2007. SHAPIRO, D.I.; MCCOY, C.W.; FARES, A.; OBREZA, T.;
DOU, H. Effects of soil type on virulence and persistence
of entomopathogenic nematodes in relation to control of
Diaprepes abbreviatus (Coleoptera: Curculionidae). Environmental
Entomology, v.29, n.5, p.1083-1087, 2000. https://doi. org/10.1603/0046-225X-29.5.1083 QGIS Development Team. QGIS Geographic Information System. Open Source Geospatial Foundation Project 2019. Available
from: http://qgis.osgeo.org. Access on: May 25, 2019. SHAPIRO, D.I.; GOUGE, D.H.; PIGGOTT, S.; FIFE, J.P. Application
technology and environmental considerations for use of
entomopathogenic nematodes in biological control. Biological
Control, v.38, n.1, p.124-133. 2006. https://doi.org/10.1016/j. biocontrol.2005.09.005 SHAPIRO, D.I.; GOUGE, D.H.; PIGGOTT, S.; FIFE, J.P. Application
technology and environmental considerations for use of
entomopathogenic nematodes in biological control. Biological
Control, v.38, n.1, p.124-133. 2006. https://doi.org/10.1016/j. biocontrol.2005.09.005 RIVERA, M.J.; RODRIGUEZ-SAONA, C.; EGIZI, A.; FONSECA,
D.M.; JENNINGS, D.E.; KOPPENHÖFER, A.M. Cultivation and
domestication of highbush blueberry (Vaccinium corymbosum)
alters abundance, diversity and virulence of entomopathogenic
nematodes. Agriculture, Ecosystems & Environment, v.222, p.148-
155, 2016. https://doi.org/10.1016/j.agee.2016.02.013 RIVERA, M.J.; RODRIGUEZ-SAONA, C.; EGIZI, A.; FONSECA,
D.M.; JENNINGS, D.E.; KOPPENHÖFER, A.M. Cultivation and
domestication of highbush blueberry (Vaccinium corymbosum)
alters abundance, diversity and virulence of entomopathogenic
nematodes. © 2020 Instituto Biológico
This is an open access article distributed under the terms of the Creative Commons license. REFERENCES Agriculture, Ecosystems & Environment, v.222, p.148-
155, 2016. https://doi.org/10.1016/j.agee.2016.02.013 UHLMANN, A.; GASPER, A.L.; SEVEGNANI, L.; VIBRANS, A.C.;
MEYER, L.; LINGNER, D.V. Fitogeografia de Santa Catarina. In:
VIBRANS, A.C.; SEVEGNANI, L.; GASPER, A.L.; LINGNER, D.V. (Eds.). Inventário florístico florestal de Santa Catarina. Blumenau:
EDIFURB, 2012. v. 1. p. 113-123. SALAME, L.; GLAZER, I. Stress avoidance: vertical movement
of entomopathogenic nematodes in response to soil moisture
gradient. Phytoparasitica, v.43, n.5, p.647-655, 2015. https://
doi.org/10.1007/s12600-015-0488-8 Arq. Inst. Biol., v.87, 1-6, e2019032, 2020 6
|
https://openalex.org/W2766747008
|
https://www.mdpi.com/1422-0067/18/10/2163/pdf?version=1508243996
|
English
| null |
The Specific Mitogen- and Stress-Activated Protein Kinase MSK1 Inhibitor SB-747651A Modulates Chemokine-Induced Neutrophil Recruitment
|
International journal of molecular sciences
| 2,017
|
cc-by
| 10,490
|
Mokarram Hossain, Entesar Omran, Najia Xu and Lixin Liu * Abstract: Mitogen-activated protein kinase (MAPK) signaling is involved in a variety of cellular
functions. MAPK-dependent functions rely on phosphorylation of target proteins such as mitogen-
and stress-activated protein kinase 1 (MSK1). MSK1 participates in the early gene expression and in
the production of pro- and anti-inflammatory cytokines. However, the role of MSK1 in neutrophil
recruitment remains elusive. Here, we show that chemokine macrophage inflammatory protein-2
(CXCL2) enhances neutrophil MSK1 expression. Using intravital microscopy and time-lapsed
video analysis of cremasteric microvasculature in mice, we studied the effect of pharmacological
suppression of MSK1 by SB-747651A on CXCL2-elicited neutrophil recruitment. SB-747651A
treatment enhanced CXCL2-induced neutrophil adhesion while temporally attenuating neutrophil
emigration. CXCL2-induced intraluminal crawling was reduced following SB-747651A treatment. Fluorescence-activated cell sorting analysis of integrin expression revealed that SB-747651A treatment
attenuated neutrophil integrin αMβ2 (Mac-1) expression following CXCL2 stimulation. Both the
transmigration time and detachment time of neutrophils from the venule were increased following
SB-747651A treatment. It also decreased the velocity of neutrophil migration in cremasteric tissue
in CXCL2 chemotactic gradient. SB-747651A treatment enhanced the extravasation of neutrophils
in mouse peritoneal cavity not at 1–2 h but at 3–4 h following CXCL2 stimulation. Collectively,
our data suggest that inhibition of MSK1 by SB-747651A treatment affects CXCL2-induced neutrophil
recruitment by modulating various steps of the recruitment cascade in vivo. Keywords: MSK1; SB-747651A; neutrophil recruitment; intravital microscopy; chemokine The Specific Mitogen- and Stress-Activated Prote
Kinase MSK1 Inhibitor SB-747651A Modulates
Chemokine-Induced Neutrophil Recruitment Mokarram Hossain, Entesar Omran, Najia Xu and Lixin Liu * International Journal of
Molecular Sciences International Journal of
Molecular Sciences 1. Introduction During acute inflammation, neutrophils are recruited to the afflicted site by a well-defined and
dynamic multi-step process that is regulated by a myriad of molecules and signaling cascades elicited
by the cross-talk between neutrophils and endothelium [1,2]. The initial step of neutrophil rolling on the
endothelium is followed by β2 integrin–ICAM-1-dependent adhesion of neutrophils to endothelium [3]. Adherent neutrophils then crawl in the vascular lumen to reach optimal emigration sites at endothelial
junctions independently of hemodynamic forces, a process mediated by the αMβ2 integrin Mac-1 [4]. Transendothelial migration of neutrophils is regulated by the interactions between integrins, PECAM-1
as well as junctional adhesion molecules and their respective ligands [1–3]. Neutrophil recruitment
in vivo can be induced by CXC chemokines such as macrophage inflammatory protein-2 (CXCL2) [5]. Signaling mechanisms that regulate different steps of neutrophil recruitment such as intraluminal
crawling and subsequent transendothelial migration of neutrophils are not completely understood. Int. J. Mol. Sci. 2017, 18, 2163; doi:10.3390/ijms18102163 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2017, 18, 2163
Int J Mol Sci 2017, 18, 2163 2 of 12
2 of 12 Mitogen-activated protein kinases (MAPKs) are involved in a wide variety of cellular functions,
such as differentiation, survival and apoptosis [6,7]. They are known to participate in the
pathophysiology of neuronal and inflammatory diseases [7,8]. MAPK-dependent functions rely
on phosphorylation of target proteins such as the closely related mitogen- and stress-activated protein
kinases MSK1 and MSK2 [9]. Both kinases are phosphorylated by extracellular signal-regulated kinase
ERK1/2 and by p38 MAPK and are, thus, activated by a wide range of physiological and pathological
stimuli [9]. MSKs are homologous with the p90 ribosomal S6 kinase (RSK) family of kinases where the
N-terminal kinase domains of both MSKs and RSKs are members of the AGC (protein kinase A, protein
kinase G and protein kinase C) family of protein kinases [10]. Cellular functions of MSK1 include
early gene expression [11] and apoptosis [12]. MSK1 regulates the activation of nuclear factor-κB
(NF-κB) [13–15] and cyclic AMP response element-binding protein (CREB) [16], two transcription
factors that are important in mediating inflammatory responses. MSK1 also regulates the production of
pro- and anti-inflammatory cytokines [16–20] as well as endogenous mediators such as prostaglandin
E2 [21]. However, the role of MSK1 in innate immunity remains elusive. Mitogen-activated protein kinases (MAPKs) are involved in a wide variety of cellular functions,
such as differentiation, survival and apoptosis [6,7]. 1. Introduction They are known to participate in the
pathophysiology of neuronal and inflammatory diseases [7,8]. MAPK-dependent functions rely on
phosphorylation of target proteins such as the closely related mitogen- and stress-activated protein
kinases MSK1 and MSK2 [9]. Both kinases are phosphorylated by extracellular signal-regulated kinase
ERK1/2 and by p38 MAPK and are, thus, activated by a wide range of physiological and pathological
stimuli [9]. MSKs are homologous with the p90 ribosomal S6 kinase (RSK) family of kinases where the
N-terminal kinase domains of both MSKs and RSKs are members of the AGC (protein kinase A, protein
kinase G and protein kinase C) family of protein kinases [10]. Cellular functions of MSK1 include early
gene expression [11] and apoptosis [12]. MSK1 regulates the activation of nuclear factor-κB (NF-κB)
[13–15] and cyclic AMP response element-binding protein (CREB) [16], two transcription factors that
are important in mediating inflammatory responses. MSK1 also regulates the production of pro- and
anti-inflammatory cytokines [16–20] as well as endogenous mediators such as prostaglandin E2 [21]. However, the role of MSK1 in innate immunity remains elusive. C ll l
f
i
f MSK1
i
l
l
id
d b
i
li
i
l
i
[20] Cellular functions of MSK1 were previously elucidated by murine germline manipulation [20]
and by inhibitors such as Ro 31-8220 and H89 [22,23]. However, these compounds are less selective
and inhibit many other kinases [24]. Recently, SB-747651A was shown to be a highly selective and
cell-active inhibitor of MSK1 with properties superior to H89 and Ro 31-8220 [24], thus enabling us to
dissect the putative functions of MSK1 in vitro and in vivo. Cellular functions of MSK1 were previously elucidated by murine germline manipulation [20]
and by inhibitors such as Ro 31-8220 and H89 [22,23]. However, these compounds are less selective
and inhibit many other kinases [24]. Recently, SB-747651A was shown to be a highly selective and
cell-active inhibitor of MSK1 with properties superior to H89 and Ro 31-8220 [24], thus enabling us
to dissect the putative functions of MSK1 in vitro and in vivo. In the present study, we explored the effect of pharmacological inhibition of MSK1 using
SB-747651A on chemokine CXCL2-induced neutrophil recruitment in vivo. By using real-time
intravital microscopy and time-lapsed video analysis, we simultaneously determined the multiple
neutrophil recruitment parameters such as rolling, adhesion, emigration, intraluminal crawling velocity,
transmigration time, detachment time, migration velocity, and chemotaxis index in tissue. 2. Results
2. Results First, we examined whether the treatment of neutrophils with CXC chemokine CXCL2 affects
MSK1 protein expression. As shown in Figure 1, treatment of mouse bone marrow neutrophils with
CXCL2 significantly enhanced MSK1 protein expression in neutrophils. First, we examined whether the treatment of neutrophils with CXC chemokine CXCL2 affects
MSK1 protein expression. As shown in Figure 1, treatment of mouse bone marrow neutrophils with
CXCL2 significantly enhanced MSK1 protein expression in neutrophils. Figure 1. Effect of chemokine macrophage inflammatory protein-2 (CXCL2) on mitogen- and stress-
activated protein kinase 1 (MSK1) expression in neutrophils. (A) Representative original Western blot
and (B) means ± SEM (n = 4) showing total MSK1 expression determined in 1-h saline-treated
(Control) or CXCL2-treated (30 nM at 37 °C for 1 h) bone marrow neutrophils (relative to β-actin). ***
(p < 0.001) from the Control. Figure 1. Effect of chemokine macrophage inflammatory protein-2 (CXCL2) on mitogen- and
stress-activated protein kinase 1 (MSK1) expression in neutrophils. (A) Representative original Western
blot and (B) means ± SEM (n = 4) showing total MSK1 expression determined in 1-h saline-treated
(Control) or CXCL2-treated (30 nM at 37 ◦C for 1 h) bone marrow neutrophils (relative to β-actin). *** (p < 0.001) from the Control. Figure 1. Effect of chemokine macrophage inflammatory protein-2 (CXCL2) on mitogen- and stress-
activated protein kinase 1 (MSK1) expression in neutrophils. (A) Representative original Western blot
and (B) means ± SEM (n = 4) showing total MSK1 expression determined in 1-h saline-treated
(Control) or CXCL2-treated (30 nM at 37 °C for 1 h) bone marrow neutrophils (relative to β-actin). ***
(p < 0.001) from the Control. Figure 1. Effect of chemokine macrophage inflammatory protein-2 (CXCL2) on mitogen- and
stress-activated protein kinase 1 (MSK1) expression in neutrophils. (A) Representative original Western
blot and (B) means ± SEM (n = 4) showing total MSK1 expression determined in 1-h saline-treated
(Control) or CXCL2-treated (30 nM at 37 ◦C for 1 h) bone marrow neutrophils (relative to β-actin). *** (p < 0.001) from the Control. To test whether CXCL2-sensitive MSK1 participates in neutrophil recruitment in vivo, we
studied the effect of the specific MSK1 inhibitor SB-747651A on neutrophil-endothelial cell
interactions using intravital microscopy of post-capillary venule in mouse cremaster muscle. 1. Introduction In the present study, we explored the effect of pharmacological inhibition of MSK1 using SB-
747651A on chemokine CXCL2-induced neutrophil recruitment in vivo. By using real-time intravital
microscopy and time-lapsed video analysis, we simultaneously determined the multiple neutrophil
recruitment parameters such as rolling, adhesion, emigration, intraluminal crawling velocity,
transmigration time, detachment time, migration velocity, and chemotaxis index in tissue. 2. Results
2. Results To this
end, superfusion of murine cremaster muscle with SB-747651A (5 µM) for 30 min prior to and for 1 h
following the placement of CXCL2-containing gel significantly enhanced leukocyte rolling flux (83.7
± 3.4 cells/min with SB-747651A treatment versus 48.3 ± 3.1 cells/min without SB-747651A treatment;
To test whether CXCL2-sensitive MSK1 participates in neutrophil recruitment in vivo, we studied
the effect of the specific MSK1 inhibitor SB-747651A on neutrophil-endothelial cell interactions using
intravital microscopy of post-capillary venule in mouse cremaster muscle. To this end, superfusion
of murine cremaster muscle with SB-747651A (5 µM) for 30 min prior to and for 1 h following the
placement of CXCL2-containing gel significantly enhanced leukocyte rolling flux (83.7 ± 3.4 cells/min Int. J. Mol. Sci. 2017, 18, 2163 3 of 12 with SB-747651A treatment versus 48.3 ± 3.1 cells/min without SB-747651A treatment; n = 4, p < 0.01)
and rolling velocity (64.4 ± 2.8 µm/s with SB-747651A treatment versus 46.9 ± 5.8 µm/s without
SB-747651A treatment; n = 4, p < 0.05) in comparison to CXCL2-treated group in the absence of
SB-747651A treatment. Int. J. Mol. Sci. 2017, 18, 2163
3 of 12
without SB-747651A treatment; n = 4, p < 0.05) in comparison to CXCL2-treated group in the absence
of SB-747651A treatment. During neutrophil recruitment, not all rolling neutrophils became adherent and emigrated in the
microvasculature. To analyze the subsequent steps of neutrophil recruitment in the same cremaster
muscle, we visualized the neutrophil recruitment process and determined the number of adherent and
emigrated neutrophils. As depicted in Figure 2A, the number of adherent neutrophils was significantly
increased at 30–60 min after the placement of CXCL2-containing gel on the cremaster muscle, an effect
significantly more pronounced in the presence of SB-747651A treatment. Similarly, the number of
emigrated neutrophil was significantly increased at 30–60 min after placement of CXCL2-containing
gel on the cremaster muscle, an effect that was significantly reduced by the SB-747651A treatment
(Figure 2B). Additional experiments were conducted to explore the consequence of SB-747651A
treatment on prolonged stimulation with CXCL2. To this end, SB-747651A treatment (3 mg/kg
intrascrotal injection, 1 h prior to the administration of CXCL2) resulted in increased neutrophil
adhesion 3.5–4.5 h following stimulation with CXCL2 (0.2 µg intrascrotal injection) as compared to the
effect of CXCL2 stimulation alone (Figure 2C). 2. Results
2. Results (C) Time course of
the number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or
in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2
treatment) at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM
(n = 3). *** indicates significant difference (p < 0.001) from the Control. (E) Representative images
from intravital video microscopy showing a postcapillary venule (Left) and the surrounding cremaster
muscle with emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel (Right). Reduced emigration of neutrophils following treatment with SB-747651A at early time points
following CXCL2 stimulation could be due to the impairment in the early neutrophil recruitment
steps subsequent to adhesion. To explore the effect of SB-747651A treatment on neutrophil
intraluminal crawling and transendothelial migration, we analyzed neutrophil intraluminal crawling
using time-lapsed video photography. As shown in Figure 3A, the velocity of intraluminal crawling
in response to CXCL2 chemotactic gradient was significantly lower following SB-747651A treatment
as compared to the CXCL2 control. These data suggest that SB-747651A treatment thwarts the
intraluminal crawling of adherent neutrophils to optimal sites of emigration. Intraluminal crawling
of adherent neutrophils is dictated by neutrophil αMβ2 integrin Mac-1 [4]. Flow cytometry analysis
revealed that neutrophil Mac-1 expression was significantly increased following the treatment of
bone marrow neutrophils with CXCL2, an effect significantly blunted by SB-747651A treatment
(Figure 3B and Figure S1). We also determined the effect of SB-747651A on the expression of integrin
αLβ2, LFA-1, another important β2 integrin on CXCL2-treated neutrophils and found that CXCL2 only
marginally enhanced LFA-1 expression on neutrophils and SB-747651A was completely ineffective
on the LFA-1 expression level in the presence or absence of CXCL2 (Figure S2). These results suggest
that SB-747651A treatment affects CXCL2-induced intraluminal crawling of neutrophils in a Mac-1-
d
d
Reduced emigration of neutrophils following treatment with SB-747651A at early time points
following CXCL2 stimulation could be due to the impairment in the early neutrophil recruitment steps
subsequent to adhesion. To explore the effect of SB-747651A treatment on neutrophil intraluminal
crawling and transendothelial migration, we analyzed neutrophil intraluminal crawling using
time-lapsed video photography. 2. Results
2. Results (C) Time course of the
number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2 treatment)
at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM (n = 3). ***
indicates significant difference (p < 0.001) from the Control. (E) Representative images from intravital
video microscopy showing a postcapillary venule (Left) and the surrounding cremaster muscle with
emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement
of CXCL2-containing gel (Right). Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. (C) Time course of
the number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or
in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2
treatment) at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM
(n = 3). *** indicates significant difference (p < 0.001) from the Control. (E) Representative images
from intravital video microscopy showing a postcapillary venule (Left) and the surrounding cremaster
muscle with emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel (Right). Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. 2. Results
2. Results (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control) or in
the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. (C) Time course of the
number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2 treatment)
at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM (n = 3). ***
indicates significant difference (p < 0.001) from the Control. (E) Representative images from intravital
video microscopy showing a postcapillary venule (Left) and the surrounding cremaster muscle with
emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement
of CXCL2-containing gel (Right). Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. (C) Time course of
the number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or
in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2
treatment) at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM
(n = 3). *** indicates significant difference (p < 0.001) from the Control. 2. Results
2. Results The number of emigrated neutrophils was significantly
increased after SB-747651A treatment at 3.5–4.5 h following CXCL2 stimulation as compared to the
CXCL2 treatment alone (Figure 2D). During neutrophil recruitment, not all rolling neutrophils became adherent and emigrated in
the microvasculature. To analyze the subsequent steps of neutrophil recruitment in the same
cremaster muscle, we visualized the neutrophil recruitment process and determined the number of
adherent and emigrated neutrophils. As depicted in Figure 2A, the number of adherent neutrophils
was significantly increased at 30–60 min after the placement of CXCL2-containing gel on the
cremaster muscle, an effect significantly more pronounced in the presence of SB-747651A treatment. Similarly, the number of emigrated neutrophil was significantly increased at 30–60 min after
placement of CXCL2-containing gel on the cremaster muscle, an effect that was significantly reduced
by the SB-747651A treatment (Figure 2B). Additional experiments were conducted to explore the
consequence of SB-747651A treatment on prolonged stimulation with CXCL2. To this end, SB-
747651A treatment (3 mg/kg intrascrotal injection, 1 h prior to the administration of CXCL2) resulted
in increased neutrophil adhesion 3.5–4.5 h following stimulation with CXCL2 (0.2 µg intrascrotal
injection) as compared to the effect of CXCL2 stimulation alone (Figure 2C). The number of emigrated
neutrophils was significantly increased after SB-747651A treatment at 3.5–4.5 h following CXCL2
stimulation as compared to the CXCL2 treatment alone (Figure 2D). Figure 2. Cont. Figure 2. Cont. 4 of 12
4 of 12 Int. J. Mol. Sci. 2017, 18, 2163
Int J Mol Sci 2017 18 2163 Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control) or in
the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. 2. Results
2. Results (E) Representative images
from intravital video microscopy showing a postcapillary venule (Left) and the surrounding cremaster
muscle with emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel (Right). Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control) or in
the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. (C) Time course of the
number of adherent neutrophils (cells/100-µm venule) and (D) time course of the number of
emigrated neutrophils (cells/443 × 286 µm2 field) induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (3 mg/kg, intrascrotal injection, 1-h prior to CXCL2 treatment)
at 3.5–4.5 h following an intrascrotal injection of 0.2 µg CXCL2. Data are means ± SEM (n = 3). ***
indicates significant difference (p < 0.001) from the Control. (E) Representative images from intravital
video microscopy showing a postcapillary venule (Left) and the surrounding cremaster muscle with
emigrated neutrophils (arrow head) at 60 min induced by CXCL2 in the absence (Control) or in the
presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement
of CXCL2-containing gel (Right). Figure 2. Effect of SB-747651A on CXCL2-induced neutrophil adhesion and emigration. (A) Time
course of the number of adherent neutrophils (cells/100-µm venule) and (B) time course of the number
of emigrated neutrophils (cells/235 × 208 µm2 field) induced by CXCL2 in the absence (Control)
or in the presence of MSK1 inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the
placement of CXCL2-containing gel. Data are means ± SEM (n = 4). *, ** and *** indicate significant
difference (p < 0.05, p < 0.01 and p < 0.001, respectively) from the Control. 2. Results
2. Results Data are means ± SEM
(n = 4). ** indicates significant difference (p < 0.01) from the Control. (B) Means ± SEM (n = 3) of
Mac-1-dependent fluorescence expressed as geomeans in untreated neutrophils (Control; white bar)
and in neutrophils treated with SB-747651A (5 µM, 30 min prior to addition of CXCL2; black bar) in
the absence (left bars) or in the presence (right bars) of stimulation with CXCL2 (30 nM for 10 min). *, **, and *** indicate significant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) from the
Control without CXCL2. # and ### indicate significant difference (p < 0.05 and p < 0.001, respectively)
from the group without SB-747651A. (C) The duration (min) of neutrophil transmigration across the
endothelium and (D) the detachment time (min) of neutrophils from the venule upon stimulation with
CXCL2 in the absence (Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A
(5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing gel. Data are means
± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Next, we performed an additional series of experiments to elucidate whether SB-747651A treatment
modulates extravascular migration of neutrophils in tissue. Time-lapsed video microscopy analysis
revealed that in response to CXCL2 chemotactic gradient, the speed of neutrophil migration was
significantly reduced following SB-747651A treatment compared to the CXCL2 control group (Figure 4A). Chemotaxis index, a parameter of migration directionality, measures the ratio of the distance in the
direction toward CXCL2-gel to the total migration distance the cell moved in the tissue. In response to the
CXCL2 chemotactic gradient, chemotaxis index of migrating neutrophils was, however, not altered
following SB-747651A treatment as compared to the CXCL2 control (Figure 4B). These data indicate that
SB-747651A treatment inhibits the migration speed of extravascular chemotaxing neutrophils but does
not affect their directionality in response to CXCL2 chemotactic gradient. Next, we performed an additional series of experiments to elucidate whether SB-747651A
treatment modulates extravascular migration of neutrophils in tissue. Time-lapsed video microscopy
analysis revealed that in response to CXCL2 chemotactic gradient, the speed of neutrophil migration
was significantly reduced following SB-747651A treatment compared to the CXCL2 control group
(Figure 4A). 2. Results
2. Results (C) The duration (min) of neutrophil transmigration across the
endothelium and (D) the detachment time (min) of neutrophils from the venule upon stimulation with
CXCL2 in the absence (Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A
(5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing gel. Data are means
± SEM (n = 4) ** indicates significant difference (p < 0 01) from the Control Figure 3. Effect of SB-747651A on CXCL2-induced Mac-1-dependent intraluminal crawling and
transendothelial migration. (A) The velocity of intraluminal crawling (µm/min) of neutrophils
crawling in the luminal surface of the endothelium upon stimulation with CXCL2 in the absence
(Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A (5 µM) 30 min prior
to and 60 min following the placement of CXCL2-containing gel. Data are means ± SEM (n = 4). **
indicates significant difference (p < 0.01) from the Control. (B) Means ± SEM (n = 3) of Mac-1-
dependent fluorescence expressed as geomeans in untreated neutrophils (Control; white bar) and in
neutrophils treated with SB-747651A (5 µM, 30 min prior to addition of CXCL2; black bar) in the
absence (left bars) or in the presence (right bars) of stimulation with CXCL2 (30 nM for 10 min). *, **,
and *** indicate significant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) from the Control
without CXCL2. # and ### indicate significant difference (p < 0.05 and p < 0.001, respectively) from the
group without SB-747651A. (C) The duration (min) of neutrophil transmigration across the
endothelium and (D) the detachment time (min) of neutrophils from the venule upon stimulation
with CXCL2 in the absence (Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-
747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing gel. Data
are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Figure 3. Effect of SB-747651A on CXCL2-induced Mac-1-dependent intraluminal crawling and
transendothelial migration. (A) The velocity of intraluminal crawling (µm/min) of neutrophils
crawling in the luminal surface of the endothelium upon stimulation with CXCL2 in the absence
(Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A (5 µM) 30 min
prior to and 60 min following the placement of CXCL2-containing gel. 2. Results
2. Results As shown in Figure 3A, the velocity of intraluminal crawling in
response to CXCL2 chemotactic gradient was significantly lower following SB-747651A treatment
as compared to the CXCL2 control. These data suggest that SB-747651A treatment thwarts the
intraluminal crawling of adherent neutrophils to optimal sites of emigration. Intraluminal crawling
of adherent neutrophils is dictated by neutrophil αMβ2 integrin Mac-1 [4]. Flow cytometry analysis
revealed that neutrophil Mac-1 expression was significantly increased following the treatment of bone
marrow neutrophils with CXCL2, an effect significantly blunted by SB-747651A treatment (Figure 3B
and Figure S1). We also determined the effect of SB-747651A on the expression of integrin αLβ2, LFA-1,
another important β2 integrin on CXCL2-treated neutrophils and found that CXCL2 only marginally
enhanced LFA-1 expression on neutrophils and SB-747651A was completely ineffective on the LFA-1
expression level in the presence or absence of CXCL2 (Figure S2). These results suggest that SB-747651A
treatment affects CXCL2-induced intraluminal crawling of neutrophils in a Mac-1-dependent manner. dependent manner. To further define the cause of the reduced early emigration following SB-747651A treatment, we
analyzed transmigration time and detachment time of neutrophils in response to CXCL2 chemotactic
gradient. As a result, SB-747651A treatment significantly increased transmigration time and
detachment time as compared to the control without this inhibitor (Figure 3C,D) indicating a slower
process of neutrophil emigration. These data suggest that SB-747651A treatment affects mechanisms
that regulate transendothelial migration of neutrophils in response to CXCL2 chemotactic gradient
To further define the cause of the reduced early emigration following SB-747651A treatment,
we analyzed transmigration time and detachment time of neutrophils in response to CXCL2
chemotactic gradient. As a result, SB-747651A treatment significantly increased transmigration time
and detachment time as compared to the control without this inhibitor (Figure 3C,D) indicating a slower
process of neutrophil emigration. These data suggest that SB-747651A treatment affects mechanisms
that regulate transendothelial migration of neutrophils in response to CXCL2 chemotactic gradient. 5 of 12
5 of 12 Int. J. Mol. Sci. 2017, 18, 2163
Int J Mol Sci 2017 18 2163 Figure 3. Effect of SB-747651A on CXCL2-induced Mac-1-dependent intraluminal crawling and
transendothelial migration. (A) The velocity of intraluminal crawling (µm/min) of neutrophils
crawling in the luminal surface of the endothelium upon stimulation with CXCL2 in the absence
(Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A (5 µM) 30 min prior
to and 60 min following the placement of CXCL2-containing gel. 2. Results
2. Results Data are means ± SEM (n = 4). **
indicates significant difference (p < 0.01) from the Control. (B) Means ± SEM (n = 3) of Mac-1-
dependent fluorescence expressed as geomeans in untreated neutrophils (Control; white bar) and in
neutrophils treated with SB-747651A (5 µM, 30 min prior to addition of CXCL2; black bar) in the
absence (left bars) or in the presence (right bars) of stimulation with CXCL2 (30 nM for 10 min). *, **,
and *** indicate significant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) from the Control
without CXCL2. # and ### indicate significant difference (p < 0.05 and p < 0.001, respectively) from the
group without SB-747651A. (C) The duration (min) of neutrophil transmigration across the
endothelium and (D) the detachment time (min) of neutrophils from the venule upon stimulation
with CXCL2 in the absence (Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-
747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing gel. Data
Figure 3. Effect of SB-747651A on CXCL2-induced Mac-1-dependent intraluminal crawling and
transendothelial migration. (A) The velocity of intraluminal crawling (µm/min) of neutrophils
crawling in the luminal surface of the endothelium upon stimulation with CXCL2 in the absence
(Control, white bar) or in the presence (black bar) of MSK1 inhibitor SB-747651A (5 µM) 30 min
prior to and 60 min following the placement of CXCL2-containing gel. Data are means ± SEM
(n = 4). ** indicates significant difference (p < 0.01) from the Control. (B) Means ± SEM (n = 3) of
Mac-1-dependent fluorescence expressed as geomeans in untreated neutrophils (Control; white bar)
and in neutrophils treated with SB-747651A (5 µM, 30 min prior to addition of CXCL2; black bar) in
the absence (left bars) or in the presence (right bars) of stimulation with CXCL2 (30 nM for 10 min). *, **, and *** indicate significant difference (p < 0.05, p < 0.01, and p < 0.001, respectively) from the
Control without CXCL2. # and ### indicate significant difference (p < 0.05 and p < 0.001, respectively)
from the group without SB-747651A. 2. Results
2. Results As shown in Figure 5, SB-747651A treatment did not enhance neutrophil emigration at 1–2 h after
CXCL2 treatment but significantly increased the number of emigrated neutrophils in the peritoneal
lavage fluid following 3 and 4 h of CXCL2 injection, indicating that SB-747651A treatment affects
neutrophil extravasation by increasing neutrophil emigration only at 3 and 4 h in mouse peritonitis
model of acute inflammation. To corroborate the effects of SB-747651A treatment on CXCL2-induced transendothelial
migration of neutrophils in vivo, an additional series of experiments were performed to explore the
effect of SB-747651A treatment on CXCL2-triggered infiltration of neutrophils to the peritoneal
cavity. As shown in Figure 5, SB-747651A treatment did not enhance neutrophil emigration at 1–2 h
after CXCL2 treatment but significantly increased the number of emigrated neutrophils in the
peritoneal lavage fluid following 3 and 4 h of CXCL2 injection, indicating that SB-747651A treatment
affects neutrophil extravasation by increasing neutrophil emigration only at 3 and 4 h in mouse
peritonitis model of acute inflammation. Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2 (0.5
µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from the Control. Discussion
Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2 (0.5
µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from the Control. Discussion
Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 2. Results
2. Results (A)
The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. (A) The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. (A)
The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. To corroborate the effects of SB-747651A treatment on CXCL2-induced transendothelial
migration of neutrophils in vivo, an additional series of experiments were performed to explore the
effect of SB-747651A treatment on CXCL2-triggered infiltration of neutrophils to the peritoneal
cavity. As shown in Figure 5, SB-747651A treatment did not enhance neutrophil emigration at 1–2 h
after CXCL2 treatment but significantly increased the number of emigrated neutrophils in the
peritoneal lavage fluid following 3 and 4 h of CXCL2 injection, indicating that SB-747651A treatment
affects neutrophil extravasation by increasing neutrophil emigration only at 3 and 4 h in mouse
peritonitis model of acute inflammation. To corroborate the effects of SB-747651A treatment on CXCL2-induced transendothelial migration
of neutrophils in vivo, an additional series of experiments were performed to explore the effect
of SB-747651A treatment on CXCL2-triggered infiltration of neutrophils to the peritoneal cavity. 2. Results
2. Results Chemotaxis index, a parameter of migration directionality, measures the ratio of
the distance in the direction toward CXCL2-gel to the total migration distance the cell moved
in the tissue. In response to the CXCL2 chemotactic gradient, chemotaxis index of migrating
neutrophils was, however, not altered following SB-747651A treatment as compared to the CXCL2
control (Figure 4B). These data indicate that SB-747651A treatment inhibits the migration speed of
extravascular chemotaxing neutrophils but does not affect their directionality in response to CXCL2
chemotactic gradient. 6 of 12
6 of 12 Int. J. Mol. Sci. 2017, 18, 2163
I t J M l S i 2017 18 2163 Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. (A)
The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. (A) The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. J Mo S i
0
,
,
63
6 o
Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. (A)
The neutrophil migration speed (µm/min) and (B) the chemotaxis index of migrating neutrophils in
response to CXCL2 stimulation in the absence (Control, white bar) or in the presence (black bar) of MSK1
inhibitor SB-747651A (5 µM) 30 min prior to and 60 min following the placement of CXCL2-containing
gel. Data are means ± SEM (n = 4). ** indicates significant difference (p < 0.01) from the Control. Figure 4. Effect of SB-747651A on CXCL2-induced neutrophil migration and chemotaxis in tissue. 3. Discussion Neutrophil-endothelial cell interactions during acute inflammation generate molecular signals that
are decisive in the recruitment of neutrophils to the site of inflammation. The present study discloses
the effect of pharmacological inhibition of MSK1 on different steps of chemokine CXCL2-induced
neutrophil recruitment. We show that in response to chemokine CXCL2, MSK1 protein expression was
upregulated in neutrophils. Pharmacological inhibition of MSK1 by using selective MSK1 inhibitor
SB-747651A enhanced CXCL2-induced adhesion of neutrophils to the microvascular lumen while
temporarily curtailing transendothelial migration of neutrophils. SB-747651A treatment thwarted
Mac-1-dependent intraluminal crawling, while increasing both transmigration time and detachment
time, effects favoring reduced transendothelial migration. SB-747651A treatment further mitigated the
migration speed of neutrophils in extravascular tissue. Mechanistically, MSK1 targets both pro- and anti-inflammatory genes [17]. Molecules upstream
of MSK1 signaling, ERK1/2 and p38 MAPK, are important in the production of inflammatory
cytokines [17]. These signaling molecules also activate negative feedback pathways via MSK1/2 to
suppress the proinflammatory effects of Toll-like receptor 4 (TLR4) signaling [20]. Mice deficient
in MSK1/2 were shown to be more susceptible to endotoxic shock and showed enhanced
myeloperoxidase activity following phorbol ester-triggered eczema [20]. Similarly, skin inflammation
was shown to be enhanced in MSK1/2-deficient mice with elevated infiltration of neutrophils in
response to oxazolone-induced allergic contact dermatitis [25]. Furthermore, MSK1/2 activation was
also shown to be involved in the pathogenesis of psoriatic skin lesions [26]. Discordantly, however,
suppression of MSK1 by inhibitors such as H89 showed amelioration of airway inflammation [27]. In another study, MSK1 is documented to participate in airway inflammation elicited by respiratory
syncytial virus [19]. Discrepancies in the effect of MSK1 inhibitors and the anti-inflammatory
phenotype of MSK1-deficient mice may well be explained by the non-specificity of the inhibitors
Ro 31-8220 and H89 to different cellular kinases reported in the earlier studies. The role of MSK1 in neutrophil-endothelial cell interactions remains elusive. In addition to
neutrophils, endothelial cells also express MSK1, which participates in the activation of CREB [28] and
in the regulation of synthesis of platelet-activating factor [29]. In the present study, however, the role
of endothelial MSK1 in the observed effects of SB-747651A treatment on CXCL2-induced neutrophil
recruitment cannot be ruled out. Thus, further investigations are warranted to examine the role of
cell-specific regulation of neutrophil recruitment by MSK1 using MSK1 knockout mice. 2. Results
2. Results 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2
(0.5 µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from
the Control. Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2 (0.5
µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from the Control. Discussion
Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2 (0.5
µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from the Control. Discussion
Figure 5. Effect of SB-747651A on CXCL2-induced neutrophil emigration in peritoneum. Time course
of the number of emigrated neutrophils (×106 cells) counted in the peritoneal lavage fluid in the
absence (Control, open square) or in the presence of MSK1 inhibitor SB-747651A (3 mg/kg, i.p. 30 min
prior to CXCL2 injection, solid circle) collected at 1, 2, 3, 4, and 6 h after an i.p. injection of CXCL2
(0.5 µg/mouse). Data are means ± SEM (n = 3). * indicates significant difference (p < 0.05) from
the Control. Int. J. Mol. Sci. 2017, 18, 2163 7 of 12 7 of 12 3. Discussion While p38 MAPK was previously shown to regulate neutrophil adhesion and transendothelial
migration [30], more recent work has suggested that p38 MAPK also contributes to other steps
of neutrophil recruitment, such as Mac-1-dependent intraluminal crawling and extravascular
migration [5]. Intracellular signals regulating neutrophil intraluminal crawling involve Vav guanine
nucleotide exchange factor 1 (Vav1) and mammalian-actin binding protein 1 downstream of spleen
tyrosine kinase [31,32]. However, the role of MSK1 in neutrophil intravascular crawling and
extravascular chemotaxis was not investigated in the previous studies. Enhanced adhesion has
been expected to be translated into increased transendothelial migration. Transendothelial migration
is effectively accomplished by subsequent recruitment steps following neutrophil adhesion and
intraluminal crawling. We observed that SB-747651A treatment attenuated CXCL2-stimulated Mac-1
expression and CXCL2-induced intraluminal crawling of neutrophils. Surprisingly, despite increased
neutrophil adhesion to the vascular endothelium, we found that SB-747651A treatment effectively
decreased intraluminal crawling and decreased transendothelial migration at the early time points,
the latter was evidenced by the decreased emigration, prolonged transmigration time and detachment
time of neutrophils at early time points during transendothelial migration following SB-747651A
treatment. In contrast to the role of p38 MAPK on the directionality of chemotaxing neutrophils [5],
inhibition of MSK1 did not affect the chemotaxis index of extravascular migrating neutrophils but
attenuated the migration speed of neutrophils. SB-747651A treatment presumably affects the expression
of adhesion molecules that regulate the passage of neutrophils into extravascular tissue. It is intriguing 8 of 12 Int. J. Mol. Sci. 2017, 18, 2163 to speculate that differential effects of SB-747651A treatment on neutrophil and endothelial adhesion
molecules may have accounted for the increased adhesion and decreased emigration of neutrophils
during the very early stage of recruitment in acute inflammation. It is also possible that the attenuation
of the migration speed of neutrophils in tissue by SB-747651A increases the accumulation of emigrated
neutrophils in the inflammatory sites at time points later than 1–2 h after CXCL2 stimulation. It is
interesting to note that SB-747651A did not change neutrophil emigration in early time points but
increased neutrophil emigration until 3–4 h in peritoneum stimulated by CXCL2. This suggests that
SB-747651A treatment may only result in enhanced neutrophil recruitment in peritoneum after 3–4 h
of CXCL2 treatment, in a pattern different from the two-phase recruitment in cremaster muscle. 3. Discussion Collectively, our data suggest that inhibition of MSK1 by SB-747651A treatment affects
CXCL2-induced neutrophil recruitment by modulating various steps of the recruitment cascade
in vivo. 4.1. Mice Male C57BL/6N mice between 8- and 16-weeks-old, purchased from Charles River Canada
(Saint-Constant, QC, Canada), were used in experiments. This study was carried out with the approved
animal protocols (#20070028; 28 November 2012 and 7 June 2013) from the University Committee
on Animal Care and Supply (UCACS) at the University of Saskatchewan following the standards of
Canadian Association of Animal Care. 4.6. Induced Peritonitis Acute mouse peritonitis was induced to obtain emigrated neutrophils after an i.p. injection of
murine CXCL2 (0.5 µg in sterile saline). Cells were then lavaged and harvested from the peritoneum
at different time points and the emigrated neutrophils were counted. 4.2. Intravital Microscopy In this approach, where indicated, SB-747651A was administered at 3 mg/kg by
intrascrotal injection 1 h prior to CXCL2 injection. CXCL2 treatment. In this approach, where indicated, SB-747651A was administered at 3 mg/kg by
intrascrotal injection 1 h prior to CXCL2 injection. 4.5. Fluorescence-Activated Cell Sorting (FACS) Analysis of Mac-1 and LFA-1 Expression The expression of β2 integrins Mac-1 and LFA-1 on neutrophils was determined using a
previously described method with slight modifications [5,38]. Following lysis of red blood cells,
bone marrow-derived neutrophils were incubated at 37 ◦C for 30 min in the presence or absence of
5 µM SB-747651A in vitro. The cells were stimulated with CXCL2 (30 nM at 37 ◦C for 10 min) to
upregulate Mac-1 and LFA-1 expression. Aliquots of the neutrophil suspension (106/mL) were washed
in ice-cold PBS containing 1% BSA, stained with a fluorescent anti-Mac-1 or anti-LFA-1 antibody
(Anti-mouse CD11b FITC; clone M1/70; anti-mouse CD11a FITC; clone M17/4, both from eBioscience,
San Diego, CA, USA) or the isotype control (Rat IgG2bκ FITC; eBioscience) and incubated for 30 min at
4 ◦C. The samples were then centrifuged (1200 rpm, 3 min, 4 ◦C) and washed twice with ice-cold PBS
containing 1% BSA and analyzed in the FL-1 channel of an Epics XL flow cytometer (Beckman Coulter,
Miami, FL, USA) with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. 4.3. Cell Tracking Using ImageJ software (Version 1.48, National Institutes of Health, Bethesda, MD, USA),
neutrophil intraluminal crawling, transmigration, and chemotaxis in cremasteric microvasculature
were analyzed using the time-lapsed movie converted from the real-time video recording of the
experiment as described previously [4,5,35,36]. The following recruitment parameters were quantified
from tracking and analyzing at least 40 cells for each treatment group: (a) velocity of intraluminal
crawling (µm/min): the total distance the neutrophil crawled from the initial site of adhesion to the
transmigration site (µm) divided by the duration of neutrophils undergoing intraluminal crawling
(min); (b) transmigration time (min): from the time the neutrophil stopped crawling and started to
transmigrate to the time the whole neutrophil body was just outside the venule; (c) detachment time
(min): from the time the neutrophil body was just outside the venule after its transmigration to the
time when the neutrophil migrated away and lost contact to the venule; (d) speed of migration in
tissue (µm/min): neutrophil migration distance in tissue (µm) divided by the time that the neutrophil
migrated (min); (e) chemotaxis index in tissue: the ratio of the distance in the direction toward the
CXCL2-gel to the total migration distance the neutrophil moved in tissue. 4.2. Intravital Microscopy Mice were anaesthetized with an intraperitoneal (i.p.)
injection of 10 mg/kg xyalzine
(Bayer, Toronto, ON, Canada) and 200 mg/kg ketamine hydrochloride (Rogar, Montreal, QC, Canada). The mouse cremaster muscle preparation was used to study neutrophil behaviour in microcirculation
and tissue as described previously [5,33–35]. The cremaster muscle was kept warm and superfused
with 37 ◦C-warmed bicarbonate-buffered saline (pH 7.4; containing in mM 133.9 NaCl, 4.7 KCl,
1.2 MgSO4 and 20 NaHCO3). An upright microscope (model Eclipse Ci-s, Nikon, Tokyo, Japan) with a
LUCPLFLN 20× objective lens was projected to a charge-coupled device (CCD) color video camera
(DC-220, Dage, Dage-MTI, Inc., Michigan City, IN, USA) for bright-field intravital microscopy. For the
induction of neutrophil recruitment, two approaches were taken. In the first approach, an agarose gel at
1-mm3 size containing murine CXC chemokine CXCL2 (0.5 µM; R&D Systems, Minneapolis, MN, USA)
was placed on the surface of the cremaster muscle in a preselected area 350-µm distant from
and parallel to the observed postcapillary venule. After placing a glass coverslip to hold the
gel, the cremaster muscle was superfused with bicarbonate-buffered saline at a very slow rate
(≤10 µL/min) to allow the formation of CXCL2 chemotactic gradient. Throughout the experiment,
neutrophil behaviour and hemodynamic changes in the selected cremasteric postcapillary venule
(25–40 µm diameter) were visualized on a TV monitor and recorded at real time on a DVD recorder
before (for time 0 min) and after the addition of CXCL2-containing gel (recorded for 60 min). During
recording, all efforts were made to adjust and keep the microscope images focused on the adhering,
crawling, transmigrating and chemotaxing neutrophil inside the venule and in the muscle tissue. The number of rolling, adherent, and emigrated neutrophils was determined in the cremasteric
microvasculature during offline playback analysis of the recorded video as described previously [34]. Where indicated, the specific MSK1 inhibitor 5 µM SB-747651A (Axon Medchem BV, Groningen,
The Netherlands) was superfused on the cremaster muscle 30 min prior to and remained superfused
for 60 min after the addition of CXCL2-containing gel. The second approach was to induce neutrophil
recruitment at later time points by intrascrotal injection of CXCL2 (0.2 µg in 100 µL sterile saline) and
by determining the parameters of neutrophil recruitment under intravital microscopy at 3.5–4.5 h after Int. J. Mol. Sci. 2017, 18, 2163 9 of 12 CXCL2 treatment. 4.4. Isolation of Murine Neutrophils Bone marrow cells were freshly harvested from mouse femurs and tibias, and the marrow was
flushed with ice-cold Ca2+- and Mg2+-free phosphate-buffered saline (PBS) solution. Neutrophils were
isolated using a Percoll (GE Healthcare, Uppsala, Sweden) gradient (72%, 64%, and 52%) centrifugation
at 1060× g at room temperature for 30 min as described previously [37] and subsequently washed
with PBS. The isolated cells had >85% purity of morphologically mature neutrophils. 4.8. Statistical Analysis Data are expressed as means ± SEM. n denotes the number of mice studied in each group or the
number of mice used to derive bone marrow neutrophils for in vitro studies. Statistical analysis was
performed using two-tailed Student’s t-test and p values < 0.05 were considered statistically significant. Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/10/2163 Acknowledgments: This study was supported by a research grant from the Natural Sciences and Engineering
Research Council of Canada (NSERC, #386732-2010 to Lixin Liu). Lixin Liu was a recipient of the CIHR New
Investigator Award. The authors thank S.M. Qadri (University of Saskatchewan, Saskatoon, SK, Canada) for
critical reading of the manuscript and for valuable suggestions and Y. Su and L. Hao (University of Saskatchewan,
Saskatoon, SK, Canada) for technical support. Author Contributions: Mokarram Hossain and Entesar Omran designed and performed the experiments. Mokarram Hossain, Entesar Omran, and Najia Xu analyzed the data. Mokarram Hossain wrote the manuscript. Lixin Liu conceived the study and wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. 4.7. Western Blotting After the indicated treatment, bone marrow neutrophils were lysed in lysis buffer (pH 8.0;
containing 50 mM Tris–HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and
protease and phosphatase inhibitor cocktails, purchased from Fisher Scientific, Toronto, ON, Canada). Proteins (40 µg) were solubilized in Laemmli sample buffer at 95◦C for 5 min and resolved by 10% 10 of 12 Int. J. Mol. Sci. 2017, 18, 2163 SDS–PAGE. For immunoblotting, proteins were transferred onto a nitrocellulose membrane and
blocked with 5% BSA in Tris-buffered saline-Tween 20 at room temperature for 1 h. Then, the membrane
was incubated with anti-MSK1 antibody (1:1000; Cell Signaling Technology, Danvers, MA, USA) at
4 ◦C overnight. After incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary
antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature,
antibody binding was detected with the ECL detection reagent (GE Healtcare, Baie d’Urfe, QC, Canada). β-actin (mouse anti-β-actin antibody, 1:1000, Santa Cruz Biotechnology) was detected after stripping
with a buffer (pH 6.8; containing 0.5 M Tris–HCl, 2% SDS and 0.7% 2-β-mercaptoethanol). Densitometric quantification of the detected bands was performed using Gene Snap Software (Syngene,
Frederick, MD, USA). References 1. Phillipson, M.; Kubes, P. The neutrophil in vascular inflammation. Nat. Med. 2011, 17, 1381–1390. [CrossRef]
[PubMed] 1. Phillipson, M.; Kubes, P. The neutrophil in vascular inflammation. Nat. Med. 2011, 17, 1381–1390. [CrossRef]
[PubMed] 2. Langer, H.F.; Chavakis, T. Leukocyte—Endothelial interactions in inflammation. J. Cell. Mol. Med. 2009, 13,
1211–1220. [CrossRef] [PubMed] 2. Langer, H.F.; Chavakis, T. Leukocyte—Endothelial interactions in inflammation. J. Cell. Mol. Med. 2009, 13,
1211–1220. [CrossRef] [PubMed] 3. Petri, B.; Phillipson, M.; Kubes, P. The physiology of leukocyte recruitment: An in vivo perspective. J. Immunol. 2008, 180, 6439–6446. [CrossRef] [PubMed] 3. Petri, B.; Phillipson, M.; Kubes, P. The physiology of leukocyte recruitment: An in vivo perspective. J. Immunol. 2008, 180, 6439–6446. [CrossRef] [PubMed] 4. Phillipson, M.; Heit, B.; Colarusso, P.; Liu, L.; Ballantyne, C.M.; Kubes, P. Intraluminal crawling of neutrophils
to emigration sites: A molecularly distinct process from adhesion in the recruitment cascade. J. Exp. Med. 2006, 203, 2569–2575. [CrossRef] [PubMed] 4. Phillipson, M.; Heit, B.; Colarusso, P.; Liu, L.; Ballantyne, C.M.; Kubes, P. Intraluminal crawling of neutrophils
to emigration sites: A molecularly distinct process from adhesion in the recruitment cascade. J. Exp. Med. 2006, 203, 2569–2575. [CrossRef] [PubMed] 5. Xu, N.; Hossain, M.; Liu, L. Pharmacological inhibition of p38 mitogen-activated protein kinases affects
KC/CXCL1-induced intraluminal crawling, transendothelial migration, and chemotaxis of neutrophils
in vivo. Mediat. Inflamm. 2013. [CrossRef] [PubMed] 5. Xu, N.; Hossain, M.; Liu, L. Pharmacological inhibition of p38 mitogen-activated protein kinases affects
KC/CXCL1-induced intraluminal crawling, transendothelial migration, and chemotaxis of neutrophils
in vivo. Mediat. Inflamm. 2013. [CrossRef] [PubMed] 6. Plotnikov, A.; Zehorai, E.; Procaccia, S.; Seger, R. The MAPK cascades: Signaling components, nuclear roles
and mechanisms of nuclear translocation. Biochim. Biophys. Acta 2011, 1813, 1619–1633. [CrossRef] [PubMed] 6. Plotnikov, A.; Zehorai, E.; Procaccia, S.; Seger, R. The MAPK cascades: Signaling components, nuclear roles
and mechanisms of nuclear translocation. Biochim. Biophys. Acta 2011, 1813, 1619–1633. [CrossRef] [PubMed]
7. Kim,
E.K.;
Choi,
E.J. Pathological
roles
of
MAPK
signaling
pathways
in
human
diseases. p y
,
,
[
] [
]
7. Kim,
E.K.;
Choi,
E.J. Pathological
roles
of
MAPK
signaling
pathways
in
human
diseases. Biochim. Biophys. Acta 2010, 1802, 396–405. [CrossRef] [PubMed] 7. Kim,
E.K.;
Choi,
E.J. Pathological
roles
of
MAPK
signaling
pathways
in
human
diseases. Biochim. Biophys. Acta 2010, 1802, 396–405. [CrossRef] [PubMed] 8. Arthur, J.S.; Ley, S.C. Mitogen-activated protein kinases in innate immunity. Nat. Rev. Immunol. 2013, 13,
679–692. [CrossRef] [PubMed] 8. References Arthur, J.S.; Ley, S.C. Mitogen-activated protein kinases in innate immunity. Nat. Rev. Immunol. 2013, 13,
679–692. [CrossRef] [PubMed]
9. McCoy, C.E.; Campbell, D.G.; Deak, M.; Bloomberg, G.B.; Arthur, J.S.C. MSK1 activity is controlled by 9. McCoy, C.E.; Campbell, D.G.; Deak, M.; Bloomberg, G.B.; Arthur, J.S.C. MSK1 activity is contro
multiple phosphorylation sites. Biochem. J. 2005, 387, 507–517. [CrossRef] [PubMed] 10. Frodin, M.; Gammeltoft, S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell. Endocrinol. 1999, 151, 65–77. [CrossRef] 10. Frodin, M.; Gammeltoft, S. Role and regulation of 90 kDa ribosomal S6 kinase (RSK) in signal transduction. Mol. Cell. Endocrinol. 1999, 151, 65–77. [CrossRef] 11. Dunn, K.L.; Espino, P.S.; Drobic, B.; He, S.; Davie, J.R. The Ras-MAPK signal transduction pathway, cancer
and chromatin remodeling. Biochem. Cell. Biol. 2005, 83, 1–14. [CrossRef] [PubMed] 11 of 12 Int. J. Mol. Sci. 2017, 18, 2163 12. Van der Heide, L.P.; van Dinther, M.; Moustakas, A.; ten Dijke, P. TGFβ activates mitogen- and stress-activated
protein kinase-1 (MSK1) to attenuate cell death. J. Biol. Chem. 2011, 286, 5003–5011. [CrossRef] [PubMed] 13. Vermeulen, L. Transcriptional activation of the NF-κB p65 subunit by mitogen- and stress-activated protein
kinase-1 (MSK1). EMBO J. 2003, 22, 1313–1324. [CrossRef] [PubMed] 14. Beck, I.M.; vanden Berghe, W.; Vermeulen, L.; Bougarne, N.; vander Cruyssen, B.; Haegeman, G.;
de Bosscher, K. Altered subcellular distribution of MSK1 induced by glucocorticoids contributes to NF-κB
inhibition. EMBO J. 2008, 27, 1682–1693. [CrossRef] [PubMed] 15. Olson, C.M.; Hedrick, M.N.; Izadi, H.; Bates, T.C.; Olivera, E.R.; Anguita, J. p38 mitogen-activated protein
kinase controls NF-κB transcriptional activation and tumor necrosis factor alpha production through
RelA phosphorylation mediated by mitogen- and stress-activated protein kinase 1 in response to Borrelia
burgdorferi antigens. Infect. Immun. 2007, 75, 270–277. [CrossRef] [PubMed] 16. Mayer, T.Z.; Simard, F.A.; Cloutier, A.; Vardhan, H.; Dubois, C.M.; McDonald, P.P. The p38-MSK1 signaling
cascade influences cytokine production through CREB and C/EBP factors in human neutrophils. J. Immunol. 2013, 191, 4299–4307. [CrossRef] [PubMed] 17. Moens, U.; Kostenko, S.; Sveinbjornsson, B. The Role of Mitogen-Activated Protein Kinase-Activated Protein
Kinases (MAPKAPKs) in Inflammation. Genes 2013, 4, 101–133. [CrossRef] [PubMed] 8. Darragh, J.; Ananieva, O.; Courtney, A.; Elcombe, S.; Arthur, J.S.C. MSK1 regulates the transcription of IL
in response to TLR activation in macrophages. Biochem. J. 2010, 425, 595–602. [CrossRef] [PubMed] 19. Jamaluddin, M.; Tian, B.; Boldogh, I.; Garofalo, R.P.; Brasier, A.R. References Respiratory syncytial virus infection
induces a reactive oxygen species-MSK1-phospho-Ser-276 RelA pathway required for cytokine expression. J. Virol. 2009, 83, 10605–10615. [CrossRef] [PubMed] 20. Ananieva, O.; Darragh, J.; Johansen, C.; Carr, J.M.; McIlrath, J.; Park, J.M.; Wingate, A.; Monk, C.E.; Toth, R.;
Santos, S.G.; et al. The kinases MSK1 and MSK2 act as negative regulators of Toll-like receptor signaling. Nat. Immunol. 2008, 9, 1028–1036. [CrossRef] [PubMed] 21. MacKenzie, K.F.; van Den Bosch, M.W.M.; Naqvi, S.; Elcombe, S.E.; McGuire, V.A.; Reith, A.D.; Blackshear, P.J.;
Dean, J.L.E.; Arthur, J.S.C. MSK1 and MSK2 inhibit lipopolysaccharide-induced prostaglandin production
via an interleukin-10 feedback loop. Mol. Cell. Biol. 2013, 33, 1456–1467. [CrossRef] [PubMed] 22. Fernandez-Martinez, A.B.; Lucio Cazana, F.J. Prostaglandin E2 induces retinoic acid receptor-β up-re
through MSK1. Biochim. Biophys. Acta 2014, 1843, 1997–2004. [CrossRef] [PubMed] 23. Caivano,
M.;
Cohen,
P. Role
of
mitogen-activated
protein
kinase
cascades
in
mediating
lipopolysaccharide-stimulated induction of cyclooxygenase-2 and IL-1 β in RAW264 macrophages. J. Immunol. 2000, 164, 3018–3025. [CrossRef] [PubMed] 24. Naqvi, S.; Macdonald, A.; McCoy, C.E.; Darragh, J.; Reith, A.D.; Arthur, J.S.C. Characterization of the cellular
action of the MSK inhibitor SB-747651A. Biochem. J. 2012, 441, 347–357. [CrossRef] [PubMed] 25. Bertelsen, T.; Iversen, L.; Riis, J.L.; Arthur, J.S.C.; Bibby, B.M.; Kragballe, K.; Johansen, C. The role of mitogen-
and stress-activated protein kinase 1 and 2 in chronic skin inflammation in mice. Exp. Dermatol. 2011, 20,
140–145. [CrossRef] [PubMed] 26. Gesser, B.; Johansen, C.; Rasmussen, M.K.; Funding, A.T.; Otkjaer, K.; Kjellerup, R.B.; Kragballe, K.;
Iversen, L. Dimethylfumarate specifically inhibits the mitogen and stress-activated kinases 1 and 2 (MSK1/2):
Possible role for its anti-psoriatic effect. J. Investig. Dermatol. 2007, 127, 2129–2137. [CrossRef] [PubMed] 27. Reber, L.L.; Daubeuf, F.; Nemska, S.; Frossard, N. Thehe AGC kinase inhibitor H89 attenuates
inflammation in mouse models of asthma. PLoS ONE 2012, 7, e49512. [CrossRef] [PubMed] 28. Gustin, J.A. Tumor necrosis factor activates CRE-binding protein through a p38 MAPK/MSK1 signaling
pathway in endothelial cells. Am. J. Physiol. Cell. Physiol. 2004, 286, C547–C555. [CrossRef] [PubMed] 29. Marchand, C.; Favier, J.; Sirois, M.G. Role of MSK1 in the signaling pathway leading to VEGF-mediated PAF
synthesis in endothelial cells. J. Cell. Biochem. 2006, 98, 1095–1105. [CrossRef] [PubMed] 30. Cara, D.C.; Kaur, J.; Forster, M.; McCafferty, D.-M.; Kubes, P. Role of p38 mitogen-activated protein kinase
in chemokine-induced emigration and chemotaxis in vivo. J. Immunol. 2001, 167, 6552–6558. [CrossRef]
[PubMed] 31. References Hepper, I.; Schymeinsky, J.; Weckbach, L.T.; Jakob, S.M.; Frommhold, D.; Sixt, M.; Laschinger, M.;
Sperandio, M.; Walzog, B. The mammalian actin-binding protein 1 is critical for spreading and intraluminal
crawling of neutrophils under flow conditions. J. Immunol. 2012, 188, 4590–4601. [CrossRef] [PubMed] 12 of 12 12 of 12 Int. J. Mol. Sci. 2017, 18, 2163 32. Schymeinsky, J.; Sindrilaru, A.; Frommhold, D.; Sperandio, M.; Gerstl, R.; Then, C.; Mocsai, A.;
Scharffetter-Kochanek, K.; Walzog, B. The Vav binding site of the non-receptor tyrosine kinase Syk at
Tyr 348 is critical for β2 integrin (CD11/CD18)-mediated neutrophil migration. Blood 2006, 108, 3919–3927. [CrossRef] [PubMed] 33. Lei, X.; Hossain, M.; Qadri, S.M.; Liu, L. Different microvascular permeability responses elicited by the CXC
chemokines MIP-2 and KC during leukocyte recruitment: Role of LSP1. Biochem. Biophys. Res. Commun. 2012, 423, 484–489. [CrossRef] [PubMed] 34. Liu, L.; Cara, D.C.; Kaur, J.; Raharjo, E.; Mullaly, S.C.; Jongstra-Bilen, J.; Jongstra, J.; Kubes, P. LSP1 is an
endothelial gatekeeper of leukocyte transendothelial migration. J. Exp. Med. 2005, 201, 409–418. [CrossRef]
[PubMed] 35. Xu, N.; Lei, X.; Liu, L. Tracking neutrophil intraluminal crawling, transendothelial migration and chemotaxis
in tissue by intravital video microscopy. J. Vis. Exp. 2011, 55. [CrossRef] [PubMed] 36. Phillipson, M.; Heit, B.; Parsons, S.A.; Petri, B.; Mullaly, S.C.; Colarusso, P.; Gower, R.M.; Neely, G.; Simon, S.I.;
Kubes, P. Vav1 is essential for mechanotactic crawling and migration of neutrophils out of the inflamed
microvasculature. J. Immunol. 2009, 182, 6870–6878. [CrossRef] [PubMed] 37. Lieber, J.G. The in vitro production and characterization of neutrophils from embryonic stem cells. Blood
2004, 103, 852–859. [CrossRef] [PubMed] 38. Buschmann, K.; Koch, L.; Braach, N.; Mueller, H.; Frommhold, D.; Poeschl, J.; Ruef, P. CXCL1-triggered
interaction of LFA1 and ICAM1 control glucose-induced leukocyte recruitment during inflammation in vivo. Mediat. Inflamm. 2012, 2012, 739176. [CrossRef] [PubMed] © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
|
https://openalex.org/W4311101114
|
https://zenodo.org/records/7424679/files/2.%20Cinema%20and%20Geopolitics.pdf
|
English
| null |
Cinema, Geopolitics, and Power Surveillance: Screening Middle East in Post-9/11 Hollywood Films
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,022
|
cc-by
| 11,798
|
Cinema, Geopolitics, and Power Surveillance:
Screening Middle East in Post-9/11 Hollywood Films Mohamed Bataoui
Hassan Premier University, Morocco © Arab Journal of International Law, Morocco, Marrakech, 2022.
10.5281/zenodo.7424679
https://doi.org/ Abstract This paper tries to investigate the ways in which American geopolitical
hegemony operates through popular culture produced in the wake of 9/11. Specifically, it examines how American geopolitical anxieties and sensibilities
are aesthetically, culturally and politically mediated and maintained via the
cultural and politicized medium of film. It chooses The Kingdom (2007), a
geographically informed and geopolitically framed film, as its primary source of
analysis to interrogate the ways in which geopolitical anxieties and sensibilities
manifest in the film's narrative through the use of geopoliticized rhetoric of
screened landscapes and the specific narration of "Self"/"Other" identities. Drawing on literature from popular geopolitics, geocriticism, visual politics, and
postmodernism, this research analyzes how the various interdependent registers
of mapping intersect to geopolitically produce and imagine the Middle East. It
maintains that the rhetoric discourse of "war on terrorism" is cinematically
appropriated and geopolitically manipulated in order to diffuse and reproduce
American global hegemony on the global stage. In so doing, it moves beyond
the mere analysis of popular culture as only an object for analysis in world
politics to how cinematic texts are sites wherein specific modes of geopolitical
knowledge, geo-power and world politics are communicated and understood
globally. Keywords: Hollywood cinema; popular culture; "war on terrorism"; Middle
East; international politics; global hegemony, critical geopolitics © Arab Journal of International Law, 2022 https://doi.org/ 10.5281/zenodo.7424679 1. Introduction: Cinema, Politics, and Representation "As a technology of seeing and a form of porjectionism, film can be regarded as
eminently geo-graph-ical and geo-political."1 Throughout the history of cinema, the landscape and culture of Middle East
have been important to the discourse of Hollywood cinematic perceptions,
reproductions and codifications of mythical, political and ideological discursive
narratives. In particular, movies, as popular cultural artifacts, have played an
extremely important, if frequently controversial, role in the mobilization of
political culture, geography and propaganda. World nations and governments
have tremendously relied on the power of the moving images to spread their
soft power in the global sphere, and thus help create particular sentiments and
discourses that ultimately work for the fulfillment of specific ends, nationally
and internationally2. Given its pervasive influence and potency as a strategic
communication on the global stage, soft power is considered to be instrumental
for the United States as it consistently seeks to maintain global hegemony and
nation-branding. The medium of cinema, in other words, has been deployed to
sustain prevailing political sensibilities by dramatizing stories of nationalism,
heroism, villainy and geo-power as a narrative strategy to refashion a new sense
of American power round the globe. Films like Syriana (Stephen Gaghan,
2005), Munich (Steven Spielberg, 2005), Home of the Brave (Irwin Winkler,
2006), Redacted (De Palma, 2007), Body of Lies (Ridley Scott, 2008), or Zero
Dark Thirty (Kathryn Bigelow, 2012) are cases in point which exhibit these
tendencies and sensibilities in more subtle ways. Hence, given the power influence of popular culture in producing and
authorizing knowledge about distant cultures, peoples and lands, cinema, as a
powerful popular signifying system, is therefore able to vehicle, institute and
normalize the established socio-political constructions and mythologies it
consistently purports and perpetuates. As Mark Lacy has noted, "cinema 19 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 becomes a space where "common" sense ideas about global politics and history
are (re)produced and where stories about what is acceptable behaviour from
states and individuals are naturalized and legitimated"3. Cinema, as an art, then,
is never neutral, but is always shaped by the political, social and economic
sensibilities, anxieties, and forces that in turn shape and reshape representations
of reality. Mao Zedong postulates that, "there is in fact no such thing as art for
art's sake, art that stands above classes or art that is detached or independent of
politics"4. 1. Introduction: Cinema, Politics, and Representation What is more, "popular culture", to borrow Weldes and Rowely's
words, "not only reflects but also constitutes world politics"5. Films, as popular
cultural texts, discursively construct the world politics, the subjects and the
objects they visually introduce to the audience, therefore illustrating the
centrality of strategic cinema in shaping identities, geographies and perceptions
of surveillance and international politics. Keeping with the scope of this intervention, the complex connections between
cinema, geopolitics, power and representation matter a great deal in
contemporary Hollywood geopolitically-inclined films featuring the Middle
East after the vents of 9/11. Following the attacks on the Pentagon in
Washington and the World Trade center in New York, Pentagon officials
conducted a series of meetings with Hollywood directors, screenwriters,
scenarists and specialists in disaster movies to "solicit the help of Hollywood in
the war against terrorism"6 and put forward possible scenarios to dramatize and
respond to the attacks threatening American identity as an international
hegemon. In a similar vein, White House advisors met with Hollywood
executives to discuss the role of Hollywood in "getting the right ideological
message across not only to Americans, but also to the Hollywood public around
the globe"7. As a consequence, Hollywood mainstream cinematic responses
have immensely contributed into fashioning movies that capitalize on pro-
American sentiment to extol the virtues of the American vision of the world,
display American socio-political values and reposition American perceptions of 20 © Arab Journal of International Law, 2022 https://doi.org/ 10.5281/zenodo.7424679 geo-power on the global stage. It is to this respect that the events of 9/11 are
aesthetically and politically mobilized and manipulated in numerous cinematic
texts in order to staunch American patriotic values in the face of seemingly
undefeatable odds, implying, in the process, the point that securing American
national identity is an essential step towards redefining American identity on the
global scale. Indeed, the rhetoric discourse of the "war on terrorism" has provided fertile
grounds for Hollywood movie-makers and entertainers to reconfigure the role of
the US in world geo-politics. By moulding plots, settings and landscapes to tell
geopolitical tales, Hollywood geopolitical movies, in other words, subscribe to
American foreign policies, using "war on terrorism" as a starting point for
dramatizing American geopolitical anxieties and sensibilities in the Middle
East. 1. Introduction: Cinema, Politics, and Representation In so doing, Hollywood popular culture and cinematographic
representations of distant geographies and cultures have shifted, or have been
made to shift, from producing films preoccupied with Western cultural
supremacy to producing films framed by geopolitics and American foreign
policies. Themes of violence, melodrama, heroism and the role of American position in
world politics underpin most of post-9/11 cinematic texts. One can consider
here, for instance, movies like Fahrenheit 9/11 (2004), Rendition (Gavin Hood,
2007), Charlie Wilson's War (2007), In the Valley of Elah (2007), Redacted
(2008), The Hurt Locker (2008), and Zero Dark Thirty (2012). On their surface,
films of this period seem to reiterate the Orientalizing discourse that basically
justifies American intervention and involvement in the Middle East. Images of
American heroes who epitomize American values and who heroically face and
conquer the forces of "evil" in the "exotic" and "dangerous" lands are repeatedly
highlighted to display America as the "benevolent" hegemon. Deep down,
however, the production and codification of Hollywood cinematic texts after
9/11 are more than simply portraying Middle East through Orientalizing lenses 21 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 of danger and exoticism that denigrate and essentialize the lived reality of the
region. Indeed, the use of the locations such as Saudi Arabia, Iraq and
Afghanistan serves to emphasize the geopolitical importance of the "Other"
landscape for American quest for geo-power. Thus, the very specific geographic
contexts in which American heroism versus the Other's villainy happens tell
more about what America stands for than simply what the Other must
discursively represent. As Peter Van Ham states, "the events of 9/11not only
triggered renewed efforts to market ‘Brand USA’ and US policies, but also
generated a process of reflection on what ‘America’ actually stands for (or,
perhaps better, should stand for)"8. In fact, at the heart of Hollywood cinematic texts in contemporary time is the
branding of USA nation as a modern empire intent to dominate the world
politics. The construction of Middle Eastern spaces as "wild zones" in need of
military ethos and American violence can only be understood in the context of
how American policy-makers use the apparatus of Hollywood cinema to present
America as a unilateral force which alone takes the burden of bringing peace to
the world9. 1. Introduction: Cinema, Politics, and Representation Read in this context, the Hollywood codifications of American
adventures, surveillance technologies and disciplinary power structures inform
the way American empire and Hollywood industry harness their efforts to
redefine and remap American perceptions of global power. Indeed, the presence
of American military power policing and disciplining the Middle East has many
of the characteristics of imperial power. Using force and violence to impose
order and meaning upon the space, interfering into Middle Eastern states' affairs
coupled with America's entire war on terror in the region could all be
interpreted as an exercise in imperialism seeking to protect American imperial
geo-strategic and military interests in areas beyond its territorial boundaries. George Kieh concurs that the United States' military intervention in the Middle
East was not propelled by "the lofty ideals of democracy", but was indeed
prompted by "exigencies of imperialism". He puts it clearly that "the [US] 22 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 military intervention was ostensibly designed to maintain and expand the United
States' politico-security and economic stranglehold on the Middle East"10. military intervention was ostensibly designed to maintain and expand the United
States' politico-security and economic stranglehold on the Middle East"10. Accordingly, what intensifies much the drama of the American intervention in
the Middle East is the settings in which these dramas are played out. In
particular, given its extreme geo-circumstances, the Middle East landscape
offers dramatic possibilities for branding American nation-state and dramatizing
its geopolitical sensibilities. In other words, the territoriality of Middle East
serves as a projection zone for U.S power; a geographical space which enables
them to see themselves as a superpower while they displace their own
nationalism, geopolitics and violence upon the region. Therefore, rather than
reducing American/Middle East conflict to merely cultural and civilizational
factors, the delineation of Middle East is in fact geopolitically driven. Gearoid
Ó Tuathail notes that "to designate a conflict a civilizational one is to determine
its character in a definitive and totalizing manner. It is to impose a closure upon
events, situations, and peoples. The geographical specificity and place-based
particularity of conflicts are reduced to terms of a civilizational script"11. States politico-security and economic stranglehold on the Middle East
. Accordingly, what intensifies much the drama of the American intervention in
the Middle East is the settings in which these dramas are played out. 1. Introduction: Cinema, Politics, and Representation In
particular, given its extreme geo-circumstances, the Middle East landscape
offers dramatic possibilities for branding American nation-state and dramatizing
its geopolitical sensibilities. In other words, the territoriality of Middle East
serves as a projection zone for U.S power; a geographical space which enables
them to see themselves as a superpower while they displace their own
nationalism, geopolitics and violence upon the region. Therefore, rather than
reducing American/Middle East conflict to merely cultural and civilizational
factors, the delineation of Middle East is in fact geopolitically driven. Gearoid
Ó Tuathail notes that "to designate a conflict a civilizational one is to determine
its character in a definitive and totalizing manner. It is to impose a closure upon
events, situations, and peoples. The geographical specificity and place-based
particularity of conflicts are reduced to terms of a civilizational script"11. This paper is an extension of recent work in "critical geopolitics"12 on film as a
genre that is worth investigation. By drawing on literature from popular
geopolitics, geocriticism, visual politics and postmodernism, this study
interrogates the ways in which the movie, The Kingdom (2007), appropriates
and manipulates the discourse of "war on terror" within the broader framework
of American foreign-policy and international relations more particularly. The
geopolitical codes, the visual geopoliticized rhetoric of screened landscapes and
the discursive constructions of otherness, violence, heroism, melodrama and
trauma all provide illuminating examples of how these various interdependent
registers of mapping and reproducing the Middle East circulate and resonate
across the continuum formed by popular culture and world politics in the film
under study, The Kingdom. To do so, the research is divided into three sections. The first section begins by examining the complex interconnections between 23 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 cinema, politics and representation in order to scrutinize how "popular culture",
"world politics" and "representation" are complex and contested concepts, as the
relations between and among them are complex and dynamic. In so doing, the
research reads popular culture not only as an object for analysis, but also as a
form of political communication wherein global politics are encoded and
disseminated. The other two sections are devoted to the analysis of the
geopoliticized narratives related specifically to the ways in which The Kingdom
narrativizes a variety of anxieties associated with America's current position in
world affairs. 1. Introduction: Cinema, Politics, and Representation Moving beyond the artistic merits of landscapes and
characterization, the research examines how tropes that inform Orientalist
discourse are activated and accommodated in post-9/11 Hollywood cinema to
serve specific geopolitical agendas. Power, knowledge, surveillance and
disciplining are also examined to investigate the ways in which the film
naturalizes its violent approach to Middle East in a broadly inflicted geopolitical
narrative. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension Because “identities are constructed… in relation to what they are not”13,
producing and reproducing the "Other" which is anti-thesis of the "Self" has
been a prerequisite condition to the well-definition and formation of the "Self"
for ages. Most often, the relationship between the "Self" and the "Other" is
characterized by alterity: the "Other" is what the "Self" is not. Accordingly, the
"Other" becomes associated with negative traits, whereas the "Self" implicitly
holds positive attributes. This relational sense of identity, however, necessitates
demonization, dehumanization, and vilification of the "Other". Furthermore,
this dialectic relationship to the "Other" is enmeshed in a relationship of power
wherein the dominant "Self" constructs and defines the "Other" in ways that 24 © Arab Journal of International Law, 2022 https://doi.org/ 10.5281/zenodo.7424679 accord to specific cultural, political and ideologically-laden discourses and
interests. In his analysis of asymmetrical power relations between the "Self" and
the "Other", the East and the West, Edward Said posits that, "the relationship
between Occident and Orient is a relationship of power, domination, of varying
degrees of a complex hegemony."14 Since America, therefore, is a cultural, economic and military superpower, its
enduring constructions of the "Other" as "villainous", "threatening" and "violent
ideological zealots", while the American "Self" assumes the contrasting virtues,
becoming "civilized", "moral" and "humanitarian", promote sharp distinctions
between the Middle East and the United States. Because of such asymmetrical
power relationships, therefore, the dominant "Self" is able to theorize about and
define the "Other". For this sort of binary constructions and anxieties to be
culturally and politically validated and legitimized, they need to be
disseminated to the audiences in order to be widely accepted as true
demarcations between the "Self" and the "Other", between the Oriental
"villainy" and the American "heroism", thus justifying America's intervention
and involvement in the Middle East. The movie under study speaks volume to this sense of relationality,
ambivalence, power-knowledge asymmetrical relationships, and the desire to
project America as an international hegemon. Staring Jamie Foxx as Ronald
Fleury, Chris Cooper as Grant Sykes, Jennifer Garner as Janet Mayes, and Jason
Bateman as Adam Leavitt, The Kingdom, directed by Peter Berg, was released
on September 28, 2007 in the genre of drama, politics, and war. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The movie's
plot centers on a wave of terrorist attacks committed by Abu Hamza, a
"fundamentalist" Islamic "terrorist", and his followers against "innocent"
American civilians during a softball game at an American oil company housing
compound in Ryyad, Saudi Arabia. In response, four American FBI special
agents are dispatched to the scene to investigate the terrorist attack and bombing
of an American facility by "evil" ideologues. Upon landing on the desert of the 25 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 kingdom, the FBI team is met by Colonel Faris al-Ghazi, the commander of the
Saudi State Police Force, who advises them of how to act in a hostile
environment. Later, they are invited to the palace of Saudi Prince Ahmed bin
Khaled for dinner (Mayes is excluded because of her gender, which implies the
"backwardness" and "rigidity" ascribed to the East) in order to imply a kind of
collaboration between the two states against terrorism. From now on it will be a
showdown between the FBI heroes and the villains; the more powerful the
villains are, the greater are the heroes. Lead by Ronald Fleury and assisted by
Faris al-Ghazi, the FBI team brings down the insurmountably "evil" forces that
America must face in order to bring about peace and security to a land that is
presented as barbarian and hostile to the progressive ethos and values of the
American nation-state. Displaying the confrontation with the "evil" in this way,
the moral message of The Kingdom conferred to the audience is that anything,
and everything that can be done to murder the "villains", must be done, not only
to exterminate the threatening "Other", but more importantly to reconfigure the
role of American as "the legitimate gendarme of the world."15 Indeed, the release of The Kingdom can be interpreted as indicative of
American geopolitical anxieties regarding its military position in the world and
the search for a new threat of terrorist attack to justify this anxiety. In other
words, the need to create a new Enemy after the fall of the Soviet empire has
been a prerequisite need for American policy makers and Hollywood film
entertainers in order to rationalize the US exterritorial military intervention in
the Middle East. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The explicit terrorist attacks, military invasions and emerging
enemies in this film, therefore, demonstrate how far are geopolitical interests
and anxieties are manifest in the consciousness of American policy makers,
film producers and their audience in the post-9/11 world. To attain its
geopolitical goals, as a consequence, the movie's characterization of the "Other"
developed into worst manifestations: the constructions of the Arabs/Muslims as
the Enemy. This intensified construction which demonstrates the transition from 26 © Arab Journal of International Law, 2022 https://doi.org/ 10.5281/zenodo.7424679 the "Other" to the Enemy has been, however, a condition for the well-projection
of the American "Self", for it has become "more and more difficult to imagine
who we are without reference to our enemy […] Without him who would we
blame for the slings and arrows, the failures, the wounds, the inchoate anger, the
gnawing frustration, the injustice."16 The Kingdom begins on battlefield, in a lull in combat operations between the
forces of "good" and the "evil". Prior to this, the camera shows American
families playing baseball and enjoying barbecue, but constantly cuts to zoom in
Abu Hamza from a faraway distance on a top building controlling the terrorist
operation that his followers are tasked to destroy lives of dozens of American
residents in a nameless city in Saudi Arabia. The bombing operation is
successfully implemented; dead corpses of American civilians fill the screen,
and then the camera cuts to Abu Hamza murmuring words in Arabic thanking
God for the success of his "evil" plans. To further aggravate the feelings of the
audience, the camera depicts one of Abu Hamza's followers citing the testimony
then blows himself up and tragically kills the rest of survived people around
him while pretending to save them. The figure of Abu Hamza then is presented
as representative of everything that America stands against. He is the leader of
the terrorist group and the representative of God to his followers. He is shot
three times indoctrinating "fundamentalist" thinking into the minds of a group
of little kids. The scene implies how the terrorist network in the Middle East is
in a constant conspiracy to destroy the "ideals" of American culture. Little kids
are portrayed in the Muslim costume devoting their attention wholeheartedly to
the Abou Hamza’s "extremist" discourse, and express their willing to bomb
Americans as a response to the "call" of their faith. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The shooting of the Saudi
Abou Hamza and his faithful group in this way invokes Berg’s political ends to
personalize Abou Hamza and his ‘extremist’ preaches to Bin Laden, implying
that just like Abou Hamza Bin Laden’s "extremism" had started in the same
way before "his" attacks of September 11 took place. The prolonged scenes of The Kingdom begins on battlefield, in a lull in combat operations between the
forces of "good" and the "evil". Prior to this, the camera shows American
families playing baseball and enjoying barbecue, but constantly cuts to zoom in
Abu Hamza from a faraway distance on a top building controlling the terrorist
operation that his followers are tasked to destroy lives of dozens of American
residents in a nameless city in Saudi Arabia. The bombing operation is
successfully implemented; dead corpses of American civilians fill the screen,
and then the camera cuts to Abu Hamza murmuring words in Arabic thanking
God for the success of his "evil" plans. To further aggravate the feelings of the
audience, the camera depicts one of Abu Hamza's followers citing the testimony
then blows himself up and tragically kills the rest of survived people around
him while pretending to save them. The figure of Abu Hamza then is presented
as representative of everything that America stands against. He is the leader of
the terrorist group and the representative of God to his followers. He is shot
three times indoctrinating "fundamentalist" thinking into the minds of a group
of little kids. The scene implies how the terrorist network in the Middle East is
in a constant conspiracy to destroy the "ideals" of American culture. Little kids
are portrayed in the Muslim costume devoting their attention wholeheartedly to
the Abou Hamza’s "extremist" discourse, and express their willing to bomb
Americans as a response to the "call" of their faith. The shooting of the Saudi
Abou Hamza and his faithful group in this way invokes Berg’s political ends to
personalize Abou Hamza and his ‘extremist’ preaches to Bin Laden, implying
that just like Abou Hamza Bin Laden’s "extremism" had started in the same
way before "his" attacks of September 11 took place. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The prolonged scenes of 27 © Arab Journal of International Law, 2022 https://doi.org/ 10.5281/zenodo.7424679 the "Other" preparing bombs, intercut with significant scenes depicting
mosques, followed by extreme attacks, are not only demeaning depictions of the
"Other", but can also be defined as a threat to America’s position as a global
power. Furthermore, the enemies encountered in this film are but faces viewed through
the close-ups of the camera. They are dehumanized to the point that they are not
recognized as people with their own full agency and motivation. They act only
in response to fanatical dictations communicated to them by their leader, Abu
Hamza. Their crudeness is also manifested in their use of weaponry as the
brutality of its effects does not discriminate between its targets, implying that
the enemy are backwards, technologically and morally, as their bombs against
American civilians are incapable of discerning between friend and foe, innocent
and guilty, therefore constructing the "enemy" as entirely uncompromising in
the pursuit of their sinister goals. This suggests, however, that although the
villains are incompetent and backward, they still appear as powerful and
dangerous. This is consistent with the paradoxical view of the Orient, which is
inferior to the West, yet "it has always been endowed with greater potential to
power (usually destructive) than the West"17. This is not to say, however, as
Edward Said highlights, that the Orient possesses more power than the West but
it is because as it is "backward" and "precipitous", the little power it produces
becomes threatening and dangerous. The discursive construction of the "Other" as enemy in this film and others after
the appalling events of 9/11, threatens, in fact, the geopolitical and geo-
economic stability of the United States in the Middle East and in the
international world by extension. Nonetheless, the conflict with the "evil"
"Other" is necessary in order to bring about codes of morality, heroism,
humanity and nationalism that the film confers to the four American FBI agents
who are tasked to redefine American domestic and foreign policies in overseas. As previously noted, post-9/11 Hollywood movies tell more about what defines 28 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 American identity than simply how the "Other" must discursively and
cinematically be introduced. Nonetheless, the "violent" "Other" is inevitably
demanded for this relational meaning to take place. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The film introduces FBI
agents possessing all the heroic, humanitarian and distinguishing traits that
separate them from the forces of "evil". Throughout the most of the film, the
camera follows the movements of Ronald Fleury and his team sacrificing their
lives to bring peace and stability to Saudi Arabia, and the Middle East by large. The latter, as helpless as it is, could not save itself from the power destruction of
its terrorists, which is symptomatic of Orientalist discourse that the Middle East
cannot save itself, therefore it needs to be rescued. The heroic American
characters are then called upon to save the day. It is, therefore, this construction
of Manichean antagonism between "extremist" enemy and American "heroes"
that discursively structures the narrative and establishes the grounds for the
projection of American nationalist sensibilities and geopolitical anxieties. By constructing the protection of freedom and democratic values as definitely
an American duty, the depiction of FBI heroes in The Kingdom as "freedom
fighters" draws clear parallels with American military action around the globe. Endowed with all "positive", "humanitarian" and "heroic morals", the FBI
agents are portrayed struggling heroically in exotic landscapes "specified as in
need of pacification and administration due to the absence of such social
attributes in such places not blessed with the benefits of civilization"18. In the
process, FBI agents, displayed as bearers of civilization and order, live out the
codes of virtuous warriors who stand in for American honour, values and
power. Throughout the film, the camera follows their movements keeping them
in center frame as they fight against the forces of evil in order to establish peace
in extreme and exotic circumstances. In so doing, they physically reassert
American identity, restore its dignity and secure it as the repository virtue
against barbaric threats to the values it stands for. Responding with alacrity,
therefore, FBI agents determine to exterminate scores of terrorists and 29 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 heroically kill Abu Hamza and his followers. United and victorious, Fleury,
Mayes, Sykes and Leavitt celebrate their victory and return to America. The
victory of American agents and the defeat of the "Other" is essential to the
structure of the narrative. Seen from a relational perspective, the defeat of the
enemy resolves tensions and divisions within the "Self". The "Self" achieves its
beingness and becomes whole again only upon eliminating the "Other". 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension Pierre
Biskind posits that in order to understand the ideology upon which films derive
their significance, "it is essential to ask who lives happily even after one dies …
and why?"19. The victorious ending of The Kingdom is explicitly associated
with the eradication of Arab characters. To this context, the prevalent concept of
heroism and villainy as encapsulated by Porteaus's statement: "one murder
made a villain, millions made a hero"20 speaks volume to the workings of the
war narrative in this film. Indeed, the cinemagraphic portrayals of FBI warriors confronting perils in
strange lands for "freedom" and "security" provide the central moral message
for the "right" conduct through which their violence can morally be justifiable. In contradistinction to the discursive dehumanization of the "Other" enemy as
immoral, violent, and having no sense of restraint, the American heroes retain
positive and humanitarian traits; although willing to sacrifice their own life to
fight villainy, they certainly will never deliberately sacrifice the life of the
innocent. In conferring this sense of moral superiority to FBI agents, the film
shows their struggle as "noble" and "humane". In this regard, the moral message
of The Kingdom conferred to the audience is that American violence ought to
be understood nonetheless as noble, whereas the violence depicted against the
"enemy" is sanctioned and justified. The Kingdom doesn't show Arabs as
victims, actually it undermines this idea. The Kingdom follows an established
tradition in Hollywood films whereon Arabs are presented "only as perpetrators
of terror, never as victims."21 30 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 Moreover, the normative workings of melodrama depicted in The Kingdom
coupled with sensibilities of American nationalism they serve equally underpin
the narrative, shape the American audience's consciousness and reconfirm a
sense of national belonging. In fact, The Kingdom relies on a number of
strategies to mobilize a particular conception of identity, one that triggers
feelings of American national harmony and togetherness. First, the plot
narrative creates an emotional connection between audience and nation: a sense
of safety and contentment with the nation was then juxtaposed with feeling of
fear from the "Other". In the process, narratives of national cohesion and
togetherness are visualized through the victimization of American civilians in
order to mobilize national consciousness in the minds of American audiences. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension Simultaneously, the campaign theme developed in the narrative serves the aim
of reaching unity and harmony through diversity, reflecting and emphasizing
therefore the unified perceptions of American identity and nationhood. The
diverse representations of FBI characters in terms of race and gender, all
sacrificing their lives for the sake of their nation branding, engender a patriotic
response to who "we are" and reinforces the narrative of national togetherness. This is indeed reminiscent to George W. Bush's speech to the nation after 9/11,
in which he stated that: “these acts of mass murder were intended to frighten our
nation into chaos and retreat. But they have failed. Our country is strong [...] A
great people has been moved to defend a great nation”22. Following this line of
narrative, the film introduces American FBI characters who exhibit staunch
patriotic values in their long confrontation with the villains in faraway places. Moreover, they are all driven by a belief in what America represents, and are
ready to do whatever necessary to protect their nation. In the process, they show
resolve, strength and loyalty towards American national values they stand for. Their ability to achieve victory through adversity, to struggle with a fighting
spirit to the end against the violent and ideological zeal of their enemies is Moreover, the normative workings of melodrama depicted in The Kingdom
coupled with sensibilities of American nationalism they serve equally underpin
the narrative, shape the American audience's consciousness and reconfirm a
sense of national belonging. In fact, The Kingdom relies on a number of
strategies to mobilize a particular conception of identity, one that triggers
feelings of American national harmony and togetherness. First, the plot
narrative creates an emotional connection between audience and nation: a sense
of safety and contentment with the nation was then juxtaposed with feeling of
fear from the "Other". In the process, narratives of national cohesion and
togetherness are visualized through the victimization of American civilians in
order to mobilize national consciousness in the minds of American audiences. Simultaneously, the campaign theme developed in the narrative serves the aim
of reaching unity and harmony through diversity, reflecting and emphasizing
therefore the unified perceptions of American identity and nationhood. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The American body, in other terms, is thoroughly
privileged throughout the film. The suffering of FBI agents is visually
emphasized to reassert the mythology of the American body as "benevolent",
"vulnerable" and "victimized". In other words, melodrama, as a representational
device, is deployed repetitively in this film for the sake of augmenting
emotional intensification and sensational identification. Thus, to further
maintain a sense of the melodramatic, The Kingdom, as an action-thriller film,
makes use of fundamental conventions of melodrama: moral polarization of
"good" versus "evil", "noble" heroic characters, heightened emotion, and
sensationalism (emphasis on action and violence), are all effectively deployed
to advance the over-dramatic plot-line of the narrative, which is designed to
particularly play on the spectators' emotions. Accordingly, by exploiting the Furthermore, the mobilization of trauma discourse, victimhood and redemption
are explicitly advanced in the narrative of The Kingdom for the purposes of
prioritizing, excusing, and ultimately redeeming the American body. Painful
and horrific scenes of brutal actions of terrorists are intensified by the affective
strategies the film employs to arouse feelings of patriotism and national unity. The horrific scenes of American dead corpses, the tears of Janet Mayes, and the
crashing of the planes into the towers play an important role in triggering
emotions of fear, anxiety, and insecurity, which generate a sense of trauma and
victimization in the eyes of the audience. In addition, the graphic torture of
Adam Leavitt, while fighting against "terrorists", is melodramatically displayed
to emphasize the suffering inflicted upon the American male body in order to
generate a sense of sympathy for his victimized body. Linda Williams posits
that “the basic vernacular of American moving pictures consists of a story that
generates sympathy for a hero who is also a victim and that leads to a climax
that permits the audience, and usually other characters, to recognize the
character’s moral value”24. The American body, in other terms, is thoroughly
privileged throughout the film. The suffering of FBI agents is visually
emphasized to reassert the mythology of the American body as "benevolent",
"vulnerable" and "victimized". In other words, melodrama, as a representational
device, is deployed repetitively in this film for the sake of augmenting
emotional intensification and sensational identification. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension The
diverse representations of FBI characters in terms of race and gender, all
sacrificing their lives for the sake of their nation branding, engender a patriotic
response to who "we are" and reinforces the narrative of national togetherness. This is indeed reminiscent to George W. Bush's speech to the nation after 9/11,
in which he stated that: “these acts of mass murder were intended to frighten our
nation into chaos and retreat. But they have failed. Our country is strong [...] A
great people has been moved to defend a great nation”22. Following this line of
narrative, the film introduces American FBI characters who exhibit staunch
patriotic values in their long confrontation with the villains in faraway places. Moreover, they are all driven by a belief in what America represents, and are
ready to do whatever necessary to protect their nation. In the process, they show
resolve, strength and loyalty towards American national values they stand for. Their ability to achieve victory through adversity, to struggle with a fighting
spirit to the end against the violent and ideological zeal of their enemies is 31 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 playful in the construction of a national identity that evokes a sense of pride and
admiration for Americans.23 Furthermore, the mobilization of trauma discourse, victimhood and redemption
are explicitly advanced in the narrative of The Kingdom for the purposes of
prioritizing, excusing, and ultimately redeeming the American body. Painful
and horrific scenes of brutal actions of terrorists are intensified by the affective
strategies the film employs to arouse feelings of patriotism and national unity. The horrific scenes of American dead corpses, the tears of Janet Mayes, and the
crashing of the planes into the towers play an important role in triggering
emotions of fear, anxiety, and insecurity, which generate a sense of trauma and
victimization in the eyes of the audience. In addition, the graphic torture of
Adam Leavitt, while fighting against "terrorists", is melodramatically displayed
to emphasize the suffering inflicted upon the American male body in order to
generate a sense of sympathy for his victimized body. Linda Williams posits
that “the basic vernacular of American moving pictures consists of a story that
generates sympathy for a hero who is also a victim and that leads to a climax
that permits the audience, and usually other characters, to recognize the
character’s moral value”24. 2. American Popular Orientalism and the Reproduction of
"Self"/"Other" Identities: A Geopolitical Dimension Thus, to further
maintain a sense of the melodramatic, The Kingdom, as an action-thriller film,
makes use of fundamental conventions of melodrama: moral polarization of
"good" versus "evil", "noble" heroic characters, heightened emotion, and
sensationalism (emphasis on action and violence), are all effectively deployed
to advance the over-dramatic plot-line of the narrative, which is designed to
particularly play on the spectators' emotions. Accordingly, by exploiting the 32 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 trauma discourse, the film makes a clear-cut difference and distinction between
the American body and the Arab/Muslim body. While the pain of the American
body is prioritized and exceptionalized, the body of the "Other" is evacuated,
dehumanized and denigrated. In a nutshell, the popular rhetoric that
manipulated the events of 9/11, upon which the movie draws its plot, is in fact
mobilized as a strategy to legitimize the presence of America in the Middle
East. In so doing, the narrative assumes a geopolitical form to maintain a sense
of American exceptionalism. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East "Geopolitics produced international politics as theater: geography was the stage,
politics the drama, and geopolitics the detached observation of this
representational spectacle."25 The opening sequences of The Kingdom with a gripping montage that briefly
documents the history of the kingdom of Saudi Arabia since its foundation in
1932 are playful in the construction of a geopolitically narrative that underpins
the plot structure of the movie. Indeed, the opening scenes together with the
geopolitical, geo-economic, cultural and historical information they convey are
carefully chosen and displayed to guide and structure the understanding of the
audience towards an acceptance of the difficult circumstances faced by
American FBI government agents in their war against the "Other" enemy,
thereby justifying America's territorial invasion of the Middle East. As we
watch these introductory scenes, a voice-over narrates that Wahabis (Saudian
Islamic warriors) are fiercely anti-Western presence in the kingdom and wanted
to go back in time to a pure Islam that was not threatened by the West. Saudi
Arabia at that time was the number one country that produced oil in the world,
whereas America was the number one oil consumer in the world. The voice-
over continues to tell that America claimed that the Kingdom of Saudi Arabia 33 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 needed a security from USA. With the regulation of commercialization, the king
of Saudi Arabia accepted it. As a consequence, a partnership was produced
between Saudi Arabia and America in 1938, which was named as Arabian
American Oil Company (ARAMCO). The voiceover goes on to inform the
audience that in 1945 President Roosvelt and Ibnu Saud had a meeting which
resulted in signing a unity agreement between East and West, but the Wahhabi
movement rejected this unity on the grounds that America supported Israel in
the Arab war versus Israel in 1970. Also worth attention in these introductory
scenes is the display of the figures of Saddam Hussein and Osama bin Laden. The voice-over recounts that during the Gulf war, bin Laden offered help to the
kingdom of Saudi Arabia to destroy Iraq with his Afghan Mujahdidin (warriors)
and defeat Saddam Hussein in Kuwait, but Saudi Arabia got a good offer from
the United States army. Because of that rejection, Osama bin Laden did not like
the cooperation between the Kingdom of Saudi Arabia and the USA. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East Then, the
movie cuts to a plane crushing into a big building to imply that bin Laden's
reaction against America was the motif behind the tragedy of 9 September
2011. This short, but very geopoliticized introductory documentation, is, however,
implicitly overloaded with ideologically imbued assemblages of information,
geopolitical implications and imperial intents that the film is dedicated to
propagandizing the increasingly enfranchised masses into believing American
imperial expansionism has been in everyone’s interest in Saudi Arabia and the
Middle East region as a whole. The emphasis on the Middle East landscape in
these documentary sequences, therefore, merits further investigation as it is the
battlefield stage upon which world geopolitics are dramatically played out. The
visual rhetoric of screened landscape displayed in the movie informs the
motivations of the characters, creates the mood, and frames the film's narrative
around a valorization of the U.S military involvement in Middle East. In other
words, the landscape–in its screened depictions - moulds the place to the needs 34 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 of namely geopolitical production. Through specific unnerving camera angles,
low lighting, anguish and despair atmosphere, which produce the affect of
extreme fear in an enclose space similar to the regular view of Cold War
cinema, the opening of the film presents the Middle East as hostile to human
beings; it is a dark and foreboding space, one that borrows from the Orientalist
stock images and tropes prevalent in the literary sources outlined by Edward
Said in his Orientalism. Then, the background screen turns black, and the
featured landscape is a deserted, horrific place. The implied purpose is to
present a geopoliticized landscape of extreme otherness, thus legitimizing
American military interventionism in the region. Filled with intense violence, The Kingdom produces an imaginative geography
that seeks to orientate audiences, as well as emotional attachments and enmities
that shape what is considered to be politically possible and desirable. Moreover,
interruptions of the voice-over with landscape depictions isolate and restrict the
gaze of the viewer before they are transported to the battlefield, which is a vast,
desolate, and harsh world. Indeed, the film's construction of the locale for the
drama is instrumental to its geopolitically influenced narrative. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East It is through
such visual politics of essentializing the "Other" landscape that the movie can
provide the moral vocabulary into which narratives of "right" conduct and the
American imperial violence could be morally justifiable. In its discursive mapping of the Middle East, the film relies on a number of
established coherent strategies to draw up, spatialize and dominate the Middle
East geography. That the Middle East is configured as an ideologically rigid and
unchanging place in The Kingdom is not, however, perpetuated as being only
culturally different, but its geostrategic geography is what counts in the
American imperial perpetual struggle over space in global politics. However,
still the visualized fetishistic constructions of the Middle East as culturally
different and threatening is essential to the film's plot narrative as it seeks to
naturalize and legitimize American territorial invasion of the Middle East 35 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 region. This brings to mind Samuel Huntington's thesis of "cultural civilization"
in which he reduces East-West conflict to merely culture and civilization26. In
other words, Huntington's "clash of civilization" rhetoric propagates the idea
that the conflict between Western world and the non-Western world are over
clashes that are ideological, cultural and religious rather than political and
economic, which is indeed the same rhetoric used by the American
administration to legitimize its geopolitical efforts to remap the Middle East. Huntington's civilizational discourse, however, is remarkably simplistic and
comprehensively flawed as it deliberately overlooks the geographical and
geopolitical specificities of conflicts which overwhelmingly structure most of
the knowledge produced about the Middle East. Richard Rubenstein and Jarle
Crocker, for instance, note that “Huntington has replaced the nation-state, the
primary playing piece in the old game of realist politics, with a larger counter:
the civilization. But in crucial respects, the game itself goes on as always”27. Corroborating a similar view, Tuathail posits that "Huntington’s thesis is not
about the clash of civilizations. It is about making global politics a clash of
civilizations."28 Informed by geopolitical and geo-economic orientations, The Kingdom, as a
geopolitically-inclined text, displays geo-graphing politics and geo-power
anxieties as central to its dramatic narrative. That is, the imaginative geography
propagated in this film together with the desire to produce international politics
geographically is specifically driven by political and economic interests. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East The
"imaginative geography"29 elaborated by Edward Said in his Orientalism
illuminates the manner in which such Western Orientalist imagination shapes
the geopolitical and geo-economic discursive mapping of the Middle East. With
American Orientalism, more particularly, the imaginative geography of the
Middle East landscape is primarily motivated by economic and corporate
interests centered mainly on access to oil supply and the domination over the
region. Indeed, the Middle East is perceived as "a subsystem of acute strategic 36 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 importance to the rest of the world. For local and external powers there are
many immutable interests over which to compete and such competition is a
major contributory factor to the reputation of the Middle East as the world most
volatile and violent region"30. That the opening scenes of the film bring to view
the fact that Saudi Arabia has been one of the most rich-oil states in the world
and that America was the first oil consumer state in the world is one main
imperial motif structuring America's geo-economic struggle over the Middle
East. The display of the signed agreement between the two states in this film
was meant not only to secure American economic interests in the region, but
also to maintain the primacy of the United States over the region, thereby
countering the emergence of other external political-economic powers seeking
to project their presence in the region, namely China as an emergent threat to
U.S primacy in world affairs. Another constitutive and fundamental element lying bare America's geopolitical
sensibilities and anxieties in The Kingdom might be the dramatization of the
United States' hegemonic and unilateral sense of power, which assigns itself the
role of disciplining the "Other" landscape, and subjugating it to the regulatory
power structures of its unilateral military action, knowledge and surveillance
technology. John Ikenberry observes that "spurred by its war on terrorism, the
Bush Administration has advanced new, provocative ideas about the American
unilateral and preemptive use of force –and under this go-it-alone if necessary
banner"31. He adds that "unilateralism seeks to strengthen American power and
unashamedly deploy it on behalf of self-defined global ends"32. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East To this vein, the
mention of the Wahhabi Islamic warriors, Iraq, Afghanistan and Israel intercut
with images of Saddam Hussein and bin Laden exhibit American unilateral
military actions in the Middle East along with specific geopolitical anxieties
that the film narrativizes through practices of power, surveillance and
disciplining. That is, by narrating how these figures and countries are anti-
American democratic values, the movie legitimizes the violent and disciplinary 37 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 structures used to root out anti-voices of American presence and its interests in
the region. The audience therefore is encouraged to naturalize American
military invasion of Iraq, which ultimately resulted in the downthrown of
Saddam's regime and his eventual death four years after the release of The
Kingdom. Saddam and his political regime had to be removed from the Middle
East region not because of Iraq's involvement in the Gulf war, nor for the
nuclear weapons his country was supposed to possess as the voiceover
emphasizes in the movie, but for his threat to the interests of America and the
stability of Israel in the region. Simultaneously, the depiction of bin Laden in
the film, the "mastermind" behind the September 11, and who was dramatically
shot by American army four years later before the release of The Kingdom, is
brought to the fore to implicitly reaffirm America's role as a key geopolitical
player in the Middle East. Given that bin Laden and Saddam's voices are anti-
American geopolitical interests, they therefore must be exterminated in order to
establish peace and security in the world. Moreover, power, surveillance and punishment serve specific geopolitical
agendas in The Kingdom. While on the surface they are deployed in the context
of "war on terror", their disciplining effect, however, works to reterritorialize
the identity of the United States, rewrite the meaning of geo-power, and secure
U.S geopolitics on the global scene, thereby bringing to the fore the
complexities of power, knowledge, geography, and geopolitics. In exploring the
intricacy of these concepts, Tuathail posits that "geography is about power. Although often assumed to be innocent, the geography of the world is not a
product of nature but a product of histories of struggle between competing
authorities over the power to organize, occupy, and administer space"33. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East Thus,
the very identity of the subject position, be they "geographer", "cartographer",
or "geopolitician", coupled with the specific techniques by which geographical
and geopolitical objects are projected and presented are all effects of power-
knowledge relations. Michael Foucault postulates that power and knowledge 38 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 directly imply one another: “there is no power relation without the correlative
constitution of a field of knowledge, nor any knowledge that does not
presuppose and constitute at the same time power relations”34. His insights on
power, knowledge and surveillance are indeed instrumental for decoding the
power structures involved in the cinematization of space. For him, power,
knowledge and surveillance are three entities which support an integrated
system, for he argues that the success of disciplinary power, thus the
objectification of the individual, is carried out through three inter-related
instruments: "hierarchal observation, normalizing judgment and their
combination in a procedure that is specific to it, the examination"35. Indeed, as
Foucault highlights, discipline "presupposes a mechanism that coerces by means
of observation; an apparatus in which the techniques that make it possible to see
induce effect of power on whom they are applied clearly visible"36. In other
words, "hierarchal surveillance"37, or the technique of making subjects visible or
observable, allows extension of knowledge to sustain its hegemonic mechanism. Following this argument, observing and surveilling can be equated with
knowing, thus being dominant, and in the context of the film under study the
FBI characters are introduced as possessing the power/knowledge mechanisms,
something which allows them to surveil and control the "Other". Indeed, the issue of surveillance, power and cinema has been important to
media scholars. Drawing on Sebastian Lefait's study on the mutual implication
of cinema and surveillance, Xiaoning Lu writes that in his study of
contemporary film and television programs "[Lefait] suggests that cinema
engages surveillance structurally through its fictional creation of surveillance
microcosms. In the meantime, by being a reality-capturing device, the cinematic
apparatus "translates the problem of the ambiguity of the visible into terms of
mediated watching", which is also a matter at the heart of surveillance"38. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East In a
similar vein, Catherine Zimmer concurs that ""surveillance cinema" is not
simply the recurring tropes or iconographies of surveillance […] Rather […] 39 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 "the multiple mediations that occur through the cinematic narration of
surveillance, through which practices of surveillance become representational
and representational practices become surveillant"39. The Kingdom's violent,
disciplinary and surveillance narrative embodies and displays all such practices
of power and surveillance exerted upon the Middle East geography. In its
screened geopoliticized landscape, the film involves, exhibits, and relies on
specific disciplinary and surveillance techniques to counter the Other's severe
"threat" to American's national security and geopolitical interests in the region. The opening sequences of the film with aerial top-down views, the evocative
display of geopolitically charged panoramas of the landscape, the use of satellite
technologies, and the reliance on the FBI's expertise and knowledge to track the
Other enemy, all promoted by surveillance technologies, indicate how the
movie is structured around power and hierarchized surveillance. As a
consequence, through the literal and figurative lens of surveillance, the "Other"
becomes objectified, surveilled, recorded and controlled. It is equally through
these mechanisms of surveillance that a "scientific", "neutral", "objective", and
specialized language (knowledge/truth) about the "Other" is produced and
sustained. The film depicts the FBI agents as intelligence experts using satellite
reconnaissance technology to navigate and investigate the Middle East. In fact,
the camera centers much on the battle against the "Other" enemy in order to
emphasize the expertise of the FBI investigators mapping out the movements of
terrorists, which ultimately leads to the murder of Abou Hamza. Hence, by
engaging surveillance mechanisms in its cinematic narrative, the film attributes
the tropes of scientific authority, neutrality to FBI agents, thereby justifying
their military intervention in the Middle East. The consistency with which this
expertise and power are imagined point to the rationalization and disciplining
qualities of American imperialism regarding its intervention and involvement in
the Middle East. In other words, surveillance, as a mode of power, has been
i l
h
U i d S
'
li i
l di
d i
ili
i 40 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 the Middle East. The landscape of Saudi Arabia depicted in this film as a
geostrategic location is in fact an extension of American military base on the
real grounds. 3. Geography, Geopolitics and Power Surveillance: Staging the Middle
East The actual presence of American military camp in the region
unfolds the American real practices of surveillance, disciplining and punishing
the Middle East. In a nutshell, surveilling, disciplining and punishing the
Middle East either in fiction or in reality serve the very geopolitical sensibilities
of the United Stated in the region embodied in its global hegemony and imperial
expansionism. The link between the aforementioned modalities of power and
their effect on the imaginative geography can also be clearly found in the links
postulated by Edward Said between imperialism and geography, for he states
that imperialism is after all “an act of geographical violence through which
virtually every space in the world is explored, charted, and finally brought under
control”40. 4. Conclusion Attempting to interrogate the intersections of geopolitics, power, global
hegemony and politics of signification manifest in post-9/11 Hollywood
cinema, the conclusions reached herein have revealed the ways in which The
Kingdom, as a geopolitical film, has mediated, reproduced and geopoliticized
the Middle East landscape and culture along with the ethos of American
militarism, geo-policy-making and economic pursuits in the region. While the
research demonstrates how the movie has borrowed from Orientalist stock
images and strategies that have long been used to signify the Middle East, it
emphasized that 9/11 has manipulated and altered these strategies in substantive
ways. In the process, it has demonstrated that the construction and the
designation of the spaces of "Middle East" as "wild zones" in need of American
intervention was mobilized as an argument for American geopolitical interests
and interventionism in the region. Likewise, the analysis has illustrated the 41 https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 ways in which the film deployed the rhetoric of "war on terrorism" as a
narrative strategy to display American geopolitical anxieties, America's fear of
losing its way in the world, and its geopolitical efforts to recuperate American
perception of geo-power. It has maintained that the discursive constructions of
the "Other" as enemy and the delineation of the "Other" landscape as dangerous
and volatile in post-9/11 Hollywood cinematic texts are enframed within
geopolitics, petro-politics and the desire to project America as a world hegemon
intending to dominate international politics. More precisely, the construction of
the "Other" as enemy threatening American political and economic interests in
the region provided the background against which new forms of geo-power and
geopolitical narratives are reinstated and situated. In other words, with the
demise of the Cold War and the subsequent collapse of the Soviet Union as the
"evil empire", the search for a new threat of terrorist attack, a new geo-political
justification for American hegemonic identity and world position, was a
prerequisite condition for the reproduction of American hegemony on the global
stage. In addition, the study has examined the manner in which such
geopolitical sensibilities are mobilized through narratives of melodrama,
trauma, victimization, and the burden that American "benevolent" characters
must bear to bring peace to the world, therefore emphasizing American
exceptionalism and supporting American global hegemony as legitimate. 4. Conclusion It has
interrogated how the film's accommodation and manipulation of history through
a brief historical documentary of archival footage of the Middle East is
deployed and filtered through the events of 9/11 in order to rewrite and rescue
the long and contested history of American foreign policy, economic investment
and involvement in the Middle East. In so doing, it shows how the movie's
plotline narrative authorizes and validates American knowledge and expertise
about the Middle East, which, in other words, might explain American policies
of surveillance, discipline and punishment. Ultimately, what is at stake here is a 42 © Arab Journal of International Law, 2022
https://doi.org/ 10.5281/zenodo.7424679
geopolitical struggle over the space and the desire to reconfigure the role of
America as a world hegemony. https://doi.org/ 10.5281/zenodo.7424679 © Arab Journal of International Law, 2022 geopolitical struggle over the space and the desire to reconfigure the role of
America as a world hegemony. geopolitical struggle over the space and the desire to reconfigure the role of
America as a world hegemony. [12] According to Gearoid Ó Tuathail, "Critical geopolitics is distinguished by its
problematization of the logocentric infrastructures that make “geopolitics” or any spatialization
of the global political scene possible. It problematizes the “is” of “geography” and “geopolitics,”
their status as self-evident, natural, foundational, and eminently knowable realities … Rather Endnotes oted in Andrew Crampton and Marcus Power (2007). Cinema and Popular Geo-Politics. : Routledge. p.4. [1] Quoted in Andrew Crampton and Marcus Power (2007). Cinema and Popular Geo-Politics. London: Routledge. p.4. [2] For further discussion, see Floribert Patrick C Endong. (2018). "Cinema Globalization and
Nation Branding: An Exploration of the Impact of the Nollywood of the Nigerian Image Crisis". Journal of Globalization Studies, Volume 9, Issue 1. [3] Quoted in Klaus Dodds (2008). "Hollywood the Popular Geopolitics of the War on Terror",
Third World Quarterly, Vol.29: 8. 1621-1637. [4] Quoted in Jiong, Zhang (2017). Literature and Literary Theory in Contemporary China. Trans. Yang Limeng and Wu Yischeng. London and New York: Routledge, p.109. [5] Jutta Weldes and Christina Rowely (2015). "So, How Does Popular Culture Relates to World
Politics?" In Federica Caso And Caitlin Hamilton (Eds.). Popular Culture and World Politics: Theories,
Methods, Pedagogies. Bristol: E-International Relations Publishing, p.19. [6] Andrew Crampton and Marcus Power, Cinema and Popular Geo-Politics, p.1. [6] Andrew Crampton and Marcus Power, Cinema and Popular Geo-Politics, p.1. [7] Ibid [7] Ibid. [20] Ibid. [21] Jack Shaheen (1990). "Screen Images of Palestinians in 1980s". In Paul Loukides and Linda
K. Fuller (Eds.), Beyond the Stars: Stock characters in American popular film, Volume 1. Bowling Green
Ohio: Bowling Green State University Popular Press, p.57. [22] De Richard Jackson (2005). Writing the War on Terrorism: Language, Politics and Counter-terrorism. Manchester and New York: Manchester University Press, p.191. [23] Jake Pembroke (2015). "Constructing American Identity and the Terrorist ‘Other':
Representations of Foreign Policy and Identity in post-2007 Hollywood Cinema". Dissertation. The New Birmingham Review.Vol. 1. No. 1. pp.177-225. [24] Nestingen, Andrew. (2008). Cinema and Fantasy in Scandinavia: Fiction, Film, and Social Change. Copenhagen: Museum Tusculanum Press, p.115. [25] Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p.178. [7] Ibid. [8] Peter Van Ham (2008). "War, Lies, and Videotape: Public Diplomacy and USA’s War on
Terrorism."Sage Publications, Volume34, Issue 4, p.434. [9] See Thomas J. Cobb (2020). American Cinema and Cultural Diplomacy: The Fragmented
Kaleidoscope.UK: Palgrave Macmillan. [10] Georeg Klay Kieh (1992). "Western Imperialism in the Middle East: The Case of the United
States' Military Intervention in the Persian Gulf". Arab Studies Quarterly, Volume 14, Issue 1, p.1. [11] Gearoid Ó Tuathail (1996). Critical Geopolitics: The Politics of Writing Global Space. London:
Routledge, p.192. [12] According to Gearoid Ó Tuathail, "Critical geopolitics is distinguished by its
problematization of the logocentric infrastructures that make “geopolitics” or any spatialization
of the global political scene possible. It problematizes the “is” of “geography” and “geopolitics,”
their status as self-evident, natural, foundational, and eminently knowable realities … Rather 43 © Arab Journal of International Law, 2022 than innocent sites of declarative facts and constative statements about the world, these signs
["geography" and "geopolitics"] mark the site of space/power/knowledge production systems,
operations that script the actors, settings, and dramas of global politics in deeply geo-politicized
ways." (Ó Tuathail, Gearoid, Critical Geopolitics: The Politics of Writing Global Space, p.52.) [13] Katherin Woodward (1997), (Ed). Identity and Difference. London: Sage Publications, p.35. [14] Edward Said (1978). Orientalism. London and Henley: Routledge and Kegan Paul, p.5. [15] Floribert Patrick C. Endong (2018). "Cinema Globalization and Nation Branding: An
Exploration of the Impact of the Nollywood of the Nigerian Image Crisis", Journal of Globalization
Studies, Volume 9, Issue 1, p.79. [16] Hirshberg S. Mathew (1993). Perpetuating Patriotic Perceptions: The Cognitive Function of the Cold
War. New York: Praeger, p.47. [17] Edward Said (1981). Covering Islam: How Media and Experts Determine How See the Rest of the
World. New York: Pantheon Books, p.4. [18] Simon Dalby (2013). "Challenging Cartographies of Enmity: Empire, War and Culture in
Contemporary Militarization". In Anna Stavrianakis and Jan Selby (Eds.), Militarianism and
International Relations: Political Economy, Security, Theory. London and New York: Routledge, p.38. [19] Salen Alaswad (2000). "Hollywood Shoots the Arab: The Construction of the Arab in
American Culture". Diss. The Temple University Graduate Board. [20] Ibid. [36] Ibid., pp.170-171. [37] For an in-depth study, see Foucault's analysis of "Hierarchal surveillance", "normalizing
judgment", and the "examination", as three fundamental processes involved in discipline. Michael Foucault, Discipline and Punishment: The Birth of the Prison, pp.170-76, 177-83, 184. [37] For an in-depth study, see Foucault's analysis of "Hierarchal surveillance", "normalizing
judgment", and the "examination", as three fundamental processes involved in discipline. Michael Foucault, Discipline and Punishment: The Birth of the Prison, pp.170-76, 177-83, 184. [38] Xiaoning Lu (2017). "The Might of the People: Counter-Espionage Films and Participatory
Surveillance in the Early PRC". In Karen Fang (Ed.), Surveillance in Asian Cinema: Under Eastern
Eyes. New York and London: Routledge, p.13. [38] Xiaoning Lu (2017). "The Might of the People: Counter-Espionage Films and Participatory
Surveillance in the Early PRC". In Karen Fang (Ed.), Surveillance in Asian Cinema: Under Eastern
Eyes. New York and London: Routledge, p.13. [39] Catherine Zimmer (2015). Surveillance Cinema. New York and London: New York University
Press, p.2. [40] Edward Said (1993). Culture and Imperialism. New York: Vintage Books, p.271. [25] Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p.178. [26] In his article, The Clash of Civilizations?, published in Foreign Affairs in Summer 1993 (later
expanded in his book (1996) The Clash of Civilizations and the Remaking of World Order), Samuel
Huntington posits that "it is my hypothesis that the fundamental source of conflict in this new
world will not be primarily ideological or primarily economic. The great divisions among
humankind and the dominating source of conflict will be cultural. Nation States will remain the 44 © Arab Journal of International Law, 2022 most powerful actors in world affairs but the principal conflicts of global politics will occur
between nations and groups of different civilizations. The clash of civilizations will dominate
global politics. The fault lines between civilizations will be battle lines of the future" (Samuel
Huntigton P, (Summer 1993) ‘The Clash of Civilizations?’ Foreign Affairs Vol. 72, No. 3, p.22. most powerful actors in world affairs but the principal conflicts of global politics will occur
between nations and groups of different civilizations. The clash of civilizations will dominate
global politics. The fault lines between civilizations will be battle lines of the future" (Samuel
Huntigton P, (Summer 1993) ‘The Clash of Civilizations?’ Foreign Affairs Vol. 72, No. 3, p.22. most powerful actors in world affairs but the principal conflicts of global politics will occur
between nations and groups of different civilizations. The clash of civilizations will dominate
global politics. The fault lines between civilizations will be battle lines of the future" (Samuel
Huntigton P, (Summer 1993) ‘The Clash of Civilizations?’ Foreign Affairs Vol. 72, No. 3, p.22. most powerful actors in world affairs but the principal conflicts of global politics will occur
between nations and groups of different civilizations. The clash of civilizations will dominate
global politics. The fault lines between civilizations will be battle lines of the future" (Samuel
Huntigton P, (Summer 1993) ‘The Clash of Civilizations?’ Foreign Affairs Vol. 72, No. 3, p.22. [27] Richard Rubenstein and Jarle Crocker (Fall 1994), “Challenging Huntington.” Foreign Policy. No. 96. As cited in Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p. 194. [28] Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p.195. [28] Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p.195 [29] In his discussion of how "imaginative geography" functions in Orientalist discourse,
Edward Said writes that "It [Orientalism] is rather a distribution of geopolitical awareness into
aesthetic, scholarly, economic, sociological, historical, and philological texts; it is an elaboration
not only of a basic geographical distinction but also of a whole series of “interests” (Edward
Said. Orientalism, p.12). [30] James Wyllie (2005). "The Middle East: Arena of Competition and Conflict". In Trevor C. Salmon (Ed.), Issues in International Relations. New York: Routledge, p.189. [30] James Wyllie (2005). "The Middle East: Arena of Competition and Conflict". In Trevor C. Salmon (Ed.), Issues in International Relations. New York: Routledge, p.189. [31] Ikenberry G. John (2003). "Is American Multilateralism in Decline". Perspectives On Politics,
Volume 1, Issue 3, p.533. [31] Ikenberry G. John (2003). "Is American Multilateralism in Decline". Perspectives On Politics,
Volume 1, Issue 3, p.533. [32] Ibid. [33] Gearoid Ó Tuathail, Critical Geopolitics: The Politics of Writing Global Space, p.1. [34] Michael Foucault (1995). Discipline and Punishment: The Birth of the Prison. Trans. Alan
Sheridan. New York: Vintage Books, p.33. [35] Ibid., p.170. References 45 Alaswad, Salen (2000). "Hollywood Shoots the Arab: The Construction of the Arab in American
Culture". Diss. The Temple University Graduate Board. Berg, Peter (2007). The Kingdom. (Thriller movie)) Berg, Peter (2007). The Kingdom. (Thriller movie)) Cobb, Thomas J. (2020). American Cinema and Cultural Diplomacy: The Fragmented Kaleidoscope.UK:
Palgrave Macmillan. Crampton, Andrew and Power, Marcus (2007). Cinema and Popular Geo-Politics. Routledge. Dalby, Simon (2013). "Challenging Cartographies of Enmity: Empire, War and Culture in
Contemporary Militarization". In Anna Stavrianakis and Jan Selby (Eds). Militarianism and
International Relations: Political Economy, Security, Theory. London and New York: Routledge. 33-44. Dodds, Klaus (2008). "Hollywood the Popular Geopolitics of the War on Terror", Third World
Quarterly, Vol.29. No.8. pp.1621-1637. Endong, Floribert Patrick C. (2018). "Cinema Globalization and Nation Branding: An
Exploration of the Impact of the Nollywood of the Nigerian Image Crisis". Journal of Globalization
Studies. Vol.9. No.1.pp.77-90. Foucault, Michael (1995). Discipline and Punishment: The Birth of the Prison. Trans. Alan Sheridan. New York: Vntage Books. Huntington, Samuel P. (Summer 1993). "The Clash of Civilizations?" Foreign Affairs Vol. 72, No. 3. pp.21-49. Ikenberry, John.G. (2003). "Is American Multilateralism in Decline". Perspectives On Politics.1:3. pp.530-550. Jackson, De Richard (2005). Writing the War on Terrorism: Language, Politics and Counter-terrorism. New York: Manchester University Press. Klay Kieh, George (1992). "Western Imperialism in the Middle East: The Case of the United
States' Military Intervention in the Persian Gulf”. Arab Studies Quaterly. Lu, Xiaoning (2017). "The Might of the People: Counter-Espionage Films and Participatory
Surveillance in the Early PRC". In Karen Fang (Ed.), Surveillance in Asian Cinema: Under Eastern
Eyes. New York and London: Routledge. pp.13-32. Mathew, S. Hirshberg (1993). Perpetuating Patriotic Perceptions: The Cognitive Function of the Cold War. New York: Praeger. Nestingen, Andrew (2008). Cinema and Fantasy in Scandinavia: Fiction, Film, and Social Change. University of Washington Press 46 © Arab Journal of International Law, 2022 Pembroke, Jake (2015). "Constructing American Identity and the Terrorist ‘Other':
Representations of Foreign Policy and Identity in post-2007 Hollywood Cinema". Dissertation. The New Birmingham Review.Vol. 1. No. 1. pp.177-225. Said, Edward (1978). Orientalism. London and Henley: Routledge and Kegan Paul. ------------- (1981). Covering Islam: How Media and Experts Determine How See the Rest of the World. New York: Pantheon Books. - (1993). Culture and Imperialism. New York: Vintage Books. Jiong, Zhang (2017). Literature and Literary Theory in Contemporary China. Trans. Yang Limeng and
Wu Yischeng. London and New York: Routledge. - (1993). Culture and Imperialism. New York: Vintage Books. Shaheen, Jack (1990). "Screen Images of Palestinians in 1980s". In Paul Loukides and Linda K. Fuller (Eds.), Beyond the Stars: Stock characters in American popular film, Volume 1. Bowling Green
Ohio: Bowling Green State University Popular Press. 49-60. Skidmore, David (2005). "Understanding the Unilateralits Turn in U.S Foreign Policy”. Foreign
Policy Analysis. 2. 207-228. Tuathail, Gearoid O. (1996). Critical Geopolitics: The Politics of Writing Global Space. Routledge Van Ham, Peter (2008). "War, Lies, and Videotape: Public Diplomacy and USA’s War on
Terrorism." Sage Publications. Vol.34. No.4. pp.427-444. Weldes, Jutta and Rowely, Christina (2015). "So, How Does Popular Culture Relates to World
Politics?". In Federica Caso And Caitlin Hamilton (Eds.), (pp.11-33) Popular Culture and World
Politics: Theories, Methods, Pedagogies. Bristol: E-International Relations Publishing. Woodward, Kathrin (1997). (Ed). Identity and Difference. London. Sage Woodward, Kathrin (1997). (Ed). Identity and Difference. London. Sage Wyllie, James (2005). "The Middle East: Arena of Competition and Conflict". In Trevor C. Salmon (Ed.), (pp.189-221). Issues in International Relations. New York: Routledge. Zimmer, Catherine (2015). Surveillance Cinema. New York and London: New York University
Press. Jiong, Zhang (2017). Literature and Literary Theory in Contemporary China. Trans. Yang Limeng and
Wu Yischeng. London and New York: Routledge. 47 47
|
https://openalex.org/W3205293231
|
https://europepmc.org/articles/pmc8540800?pdf=render
|
English
| null |
Attention-Based Joint Training of Noise Suppression and Sound Event Detection for Noise-Robust Classification
|
Sensors
| 2,021
|
cc-by
| 6,863
|
Keywords: noise-robust classification; noise suppression; sound event detection; joint training; deep
neural network; attention Citation: J.-Y. Son and J.-H. Chang
Attention-Based Joint Training of
Noise Suppression and Sound Event
Detection for Noise-Robust
Classification. Sensors 2021, 21, 6718. https://doi.org/10.3390/s21206718 sensors sensors Jin-Young Son
and Joon-Hyuk Chang * Jin-Young Son
and Joon-Hyuk Chang * Department of Electronic Engineering, Hanyang University, Seoul 04763, Korea; ekwy3tp@hanyang.ac.kr
* Correspondence: jchang@hanyang.ac.kr; Tel.: +82-2-2220-0355 Abstract: Sound event detection (SED) recognizes the corresponding sound event of an incoming
signal and estimates its temporal boundary. Although SED has been recently developed and used in
various fields, achieving noise-robust SED in a real environment is typically challenging owing to the
performance degradation due to ambient noise. In this paper, we propose combining a pretrained
time-domain speech-separation-based noise suppression network (NS) and a pretrained classification
network to improve the SED performance in real noisy environments. We use group communication
with a context codec method (GC3)-equipped temporal convolutional network (TCN) for the noise
suppression model and a convolutional recurrent neural network for the SED model. The former
significantly reduce the model complexity while maintaining the same TCN module and performance
as a fully convolutional time-domain audio separation network (Conv-TasNet). We also do not update
the weights of some layers (i.e., freeze) in the joint fine-tuning process and add an attention module
in the SED model to further improve the performance and prevent overfitting. We evaluate our
proposed method using both simulation and real recorded datasets. The experimental results show
that our method improves the classification performance in a noisy environment under various
signal-to-noise-ratio conditions. 1. Introduction Sound event detection (SED) aims to detect the type of event corresponding to an
incoming sound and to obtain its onset and offset. SED is applied to various fields, and with
the development of technology, it is commonly used in fields closely related to human lives. For instance, it is being used in automatic assistance driving [1], smart meeting rooms [2],
drone detection [3], multimedia [4], social care [5], audio surveillance system [6]. Received: 6 September 2021
Accepted: 8 October 2021
Published: 9 October 2021 y
Early research in the field of SED used traditional shallow learning model approaches,
such as Gaussian mixture models [7], hidden Markov models [8], and random regression
forests [9]. Approaches based on support vector machines [10–12] and non-negative matrix
factorization [13–15] were also proposed. In recent years, deep neural network (DNN)-
based approaches such as convolutional neural network (CNN) [16–18], recurrent neural
network (RNN) [19,20], and convolutional recurrent neural network (CRNN) [18,21] have
presented high classification performance. Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. The above-mentioned recent studies were focused on improving the SED performance,
which demonstrated its potential for applications in real environments. However, such
applications are affected by ambient noise and cannot detect and classify the desired target
sound. Therefore, it is important to overcome the interference of a noise signal. In this
regard, in some fields, studies focused on the characteristics of noise have been conducted. Some studies focused on the point that noise components have non-Gaussian properties in
communication and radar systems and suggested solutions related to this, and a study to
improve the robustness of the DOA estimation against the interference and noise signals
was conducted [22–24]. Research related to noise-robust SED also has been performed as the 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/sensors Sensors 2021, 21, 6718. https://doi.org/10.3390/s21206718 Sensors 2021, 21, 6718 2 of 13 growing importance of being robust in noise signals for application in a real environment. Methods such as weak-level noise reduction approaches [25], adaptive noise reduction [26],
and optimally modified log-spectral amplitude-based noise filtering [27] were presented. In addition, studies combining DNN-based dereverberation and beamforming at the front
end [28] were conducted. 1. Introduction However, there is limited research on noise-robust SED using
DNN-based audio enhancement at the front end. In particular, a good-performance time-
domain audio enhancement model, such as convolutional time-domain audio separation
network (Conv-TasNet) [29], has not been studied for use at the front end of SED. However,
in the field of automatic speech recognition (ASR), various studies have been conducted
on combining DNN-based speech enhancement at the front end with DNN-based ASR at
the back end in the time–frequency domain to develop a noise-robust ASR system [30–34]. In the time-domain, for enhancing the noise-robust performance by speech denoising,
combining Conv-TasNet at the front end was proposed [35]. Furthermore, using a joint
fine-tuning method after combining with Conv-TasNet was suggested to improve the
performance of music-mixed speech recognition [36]. p
p
g
Motivated by the joint DNN-based audio enhancement actively conducted in the
ASR field, this paper proposes for the first time in the field of SED combining DNN-based
time-domain noise suppression (NS) at the front end to increase the SED performance
in a low signal-to-noise ratio (SNR) environment. For the NS model at the front end, we
use a temporal convolutional network with the group communication with context codec
method (GC3-TCN) [37], which reduce the model complexity of Conv-TasNet and secures
the same performance. In [37], time-domain GC3-TCN was originally used for audio and
speech separation; however, in this study, it is modified for NS. For the SED model at the
back end, a CRNN-based classification model is employed. Subsequently, the pretrained
NS and SED models are cascaded, which are trained for different objectives, and a joint
fine-tuning method by learning with the final SED loss is proposed. During the joint fine-
tuning process, we propose to freeze some layers’ weight and add an attention module
to prevent overfitting and improve the classification performance in a noisy environment. The proposed method is evaluated using simulation and real recorded data. The remainder of this paper is organized as follows: Section 2 describes the proposed
system, which is composed of building pretrained NS and SED models, a joint training
procedure, and an attention mechanism. The experimental settings and the evaluations are
presented in Section 3. Section 4 discusses the experimental results. Finally, in Section 5,
the conclusions are drawn. 2. Proposed System In this section, we describe our proposed system, which consists of pretraining mod-
ules of each NS and SED, a joint training module of combined two models, and an attention
module used in the joint training process. Prior to the fine-tuning by joint training, the
pretrained models are NS and SED models, as shown in Figure 1. Each pretrained model
is used to create a deeper DNN model, an attention module is added in the joint training
stage, and fine-tuning is performed based on the SED loss. An input signal X undergoes
the following process: Xenh = NS(X)
(1)
ˆX = FE(Xenh)
(2)
Yclassi f ier = SED( ˆX)
(3) (1) (2) Yclassi f ier = SED( ˆX)
(3) (3) where Xenh denotes the enhanced waveform, FE(·) is the feature extraction, ˜X denotes
extracted log-mel spectrogram feature and Yclassi f ier is the classifier output. 3 of 13 Sensors 2021, 21, 6718 Figure 1. Overall structure showing pretraining process before joint training, where s and x denote the clean and enhanced
features, respectively. Additionally, m is the number of layers, Ωm(·) is the activation feature output of the mth layer, and
classifier output is the event presence probability output. Figure 1. Overall structure showing pretraining process before joint training, where s and x denote the clean and enhanced
features, respectively. Additionally, m is the number of layers, Ωm(·) is the activation feature output of the mth layer, and
classifier output is the event presence probability output. 2.2. Sound Event Detection Model A CRNN-based classification model is used for the SED model, same as the auxiliary
network used in the NS model training procedure, as described above. A noisy log-mel
spectrogram feature is fed into the CRNN model, and the output is the event presence
probability for the same number of sound event classes as that in the dataset. The label is
one-hot encoded; therefore, the output range is [0, 1]. 2.1.2. Auxiliary Model The auxiliary model used for training the NS model is a CRNN-based classification
model, which is the same as the SED model at the back end with only differences in
the clean and noisy input features as shown in Figure 1. The model is trained with a
clean log-mel spectrogram feature, and its weights are frozen in the NS model training
procedure. Three convolutional layers with learnable kernel sizes of 3 × 3 are used to
learn high-level feature representations from the log-mel spectrogram feature. Each layer
is followed by a max-pooling layer with a 3 × 3 window size. The feature passes through
each convolutional layer followed by a max-pooling layer and is subsequently fed into
three bidirectional long short-term memory (Bi-LSTM) layers, which are used to capture
the temporal context dependencies. Finally, the feature is fed into a fully connected layer
and a sigmoid activation layer to obtain the event presence probability output. 2.1.1. Deep Feature Loss The NS model is trained using deep feature loss. In [38], a pretrained auxiliary model
was used to train the enhancement model. Similar to the method in [38], in the NS model
training process in this study, a pretrained auxiliary network that was trained with a clean
dataset for classification is introduced and its weights are frozen. The deep feature loss
is the L1 loss in the difference between the activations of the clean input feature and the
predicted enhanced feature (that undergoes NS at the front-end process) through each
layer in the auxiliary model as follows: L(s, n) =
M
∑
m=1
∥Ωm(F(s)) −Ωm(F(N(n)))∥1
(4) (4) where s and n denote the clean and noisy input signals, respectively. In addition, N(·) is
the operation in the NS model, F(·) is the feature extraction, M is the number of layers, and
Ωm(·) is the activation feature output of the mth layer in the auxiliary model. 2.1. Noise Suppression Model For the NS model at the front end, which is combined with the SED model at the
back end, the GC3-TCN method is applied. Its key concept is sub-band processing, which
divides the intermediate representations into a specific number of feature groups and
processes them separately. It reduces the model size and complexity by weight sharing
across all groups (group communication), and further decrease the number of multiply-
accumulate operation using encoder-decoder-based temporal compression method (context
codec). In the encoder part of the context codec, the temporal context of local feature is
summarized into a single feature representing the global characteristics of the context [37]. After passing the group communication-equipped separation module, the compressed
feature is transformed back to the context feature through the decoder part of the context
codec and reconstructed to the estimated waveforms through a decoding transformation. Considering the model size of the joint model consisted of NS and SED, we used the
GC3-TCN for the NS model. More details are described in [37]. Sensors 2021, 21, 6718 4 of 13 2.3. Joint Training We propose joint fine-tuning to update all parameters except the last fully connected
layer by combining the GC3-TCN-based NS model and the CRNN-based SED model. The
overall structure is a network that simply cascades the pretrained NS and SED models, as
shown in Figure 2. In the input/output process, the input mixture waveform enters the NS
model to yield an estimated noise suppressed waveform output (blue section in Figure 2),
and after conversion into a log-mel spectrogram by feature extraction (purple section in
Figure 2), it is fed into the SED model to yield the event presence probability output (orange
section in Figure 2). In the joint training procedure, the loss is propagated down from the
back end to the front end by setting the SED loss as the loss of the combined network. 5 of 13 Sensors 2021, 21, 6718 Figure 2. Overall procedure of attention-based joint training. Figure 2. Overall procedure of attention-based joint training. 3. Experiment
3.1. Dataset The proposed system was evaluated using a mixture generated by mixing clean
sound events data with the noise data. In addition, to verify the SED performance in a
real environment, not only simulated noise data but also noise data recorded in a real
environment were used. As for sound events audio data, we used the TAU Spatial Sound
Events 2019 dataset [41]. The dataset consists of ambisonic and microphone array datasets. The ambisonic dataset contains four-channel first-order ambisonic recordings, and the
microphone array dataset comprises four-channel directional microphone recordings from
a tetrahedral array configuration [41]. In this study, we used the microphone array dataset. The microphone array dataset consists of a total of 500 audio data, 400 for development,
and 100 for evaluation. Each audio clip is 1-min long and has a sampling rate of 48 kHz. The recordings were synthesized using spatial room impulse response collected from five
indoor locations, with 504 unique azimuth–elevation–distance combinations [41]. The IRs
were convolved with isolated sound events from DCASE 2016 task 2 dataset. The dataset
consists of 11 classes such as clearing throat, coughing, door knock, door slam, drawer,
human laughter, keyboard, keys (put on table), page turning, phone ringing, and speech. In this study, the development dataset was used, and each 60-s audio clip was divided
into 10-s audio clips. Prior to the joint training, the divided dataset was used as the NS
model, auxiliary network, and SED model dataset. In the joint training stage, the dataset
was divided into 5-s audio clips to double the total dataset. p
For the simulated noise data, we used the DNS Challenge Noise-full band dataset [42],
which were selected from Audioset [43] and Freesound. The dataset contains approximately
150 audio classes, such as music, speech, toothbrush, creak, etc., and 60,000 clips from
Audioset and additional 10,000 noise clips from Freesound and the DEMAND dataset [44]. Each audio clip is 10-s long and has a sampling frequency of 48 kHz. For the real experimental noise data, we recorded a real sound source in a noisy
environment created using a robot vacuum cleaner manufactured by LG Electronics. We
mounted a four-channel microphone array on a robot in the form of a tetrahedron, and
only the single channel closest to the robot was used for the experiment. 3. Experiment
3.1. Dataset In the recording
environment, the robot cleaner moved freely, and there were two modes and an additional
turbo on/off mode; therefore, a total of four types of vacuum noises were recorded. All
signals were recorded at a sampling frequency of 48 kHz. To construct a dataset considering
the noisy environment, the TAU Sound Events dataset was mixed with the DNS Challenge
Noise-full band dataset, whereas the real noisy data were built by mixing with the real
recorded noise dataset. In the mixture data generation process, one of the numerous noise
data was selected and mixed in an audio clip of the divided sound event dataset, as
described above. The SNR range for the training data consisted of −10, −5, 0, 5, and 10 dB,
and that for the validation and test data comprised 2, 0, −2, −5, −7, −10, and −12 dB. The
dataset consisted of training, validation, and test sets in a ratio of 8:1:1. 2.4. Attention Mechanism In the joint training process, an attention mechanism is added between the input
in the SED module and the output passing through each convolutional layer and max-
pooling layer. We exploit an attention mechanism of a similar form used in [39,40], but
with different stride sizes of each convolutional layer, normalization, and skip-connection. As shown in Figure 3, i is set as a variable representing the order of blocks (convolutional
and max-pooling layers) to which the attention module is added. The input feature and
the ith feature from the ith convolutional and max-pooling layer output are mapped to the
two-dimensional (2-D) feature through a 2-D convolutional layer with a 3 × 3 kernel size. The 2-D features each obtained from different inputs, the input and ith output in Figure 3,
have the same dimensions as the ith output, by setting different stride sizes for the conv2D
layer before they are added. An attention mask is produced after passing through the
sigmoid activation, 2-D convolutional layer, and another sigmoid activation. The feature is
subsequently element-wise multiplied with the obtained attention mask, and the masked
feature is added to the ith convolutional and max-pooling layer output. Figure 3. Attention mechanism. Figure 3. Attention mechanism. Figure 3. Attention mechanism. Figure 3. Attention mechanism. Sensors 2021, 21, 6718 6 of 13 3. Experiment
3.1. Dataset 3.2. Metrics To evaluate the SED performance, the segment-based level F-score and error rate were
used [45], which were calculated in one-second segments without overlapping. The F-score
is calculated using P and R, where P is the precision and R is the recall, which are defined
as follows: P =
∑K
k=1 TP(k)
∑K
k=1 TP(k) + ∑K
k=1 FP(k)
,
R =
∑K
k=1 TP(k)
∑K
k=1 TP(k) + ∑K
k=1 FN(k)
(5) (5) where K is the total number of segments, TP(k), FP(k), and FN(k) denote the total num-
ber of true positives, false positives, and false negatives in the kth one-second segment,
respectively. Subsequently, the F-score is calculated as follows: Sensors 2021, 21, 6718 7 of 13 F = 2·P·R
P + R
(6) (6) In addition, error rate was measured by calculating the total number of insertions (I),
deletions (D), and substitutions (S) relative to the number of active sound events in the
reference, N, and each is defined as follows: S(k) = min(FN(k), FP(k)),
(7)
D(k) = max(0, FN(k) −FP(k)),
(8)
I(k) = max(0, FP(k) −FN(k))
(9) (7)
(8)
(9) (7) (9) Thus, the error rate is calculated as: Thus, the error rate is calculated as: ER = ∑K
k=1 S(k) + ∑K
k=1 D(k) + ∑K
k=1 I(k)
∑K
k=1 N(k)
(10) (10) where N(k) is the number of sound events active in segment k. where N(k) is the number of sound events active in segment k. where N(k) is the number of sound events active in segment k. where N(k) is the number of sound events active in segment k. 3.3. Feature Extraction for SED Model 3.3. Feature Extraction for SED Model It is important to extract feature that can be used efficiently in one domain or in
the process of converting to another domain and this technique has also been applied to
several different fields [46–49]. In this study, as the input feature for the SED network, we
used a log-mel spectrogram. We applied a window length of 40 ms, hop length of 20 ms,
Hanning window size of 40 ms, and fast Fourier transform size of 2048 points. The log-mel
spectrogram was extracted as a 128-dimensional feature. We obtain 500 frames in a single
10-s long dataset for the NS, auxiliary, and SED models before the joint training, and by
slicing whole the frame in a half, 250 frames in a single 5-s long dataset were used for the
cascaded model in the joint training. The input to the SED model was a 128 × T log-mel
spectrogram feature map, in which T denotes the number of frames and is set to 64. 3.5. Training Details and Evaluation This section describes the NS model, auxiliary model in the NS model training proce-
dure, SED model, and joint training. 3.4. Baseline Model To verify the SED performance of the proposed system, a baseline model was es-
tablished by training with noisy data. The baseline model was a CRNN-based classifi-
cation model, with the same configuration as the auxiliary and SED models. We set the
baseline as described above to compare the results based on the joint training and the
attention mechanism. 3.5.3. Joint Training Model 3.5.3. Joint Training Model As described in Section 2.3, the joint training model is a fine-tuning process that
combines the NS and SED models. In this process, to prevent overfitting and increase
the performance of the classifier output, the weights of some layers were frozen, and an
attention mechanism was applied to reflect the estimated enhanced information during
learning. The hyperparameters of the NS model, SED model, and feature extraction process
are the same as during the pretrained model construction. The combined model was trained
with binary cross entropy loss and the Adam optimizer at a learning rate of 0.0001. An
early stopping method was applied equally. 3.5.1. Noise Suppression Model The configuration of the NS model, i.e., GC3-TCN, was set using the hyperparameters
and notations (described in Table 1) in [29,37] as follows: N = 256, L = 96, H = 128, P =
3, X = 2, R = 4, K = 4, M = 16, C = 32, B = 24. The number of channels in the bottleneck
and residual paths of the one-dimensional (1-D) conv-blocks and the number of channels
in the skip-connection paths of 1-D conv-blocks was 128 each. The NS model was trained
for 200 epochs with a deep feature loss and the Adam optimizer at a learning rate of 0.0001. An early stopping method was applied when no best validation model was obtained for 15
consecutive epochs. Sensors 2021, 21, 6718 8 of 13 Table 1. Hyperparameters and notations used in GC3-TCN. Table 1. Hyperparameters and notations used in GC3-TCN. Hyperparameters
Notation
Number of encoder filters
N
Length of the filters
L
Number of channels in convolutional blocks
H
Kernel size in convolutional blocks
P
Number of convolutional blocks in each repeat
X
Number of repeats
R
Number of groups
K
Group size
M
Context size (in frames)
C
TCN block size (in frames)
B 3.5.2. Auxiliary Model and Sound Event Detection Model The two models had the same training procedure and configuration, with differences
only in the input data, regardless of them being noisy or clean. The configuration of the
classification model is summarized in Table 2. Both models were trained for 150 epochs with
binary cross entropy loss and the Adam optimizer at a learning rate of 0.001. A dropout
rate of 0.5 was applied to the output of the last convolutional layer and the Bi-LSTM layer. An early stopping method was also applied when no best validation model was found for
10 consecutive epochs. Table 2. Configuration of CRNN. Layers
Output Size
Input
1 × 128 ×T
Convolution 1
16 × 64 × T
Max-pooling 1
16 × 32 × T
Convolution 2
32 × 16 × T
Max-pooling 2
32 × 8 × T
Convolution 3
64 × 4 × T
Max-pooling 3
64 × 2 × T
Reshape
T × 128
BLSTM × 3
T × 128
Fully connected
T × 128
Output
T × 11 Table 2. Configuration of CRNN. 4.1. Simulation Results Table 3 summarizes the results of using noisy simulated data mixed with the DNS
Challenge Noise-full band dataset and the TAU Spatial Sound Events 2019-Microphone
Array Development dataset. The F-score of simply combining the NS and SED models
before the joint training was 2.3% lower than the baseline performance. Noise suppression
aims to achieve the enhanced signal close to the original clean audio. The model was
trained on a different loss function at the pretraining process independently from the SED
model. This mismatch between the NS and SED model leads to sub-optimum and degrades
the SED performance at the back end with some possible distortions in the audio signal
when simply combining both models [30,34]. This results also exhibited the effectiveness
of combining both models and optimized on the final SED loss. After joint training with
the SED loss, as expected, the F-score and the error rate were improved by 8.7% over the
baseline performance and by approximately 0.08, respectively. In addition, the result of
the joint training by freezing the weights of the last dense layer resulted in a 1.2% F-score
improvement compared to that without freezing. Furthermore, the effectiveness of using
the attention module was investigated by comparing the results obtained by the proposed
algorithm without/with the attention module. The proposed method with the attention
module achieved a superior performance that without the attention module, which verifies
its usefulness in the joint training procedure. Thus, the joint training with the NS model at
the front end is effective for increasing the SED performance in a low SNR environment. Table 3. Performance comparison using the simulated noise dataset for input mixture. JT denotes
joint training. Table 3. Performance comparison using the simulated noise dataset for input mixture. JT denotes
joint training. Method
Model
Evaluation
NS
SED
F-Score (%)
Error Rate
Baseline
-
CRNN
33.7
0.78
Before JT
(pretrained)
GC3-TCN
CRNN
31.4
0.81
After JT
(fine-tuned)
GC3-TCN
CRNN w/o attention
w/o freeze
42.4
0.70
freeze dense
43.6
0.68
+ CNN
38.0
0.74
+ RNN
34.3
0.78
CRNN w/ attention
+ freeze dense
45.2
0.66 Figure 4 presents the results of each case per SNR of the test data. Although the total
dataset ratio was fixed, the number of datasets for each SNR was not exactly the same
because the SNR was randomly selected while creating noisy data. 3.5.4. Evaluation In the evaluation, we compared the performance of three cases. First, using noisy data
as the input, we compared the results of simply combining the NS model at the front end
without joint training against those of the baseline model. Subsequently, we compared the
before and after joint training results. Finally, based on the range of layer’s weight freezing
and the application of the attention mechanism, we compared the performance with those
of the previous cases. Sensors 2021, 21, 6718 9 of 13 4.1. Simulation Results Therefore, there is
a possibility that a certain SNR may have been formed high in the evaluation as many
datasets were involved in training, and some may have been formed low. However, as
a result, when looking at each SNR, it showed improved classification performance by
combining the NS at the front end and joint training as expected. In addition, it was shown
that the fact that the performance was improved by fixing the weights of the dense layer
and applying attention during the joint training process did not change. Furthermore,
for the unseen SNR conditions, the proposed method was effective for improving the
SED performance. 10 of 13 Sensors 2021, 21, 6718 Figure 4. Performance comparison of the test data in different cases based on joint training method
and use of additional attention mechanism under different SNR environments. SNR levels of test
data are 2, 0, –2, –5, –7, –10, and –12 dB. Figure 4. Performance comparison of the test data in different cases based on joint training method
and use of additional attention mechanism under different SNR environments. SNR levels of test
data are 2, 0, –2, –5, –7, –10, and –12 dB. 4.2. Real Experimental Results Table 4 summarizes the results of the noisy data created by mixing the real recorded
noise data obtained using a robot cleaner with the TAU Spatial Sound Events 2019- Mi-
crophone Array Development dataset. Similar to the simulated results, joint training with
NS achieved better performance in terms of the F-score and lower error rate than the
baseline method. In addition, as a result of freezing the weights of the dense layer and
applying the attention mechanism, the F-score increased by 8.6% and 10%, respectively. This demonstrated that combining NS at the front end to improve the SED performance in
a noisy environment leads to enhanced results with both simulation and real data. Table 4. Performance comparison using the real recorded noise dataset for input mixture. JT denotes
joint training. Method
Model
Evaluation
NS
SED
F-Score (%)
Error Rate
Baseline
-
CRNN
41.9
0.71
Before JT
(pretrained)
GC3-TCN
CRNN
36.0
0.75
After JT
(fine-tuned)
GC3-TCN
CRNN w/o attention
w/o freeze
48.7
0.66
freeze dense
50.5
0.64
+ CNN
47.8
0.66
+ RNN
44.6
0.69
CRNN w/ attention
+ freeze dense
51.9
0.62 Table 4. Performance comparison using the real recorded noise dataset for input mixture. JT denotes
joint training. 11 of 13 11 of 13 Sensors 2021, 21, 6718 5. Conclusions In this paper, we proposed combining a time-domain sound separation-based NS
model, GC3-TCN, with a SED model, a CRNN, for noise-robust classification. First, the
two models were trained for NS and SED, respectively. Subsequently, a joint model was
constructed by combining the two pretrained models. The combined model was jointly
fine-tuned with the final SED loss. In the joint training procedure, the dense layer was
frozen, and an attention mechanism was added to reflect the enhanced features passed
through the NS model in the training. We tested the proposed method on both simulation
and real recorded datasets, which showed that using the DNN-based NS at the front end is
effective for achieving noise-robust classification. g
As for future works, we plan to employ the soft parameter sharing method, which is
widely used in multi-task learning in the field of sound event detection and localization,
during the joint training process instead of the attention mechanism. Additionally, we
consider using the knowledge distillation, also known as a teacher-student method, to
further improve the noise-robust classification. Author Contributions: Conceptualization, J.-Y.S. and J.-H.C.; methodology, J.-Y.S. and J.-H.C.; soft-
ware, J.-Y.S.; validation, J.-Y.S.; writing, original draft preparation, J.-Y.S.; writing, review and editing,
J.-H.C.; supervision, J.-H.C.; project administration, J.-H.C.; funding acquisition, J.-H.C. Both authors
have read and agreed to the published version of the manuscript. Funding: This work was supported by Institute of Information & communications Technology
Planning & Evaluation (IITP) grant funded by the Korea government(MSIT) (No.2021-0-00456, De-
velopment of Ultra-high Speech Quality Technology for Remote Multi-speaker Conference System). Institutional Review Board Statement: Not applicable. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. Barchiesi, D.; Giannoulis, D.; Stowell, D.; Plumbley, M.D. Acoustic scene classification: Classifying environments from the sounds
they produce. IEEE Signal Process. Mag. 2015, 32, 16–34. [CrossRef] 2. Butko, T.; Pla, F.G.; Segura, C.; Nadeu, C.; Hernando, J. Two-source acoustic event detection and localization: Online implemen-
tation in a smart-room. In Proceedings of the 19th European Signal Processing Conference (EUSIPCO), Barcelona, Spain, 29
August–2 September 2011; pp. 1317–1321. g
p
; pp
3. Anwar, M.Z.; Kaleem, Z.; Jamalipour, A. Machine learning inspired sound-based amateur drone detection for public safety
applications. IEEE Trans. Veh. Technol. 2019, 68, 2526–2534. [CrossRef] g
p
pp
3. Anwar, M.Z.; Kaleem, Z.; Jamalipour, A. Machine learning inspired so
applications. IEEE Trans. Veh. Technol. 2019, 68, 2526–2534. [CrossRef] pp
4. Ahmad, K.; Conci, N. How deep features have improved event recognition in multimedia: A survey. ACM Trans. Multimed. Comput. Commun. Appl. 2019, 15, 39. [CrossRef] 5. Goetze, S.; Schroder, J.; Gerlach, S.; Hollosi, D.; Appell, J.E.; Wallhoff, F. Acoustic monitoring and localization for social care. J. Comput. Sci. Eng. 2012, 6, 40–50. [CrossRef] 6. Alsina-Pagès, R.M.; Navarro, J.; Alías, F.; Hervás, M. homesound: Real-time audio event detection based on high performance
computing for behaviour and surveillance remote monitoring. Sensors 2017, 17, 854. [CrossRef] [PubMed] 7. Zhang, X.; He, Q.; Feng, X. Acoustic feature extraction by tensor-based sparse representation for sound effects classification. In Proceedings of the IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), South Brisbane,
Australia, 19–24 April 2015; pp. 166–170. p
pp
8. Niessen, M.E.; Van Kasteren, T.L.; Merentitis, A. Hierarchical modeling using automated sub-clustering for sound event recogni-
tion. In Proceedings of the IEEE Workshop on Applications of Signal Processing to Audio and Acoustics (WASPAA), New Paltz,
NY, USA, 20–23 October 2013; pp. 1–4. pp
9. Phan, H.; Maaß, M.; Mazur, R.; Mertins, A. Random regression forests for acoustic event detection and classification. IEEE/ACM
Trans. Audio Speech Lang. Process. 2015, 23, 20–31. [CrossRef] p
g
10. Lu, X.; Tsao, Y.; Matsuda, S.; Hori, C. Sparse representation based on a bag of spectral exemplars for acoustic event detection. In
Proceedings of the IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), Florence, Italy, 4–9 May
2014; pp. 6255–6259. pp
11. McLoughlin, I.; Zhang, H.; Xie, Z.; Song, Y.; Xiao, W. Robust sound event classification using deep neural networks. IEEE/ACM
Trans. Audio Speech Lang. Process. 2015, 23, 540–552. References [CrossRef] 12 of 13 Sensors 2021, 21, 6718 12 of 13 12. Tran, H.D.; Li, H. Sound event recognition with probabilistic distance SVMs. IEEE/ACM Trans. Audio Speech Lang. Process. 2010,
19, 1556–1568. [CrossRef] 13. Chin, M.L.; Burred, J.J. Audio event detection based on layered symbolic sequence representations. In Proceedings of the IEEE
International Conference on Acoustics, Speech and Signal Processing (ICASSP), Kyoto, Japan, 25–30 March 2012; pp. 1953–1956. 13. Chin, M.L.; Burred, J.J. Audio event detection based on layered symbolic sequence representations. In Proceedings of the IEEE
International Conference on Acoustics Speech and Signal Processing (ICASSP) Kyoto Japan 25 30 March 2012; pp 1953 1956 14. Gemmeke, J.F.; Vuegen, L.; Karsmakers, P.; Vanrumste, B. An exemplar-based NMF approach to audio event detection. In
Proceedings of the IEEE Workshop on Applications of Signal Processing to Audio and Acoustics (WASPAA), New Paltz, NY,
USA, 20–23 October 2013; pp. 1–4. pp
15. Mesaros, A.; Heittola, T.; Dikmen, O.; Virtanen, T. Sound event detection in real life recordings using coupled matrix factorization
of spectral representations and class activity annotations. In Proceedings of the IEEE International Conference on Acoustics,
Speech and Signal Processing (ICASSP), South Brisbane, Australia, 19–24 April 2015; pp. 151–155. 16. Zhang, H.; McLoughlin, I.; Song, Y. Robust sound event recognition using convolutional neural networks. In Proceedings of the
IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), South Brisbane, Australia, 19–24 April 2015;
pp. 559–563. pp
17. Phan, H.; Hertel, L.; Maass, M.; Mertins, A. Robust Audio Event Recognition with 1-max Pooling Convolutional Neural Networks. Available online: https://arxiv.org/pdf/1604.06338.pdf (accessed on 1 September 2021). 18. Cakır, E.; Parascandolo, G.; Heittola, T.; Huttunen, H.; Virtanen, T. Convolutional recurrent neural n
sound event detection. IEEE/ACM Trans. Audio Speech Lang. Process. 2017, 25, 1291–1303. [CrossRef] 19. Parascandolo, G.; Huttunen, H.; Virtanen, T. Recurrent neural networks for polyphonic sound event detection in real life
recordings. In Proceedings of the IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), Shanghai,
China, 20–25 March 2016; pp. 6440–6444. 20. Hayashi, T.; Watanabe, S.; Toda, T.; Hori, T.; Le Roux, J.; Takeda, K. Duration-controlled LSTM for
detection. IEEE/ACM Trans. Audio Speech Lang. Process. 2017, 25, 2059–2070. [CrossRef] 21. Adavanne, S.; Pertilä, P.; Virtanen, T. Sound event detection using spatial features and convolutional recurrent neural network. In
Proceedings of the IEEE International Conference on Acoustics, Speech and Signal Processing (ICASSP), New Orleans, LA, USA,
5–9 March 2017; pp. 771–775. References pp
22. Ahmed, Q.Z.; Alouini, M.; Aissa, S. Bit Error-Rate Minimizing Detector for Amplify-and-Forward Relaying Systems Using
Generalized Gaussian Kernel. IEEE Signal Process. Lett. 2013, 20, 55–58. [CrossRef] 23. Novey, M.; Adali, T.; Roy, A. A complex generalized Gaussian distribution—Characterization, gene
Trans. Signal Process. 2010, 58, 1427–1433. [CrossRef] ; Roy, A. A complex generalized Gaussian distribution—Characterization, generation, and estimation. IEEE
2010, 58, 1427–1433. [CrossRef] 24. Liu, Y.; Qiu, T.; Li, J. Joint estimation of time difference of arrival and frequency difference of arrival for cyclostationary signals
under impulsive noise. Digit. Signal Process. 2015, 46, 68–80. [CrossRef] 25. Choi, I.; Kwon, K.; Bae, S.H.; Kim, N.S. DNN-based sound event detection with exemplar-based approach for noise reduction. In
Proceedings of the Workshop on Detection and Classification of Acoustic Scenes and Events (DCASE2016 Workshop), Budapest,
Hungary, 3 September 2016; pp. 16–19. g
y
p
pp
26. Zhou, Q.; Feng, Z.; Benetos, E. Adaptive noise reduction for sound event detection using subband-weighted NMF. Sensors 2019,
19, 3206. [CrossRef] 27. Wan, T.; Zhou, Y.; Ma, Y.; Liu, H. Noise robust sound event detection using deep learning and audio enhancement. In Proceedings
of the IEEE International Symposium on Signal Processing and Information Technology (ISSPIT), Ajman, UAE, 10–12 December
2019; pp. 1–5. pp
28. Noh, K.; Chang, J.H. Joint optimization of deep neural network-based dereverberation and beamforming for sound event
detection in multi-channel environments. Sensors 2020, 20, 1883. [CrossRef] 29. Luo, Y.; Mesgarani, N. Conv-tasnet: Surpassing ideal time–frequency magnitude masking for speech separation. IEEE/ACM Trans. Audio Speech Lang. Process. 2019, 27, 1256–1266. [CrossRef] [PubMed] p
g
30. Wang, Z.Q.; Wang, D. A joint training framework for robust automatic speech recognition. IEEE/ACM Trans. Audio Speech Lang. Process. 2016, 24, 796–806. [CrossRef] 31. Liu, B.; Nie, S.; Liang, S.; Liu, W.; Yu, M.; Chen, L.; Peng, S.; Li, C. Jointly adversarial enhancement training for robust end-to-end
speech recognition. In Proceedings of the Interspeech, Graz, Austria, 15–19 September 2019; pp. 491–495. 32. Fan, C.; Yi, J.; Tao, J.; Tian, Z.; Liu, B.; Wen, Z. Gated recurrent fusion with joint training framework for robust end-to-end speech
recognition. IEEE/ACM Trans. Audio Speech Lang. Process. 2020, 29, 198–209. [CrossRef] 33. Pandey, A.; Liu, C.; Wang, Y.; Saraf, Y. Dual application of speech enhancement for automatic speech
of the IEEE Spoken Language Technology Workshop (SLT), Shenzhen, China, 19–22 January 2021; of the IEEE Spoken Language Technology Workshop (SLT), Shenzhen, China, 19–22 January 2021; pp. References 223–228. 34. Li, L.; Kang, Y.; Shi, Y.; Kürzinger, L.; Watzel, T.; Rigoll, G. Adversarial Joint Training with Self-Attention Mechanism for Robust
End-to-End Speech Recognition. Available online: https://arxiv.org/pdf/2104.01471.pdf (accessed on 1 September 2021). p
g
g
gy
p (
)
J
y
pp
34. Li, L.; Kang, Y.; Shi, Y.; Kürzinger, L.; Watzel, T.; Rigoll, G. Adversarial Joint Training with Self-Attention Mechanism for Robust
End-to-End Speech Recognition. Available online: https://arxiv.org/pdf/2104.01471.pdf (accessed on 1 September 2021). p
g
g
gy
p
y
pp
34. Li, L.; Kang, Y.; Shi, Y.; Kürzinger, L.; Watzel, T.; Rigoll, G. Adversarial Joint Training with Self-Attention Mechanism for Robust 35. Kinoshita, K.; Ochiai, T.; Delcroix, M.; Nakatani, T. Improving noise robust automatic speech recognition with single-channel
time-domain enhancement network. In Proceedings of the IEEE International Conference on Acoustics, Speech and Signal
Processing (ICASSP), Barcelona, Spain, 4–8 May 2020; pp. 7009–7013. g
p
y
pp
36. Woo, J.; Mimura, M.; Yoshii, K.; Kawahara, T. End-to-end music-mixed speech recognition. In Proceedings of the Asia-Pacific
Signal and Information Processing Association Annual Summit and Conference (APSIPA ASC), Auckland, New Zealand, 7–10
December 2020; pp. 800–804. 13 of 13 13 of 13 Sensors 2021, 21, 6718 37. Luo, Y.; Han, C.; Mesgarani, N. Group communication with context codec for lightweight source separation. IEEE/ACM Trans. Audio Speech Lang. Process. 2021, 29, 1752–1761. [CrossRef] p
g
38. Germain, F.G.; Chen, Q.; Koltun, V. Speech Denoising with Deep Feature Losses. Available online: https://arxiv.org/pdf/1806.1
0522.pdf (accessed on 1 September 2021). p
(
p
)
39. Kim, J.H.; Chang, J.H. Attention wave-U-net for acoustic echo cancellation. In Proceedings of the Interspeech, Shanghai, China,
25–29 October 2020; pp. 3969–3973. pp
40. Giri, R.; Isik, U.; Krishnaswamy, A. Attention wave-u-net for speech enhancement. In Proceedings of the IEEE Workshop on
Applications of Signal Processing to Audio and Acoustics (WASPAA), New Paltz, NY, USA, 20–23 October 2019; pp. 249–253. 41. Adavanne, S.; Politis, A.; Virtanen, T. A Multi-Room Reverberant Dataset for Sound Event Localization and Detection. Available
online: https://arxiv.org/pdf/1905.08546.pdf (accessed on 1 September 2021). 42. Reddy, C.K.; Beyrami, E.; Dubey, H.; Gopal, V.; Cheng, R.; Cutler, R.; Matusevych, S.; Aichner, R.; Aazami, A.; Braun, S.; et al. The
Interspeech 2020 Deep Noise Suppression Challenge: Datasets, Subjective Speech Quality and Testing Framework. Available
online: https://arxiv.org/pdf/2001.08662.pdf (accessed on 1 September 2021). p
g p
p
(
p
)
43. 49.
De Lamare, R.C.; Sampaio-Neto, R. Adaptive reduced-rank equalization algorithms based on alternating optimization design
techniques for MIMO systems. IEEE Trans. Veh. Technol. 2011, 60, 2482–2494. [CrossRef] References Gemmeke, J.F.; Ellis, D.P.; Freedman, D.; Jansen, W.; Lawrence, W.; Moore, R.C.; Plakal, M.; Ritter, M. Audio set: An ontology and
human-labeled dataset for audio events. In Proceedings of the IEEE International Conference on Acoustics, Speech and Signal
Processing (ICASSP), New Orleans, LA, USA, 5–9 March 2017; pp. 776–780. g (
)
pp
44. Thiemann, J.; Ito, N.; Vincent, E. The diverse environments multi-channel acoustic noise database (DEMAND): A database of
multichannel environmental noise recordings. Proc. Meet. Acoust. 2013, 19, 035081. 45. Mesaros, A.; Heittola, T.; Virtanen, T. Metrics for polyphonic sound event detection. Appl. Sci. 2016, 6, 162. [CrossRef]
46. Goldstein, J.S.; Reed, I.S. Subspace selection for partially adaptive sensor array processing. IEEE Trans. Aerosp. Electron. Syst. 1997,
33, 539–544. [CrossRef] p
yp
pp
46. Goldstein, J.S.; Reed, I.S. Subspace selection for partially adaptive sensor array processing. IEEE Trans. Aerosp. Electron. Syst. 1997,
33, 539–544. [CrossRef] 47. Alouini, M.-S.; Scaglione, A.; Giannakis, G.B. PCC: Principal components combining for dense correlated multipath fading
environments. In Proceedings of the IEEE Vehicular Technology Conference Fall (VTC), Boston, MA, USA, 24–28 September 2000;
pp. 2510–2517. pp
48. Husbands, R.; Ahmed, Q.; Wang, J. Transmit antenna selection for massive MIMO: A knapsack problem formulation. In
Proceedings of the IEEE International Conferennce on Communications (ICC), Paris, France, 21–25 May 2017; pp. 1–6. g
y
pp
49. De Lamare, R.C.; Sampaio-Neto, R. Adaptive reduced-rank equalization algorithms based on alternating optimization design
techniques for MIMO systems. IEEE Trans. Veh. Technol. 2011, 60, 2482–2494. [CrossRef]
|
https://openalex.org/W2811033679
|
https://journals.plos.org/plosbiology/article/file?id=10.1371/journal.pbio.2005086&type=printable
|
English
| null |
Cell-nonautonomous local and systemic responses to cell arrest enable long-bone catch-up growth in developing mice
|
PLoS biology
| 2,018
|
cc-by
| 17,382
|
RESEARCH ARTICLE OPEN ACCESS Catch-up growth after insults to growing organs is paramount to achieving robust body pro-
portions. In fly larvae, injury to individual tissues is followed by local and systemic compen-
satory mechanisms that allow the damaged tissue to regain normal proportions with other
tissues. In vertebrates, local catch-up growth has been described after transient reduction of
bone growth, but the underlying cellular responses are controversial. We developed an
approach to study catch-up growth in foetal mice in which mosaic expression of the cell
cycle suppressor p21 is induced in the cartilage cells (chondrocytes) that drive long-bone
elongation. By specifically targeting p21 expression to left hindlimb chondrocytes, the right
limb serves as an internal control. Unexpectedly, left–right limb symmetry remained normal,
revealing deployment of compensatory mechanisms. Above a certain threshold of insult, an
orchestrated response was triggered involving local enhancement of bone growth and sys-
temic growth reduction that ensured that body proportions were maintained. The local
response entailed hyperproliferation of spared left limb chondrocytes that was associated
with reduced chondrocyte density. The systemic effect involved impaired placental function
and IGF signalling, revealing bone–placenta communication. Therefore, vertebrates, like
invertebrates, can mount coordinated local and systemic responses to developmental
insults that ensure that normal body proportions are maintained. Citation: Rosello´-Dı´ez A, Madisen L, Bastide S,
Zeng H, Joyner AL (2018) Cell-nonautonomous
local and systemic responses to cell arrest enable
long-bone catch-up growth in developing mice. PLoS Biol 16(6): e2005086. https://doi.org/
10.1371/journal.pbio.2005086 Academic Editor: Caroline Hill, Lincolns Inn Fields
Laboratory, United Kingdom of Great Britain and
Northern Ireland Academic Editor: Caroline Hill, Lincolns Inn Fields
Laboratory, United Kingdom of Great Britain and
Northern Ireland Received: December 11, 2017
Accepted: May 24, 2018
Published: June 26, 2018
Copyright: © 2018 Rosello´-Dı´ez et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Received: December 11, 2017
Accepted: May 24, 2018
Published: June 26, 2018 Copyright: © 2018 Rosello´-Dı´ez et al. This is an
open access article distributed under the terms of
the Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: The datasets
generated during and/or analysed during the
current study are tabulated in the Supporting
information and archived at the following
databases: GSE97232. Alberto Rosello´-Dı´ez1¤a*, Linda Madisen2, Se´bastien Bastide1¤b, Hongkui Zeng2,
Alexandra L. Joyner1,3* 1 Developmental Biology Program, Sloan Kettering Institute, New York, New York, United States of America,
2 Allen Institute for Brain Science, Seattle, Washington, United States of America, 3 Biochemistry, Cell and
Molecular Biology Program, Weill Cornell Graduate School of Medical Sciences, New York, New York, United
States of America a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 ¤a Current address: Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
¤b Current address: Dept. of Developmental & Stem Cell Biology, Institut Pasteur, Paris, France
* alberto.rosellodiez@monash.edu (ARD); joynera@mskcc.org (ALJ) ¤a Current address: Australian Regenerative Medicine Institute, Monash University, Clayton, Australia
¤b Current address: Dept. of Developmental & Stem Cell Biology, Institut Pasteur, Paris, France
* alberto.rosellodiez@monash.edu (ARD); joynera@mskcc.org (ALJ) Introduction An important question in biology is how cells integrate intrinsic and extrinsic information
such that their combined behaviours produce higher-order processes and structures, as seen
during organogenesis and tissue repair. In Drosophila larvae, injured imaginal discs can
undergo compensatory proliferation [1] as well as secrete an alarm signal that triggers both a
systemic developmental delay and reduced growth of the spared imaginal discs [2–5]. To-
gether, these processes allow the damaged tissue(s) to catch-up with other tissues, but the role
of damaged versus undamaged cells remains controversial [6,7]. In vertebrates, systemic
growth reduction after injury in a nonessential organ has not been reported. However, sys-
temic catch-up growth has been described after transient impairment of whole-body growth
[8–10], and local growth compensation can occur after unilateral manipulation of long bones
within the limbs [11]. Tight control of inter-limb and limb–body proportions are critical for
efficient locomotion and interaction with the environment, and therefore long bones are an
excellent model for studies of growth regulation. Growth of the long bones is driven by a process called endochondral ossification (reviewed
in [12,13]). Once mesenchymal cells condense into the template of the skeletal elements, they
differentiate into collagen II–expressing chondrocytes that go through sequential differentia-
tion steps from bone ends to centre. Round resting chondrocytes give rise to flat proliferative
cells that form columns and, after a few rounds of duplication, cease proliferation and start to
differentiate into hypertrophic chondrocytes. These cells increase their volume as they prog-
ress towards the centre of the shaft, lay down a collagen X–rich extracellular matrix, and
secrete factors that recruit vasculature and bone precursors (osteoblasts) from the perichon-
drium, a fibrous layer that wraps the cartilage [14,15]. Some hypertrophic chondrocytes die by
apoptosis, while others transdifferentiate into osteoblasts [16,17]. Osteoblasts form the primary
ossification centre by replacing the original matrix with bone. This process is later repeated at
the ends of the bone (epiphyses), forming the secondary ossification centres. The so-called
growth plate remains as a cartilage disc between the primary and secondary ossification cen-
tres and responds to both intrinsic and extrinsic factors that ultimately regulate bone length. For example, indian hedgehog (IHH), secreted by pre-hypertrophic chondrocytes, and para-
thyroid hormone-like peptide, secreted by resting chondrocytes, form a negative feedback
loop that couples chondrocyte proliferation and differentiation (reviewed in [12,13]). Long-bone catch-up growth by local and systemic mechanisms (grant number LT000521/2012-L). Granted to
ARD. The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. Charles Revson
Foundation (grant number 15-34). Granted to ARD. The funder had no role in study design, data
collection and analysis, decision to publish, or
preparation of the manuscript. cycle suppressor p21 was expressed in the cartilage that drives growth of the long bones,
targeting the left limb exclusively and leaving the right limb as an internal control. By trig-
gering the insult during the last gestational week, we found that left–right limb symmetry
was maintained due to the following 2 compensatory mechanisms: (1) hyperproliferation
of the spared cells within the targeted cartilage, which indicates that these cells respond to
a signal coming from the arrested cells, and (2) a growth reduction in the rest of the body,
an effect that correlates with changes in the levels of placental insulin-like growth factor
(IGF) signalling and that can be rescued by boosting placental efficiency. These results
reveal that the response to developmental insults is quite evolutionarily conserved across
species as well as open new avenues of future research for the development of therapies to
treat growth disorders. Competing interests: The authors have declared
that no competing interests exist. Abbreviations: BSA, bovine serum albumin; Cre,
causes recombination (recombinase enzyme
derived from the P1 bacteriophage); CT,
computerized tomography; DEG, differentially
expressed gene; Dox, doxycycline; DRAGON, Dox-
controlled and Recombinase Activated Gene
OverexpressioN; E, embryonic day; EdU, 5-ethynyl-
2’-deoxyuridine; IGF, insulin-like growth factor;
IHH, indian hedgehog; LtSL, floxed tdTomato-
STOP sequence; NK, natural killer; P, postnatal day;
PFA, paraformaldehyde; PZ, proliferative zone;
qRT-PCR, quantitative real-time polymerase chain
reaction; RNA-seq, RNA sequencing; (r)tTA,
(reverse) tetracycline transactivator; tdT, tdTomato;
WT, wild-type. Author summary The coordination of organ growth is necessary to attain correct individual organ sizes and
body proportions. While extensive studies in insects have revealed that both intra-organ
and inter-organ communication mechanisms are involved in regulating organ growth,
vertebrate studies have lagged behind. Here, we developed a new mouse model to examine
cellular mechanisms underlying growth regulation after a developmental insult. The cell Funding: NIH-NICHD (grant number
R21HD083860). Granted to ALJ. The funder had
no role in study design, data collection and
analysis, decision to publish, or preparation of the
manuscript. Human Frontiers Science Program 1 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms insulin-like growth factors [IGFs]) also play crucial roles in the modulation of chondrocyte
activity and bone growth [18,19]. As per the regulation of growth after an insult, it has been
proposed that bone catch-up growth is due to a cell-autonomous delay in the normal develop-
mental decline of chondrocyte proliferation, such that when the insult is lifted, the formerly
arrested chondrocytes retain a higher proliferative potential correlating with the stage at arrest
[9,20]. It was suggested that a similar mechanism applies to other organs [21]. However, the
possible contribution of unaffected cells has not been examined, which is important because a
cell-autonomous mechanism does not account for cases in which catch-up growth is faster
than expected for the observed maturation delay (reviewed in [13,22]). Here, we developed new mouse models to transiently decrease long-bone growth in mice in
order to determine the contributions of cell-autonomous and nonautonomous regulation dur-
ing catch-up growth. Namely, we blocked proliferation in 50% of the cartilage chondrocytes
that drive long-bone elongation, specifically in the left hindlimbs, such that the right limb
remains as an internal control. Unexpectedly, left–right symmetry was maintained, revealing
the deployment of compensatory mechanisms. Locally, we observed hyperproliferation of
wild-type (WT) chondrocytes that mostly compensated for the lack of proliferation of their
arrested neighbours. Systemically, a mild growth reduction affected the rest of the body,
contributing to maintenance of limb–body proportions. In summary, our results reveal that
long-bone catch-up growth shares some similarities with the response of imaginal discs to
developmental insults in insects. We found that this response is mostly cell nonautonomous,
representing a paradigm shift in the field that opens up new research avenues for basic and
translational studies. Introduction This
loop is the main conduit through which other local signals, such as fibroblast growth factors
and bone morphogenetic proteins, exert their function, often impacting on the expression of
key transcription factors. A number of systemic and local extrinsic signals (growth hormone, PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 2 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 An intersectional genetic approach enables inducible p21 misexpression
preferentially in left limb chondrocytes A major roadblock for studies of intra- and inter-organ growth regulation in mouse embryos
has been a lack of models in which growth rate can be altered in a specific cell type within an
organ, and ideally in only one of two paired organs, leaving the unmanipulated organ as an
internal control. To address this deficiency, we devised new mouse models of inducible and
transient growth inhibition in the left limb. We generated an Igs7TRE-LtSL-p21/+ allele, a variant
of a double-conditional allele [23], to achieve doxycycline (Dox)-tuneable misexpression of the
cell cycle suppressor Cdkn1a (p21 hereafter) [24] in the cells in which activities of the bacterio-
phage recombinase Cre and of the (reverse) tetracycline transactivator ([r]tTA) intersect (Fig
1A and 1B). Due to a floxed tdTomato-STOP sequence (LtSL), expression of tdTomato (tdT)
takes place in cells expressing (r)tTA but having no history of Cre activity, whereas p21 is
expressed in the cell population with a history of Cre and current (r)tTA activity (Fig 1A). We
named the general type of allele Dox-controlled and Recombinase Activated Gene Overexpres-
sioN (DRAGON). By combining the DRAGON-p21 allele with an asymmetric-Pitx2-enhancer-
Cre line expressing Cre in the precursors of the left limb mesenchyme (S1A–S1F Fig) [25] and
a cartilage-specific Col2a1-rtTA line containing the reverse tetracycline transactivator under
the control of a type II collagen promoter [26] (Fig 1B), Dox-dependent ectopic p21 expression
was achieved specifically in non-hypertrophic chondrocytes of the left limb cartilage elements
(Fig 1C and 1C’). Consequently, any growth adjustment detected in the right limb of triple
transgenic animals (Pit-Col-p21) when compared to control littermates must be due to activa-
tion of a systemic effect or inter-organ communication. When Dox was administered from embryonic day (E) 12.5 until birth (ePit-Col-p21 model),
analysis at E14.5–E17.5 revealed the expected cartilage-exclusive expression of tdT, mainly in 3 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Fig 1. An intersectional genetic approach enables inducible p21 misexpression primarily in left limb chondrocytes. (A) DRAGON-p21 allele
in the Igs7 locus. (B) Schematic showing p21 expression driven by the left-specific Pitx2-Cre and cartilage-specific Col2a1-rtTA (Pit-Col-p21). (C–E) Expression of tdT protein and p21 mRNA (panel C and C’) and p21 protein and EdU (panel D and E) at E15.5, with Dox administered at
E12.5. n = 3. Box in panel D is magnified in panel E. Asterisk indicate endogenous p21 expression. An intersectional genetic approach enables inducible p21 misexpression
preferentially in left limb chondrocytes Arrowheads indicate rare double-positive
cells. (F) Quantification of p21+ cells in the PZ of ePit-Col-p21 proximal tibias, at E15.5 (n = 3) and E17.5 (n = 5). The average left/right fold-
change is indicated. See also S3 Data. (G) Quantification of EdU incorporation in p21+ and p21−cells of left ePit-Col-p21 PZ of the cartilage, at
E15.5 and E17.5 (n = 3 and n = 5). Comparison by 2-way ANOVA with Cell population and Stage as variables (p-values below graphs). p-Values
for Sidak’s multiple comparisons post hoc test (between cell populations) are shown on the graph. For panel F and G, see S3 Data. 2xpA,
transcriptional STOP; E, embryonic day; EdU, 5-ethynil-2’-deoxyuridine; Ins, insulator; PZ, proliferative zone; tdT, tdTomato; TRE,
Tetracycline-responsive element; W, WPRE (mRNA-stabilizing sequence) followed by pA. Fig 1. An intersectional genetic approach enables inducible p21 misexpression primarily in left limb chondrocytes. (A) DRAGON-p21 allele
in the Igs7 locus. (B) Schematic showing p21 expression driven by the left-specific Pitx2-Cre and cartilage-specific Col2a1-rtTA (Pit-Col-p21). (C–E) Expression of tdT protein and p21 mRNA (panel C and C’) and p21 protein and EdU (panel D and E) at E15.5, with Dox administered at
E12.5. n = 3. Box in panel D is magnified in panel E. Asterisk indicate endogenous p21 expression. Arrowheads indicate rare double-positive
cells. (F) Quantification of p21+ cells in the PZ of ePit-Col-p21 proximal tibias, at E15.5 (n = 3) and E17.5 (n = 5). The average left/right fold-
change is indicated. See also S3 Data. (G) Quantification of EdU incorporation in p21+ and p21−cells of left ePit-Col-p21 PZ of the cartilage, at
E15.5 and E17.5 (n = 3 and n = 5). Comparison by 2-way ANOVA with Cell population and Stage as variables (p-values below graphs). p-Values
for Sidak’s multiple comparisons post hoc test (between cell populations) are shown on the graph. For panel F and G, see S3 Data. 2xpA,
transcriptional STOP; E, embryonic day; EdU, 5-ethynil-2’-deoxyuridine; Ins, insulator; PZ, proliferative zone; tdT, tdTomato; TRE, Fig 1. An intersectional genetic approach enables inducible p21 misexpression primarily in left limb chondrocytes. (A) DRAGON-p21 allele
in the Igs7 locus. (B) Schematic showing p21 expression driven by the left-specific Pitx2-Cre and cartilage-specific Col2a1-rtTA (Pit-Col-p21). (C–E) Expression of tdT protein and p21 mRNA (panel C and C’) and p21 protein and EdU (panel D and E) at E15.5, with Dox administered at
E12.5. n = 3. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms the right skeletal elements, and p21 expression preferentially in the left limb cartilage, albeit in
a mosaic fashion. For example, 36%–67% versus 0.8%–23% of chondrocytes were found to be
p21+ in left versus right proximal tibia (S1G–S1K Fig, Fig 1C–1F; n = 3 E15.5; n = 5 E17.5
proximal tibias; n = 3 E17.5 proximal humerus). As we previously observed with the Cre trans-
gene [19], the activity of Cre and therefore p21 expression was more widespread in the left
hindlimb than in the left forelimb (S1I–S1K Fig; only 22%–38% of chondrocytes were p21+ in
the left proximal humerus). Therefore, we focused our initial analysis on the hindlimb. As
expected, proliferation was inhibited in p21+ proximal tibia chondrocytes at E15.5 and E17.5
(Fig 1D and 1E and 1G; n = 3 and n = 5, respectively). Although a potential consequence of
p21 misexpression in proliferative zone (PZ) chondrocytes could have been their premature
differentiation, we did not find precocious expression of chondrocyte maturation markers
(e.g., Ihh, Col10a1, Cdkn1c, S2A and S2B Fig). Moreover, p21 expression did not induce cell
senescence (monitored by expression of p19 and p16) by E17.5 (S2D Fig), nor did it hamper
chondrocyte survival at E15.5 or E17.5 (S2E Fig). However, the normal expression domains
of Ihh, Cola10a1, and Cdkn1c in (pre)hypertrophic chondrocytes (which do not express the
transgene) appeared slightly fainter in the distal femur and proximal tibia cartilage, and RNA
sequencing (RNA-seq) analysis of the combined proliferative and pre-hypertrophic zones
revealed that their normalized counts were diminished in the left versus right cartilage (al-
though only significantly for Cdkn1c. S2A and S2B Fig and S1 Data, S2 Data. See S6 Fig for
description of the RNA-seq experiment). These results suggest that expression of p21 causes
only a mild maturation impairment. Mosaic local proliferation blockade in the left limb cartilage slightly
reduces bone growth but does not alter left–right limb symmetry We next examined whether mosaic chondrocyte arrest altered left bone growth. As a first test,
we compared the lengths of several left forelimb and hindlimb bones of ePit-Col-p21 mice to
Pitx2-Cre; Igs7TRE-LtSL-p21/+ control mice (ePit-p21 hereafter). At E17.5, the left bones were 0.2–0.3
mm (approximately 10%) shorter in animals misexpressing p21 (Fig 2A, n = 7 for ePit-p21; n = 15
for ePit-Col-p21 femora and radii; n = 4 and 11 for humeri and tibiae), indicating that blocking
chondrocyte proliferation resulted in decreased bone growth. However, the effect was milder than
expected, given that between one-third and two-thirds of chondrocytes were being arrested. This
result suggested that compensatory mechanisms that minimized the impact of the p21 insult had
been activated in the left limbs. Indeed, at E15.5 or E17.5, no major changes in the length of the
proliferative or hypertrophic zones of the growth plate were found (S2F and S2G Fig). We next took advantage of our unilateral approach that provides an experimental and a
control limb within an animal and performed left–right intra-individual comparisons to deter-
mine the degree of asymmetry. Unexpectedly, most ePit-Col-p21 bones measured at E17.5 or
birth (P0) showed no obvious difference in their left/right length ratio compared to ePit-p21 con-
trol littermates. The one exception was a transient small reduction in the size of the left radius
compared to the right (Fig 2B and 2C, n 4 for ePit-p21; n 11 for ePit-Col-p21 at E17.5; n 5
and 6 at P0). These results suggested that the right (i.e., control) bones in ePit-Col-p21 mice
responded to the impact of p21 expression in the left limbs via a systemic effect or inter-organ
communication, such that their growth was reduced similarly to that of the left bones. An intersectional genetic approach enables inducible p21 misexpression
preferentially in left limb chondrocytes Box in panel D is magnified in panel E. Asterisk indicate endogenous p21 expression. Arrowheads indicate rare double-positive
cells. (F) Quantification of p21+ cells in the PZ of ePit-Col-p21 proximal tibias, at E15.5 (n = 3) and E17.5 (n = 5). The average left/right fold-
change is indicated. See also S3 Data. (G) Quantification of EdU incorporation in p21+ and p21−cells of left ePit-Col-p21 PZ of the cartilage, at
E15.5 and E17.5 (n = 3 and n = 5). Comparison by 2-way ANOVA with Cell population and Stage as variables (p-values below graphs). p-Values
for Sidak’s multiple comparisons post hoc test (between cell populations) are shown on the graph. For panel F and G, see S3 Data. 2xpA,
transcriptional STOP; E, embryonic day; EdU, 5-ethynil-2’-deoxyuridine; Ins, insulator; PZ, proliferative zone; tdT, tdTomato; TRE,
Tetracycline-responsive element; W, WPRE (mRNA-stabilizing sequence) followed by pA. https://doi.org/10.1371/journal.pbio.2005086.g001 4 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Fig 2. Mosaic proliferation blockade in the left limb cartilage slightly reduces bone growth but not left–right limb symmetry. (A) Absolute
length of the indicated left bones of ePit-p21 (n = 4–7 depending on the bone) and ePit-Col-p21 (n = 11–15) E17.5 embryos. (B, C) Skeletal
preparations (panel B) and quantification of the left/right ratio (panel C, mean ± SD) of the calcified region of the indicated bones at E17.5 (n =
4–7 ePit-p21 and n = 11–15 ePit-Col-p21 mice, depending on the bone) and P0 (n = 5–9, and 6–9). Dashed lines in panel B mark the ends of the
mineralized region in control bones. In panel A and C, data were analysed by 2-way ANOVA with Genotype and Bone identity as variables. p-
Values are shown below the graphs. For variables with significantly different measurements, Sidak’s post hoc test p-values are shown in the
graph. For panel A and C, see S3 Data. E, embryonic day; P, postnatal day. https://doi.org/10.1371/journal.pbio.2005086.g002 Fig 2. Mosaic proliferation blockade in the left limb cartilage slightly reduces bone growth but not left–right limb symmetry. (A) Absolute
length of the indicated left bones of ePit-p21 (n = 4–7 depending on the bone) and ePit-Col-p21 (n = 11–15) E17.5 embryos. (B, C) Skeletal
preparations (panel B) and quantification of the left/right ratio (panel C, mean ± SD) of the calcified region of the indicated bones at E17.5 (n =
4–7 ePit-p21 and n = 11–15 ePit-Col-p21 mice, depending on the bone) and P0 (n = 5–9, and 6–9). Dashed lines in panel B mark the ends of the
mineralized region in control bones. In panel A and C, data were analysed by 2-way ANOVA with Genotype and Bone identity as variables. p-
Values are shown below the graphs. For variables with significantly different measurements, Sidak’s post hoc test p-values are shown in the
graph. For panel A and C, see S3 Data. E, embryonic day; P, postnatal day. Fig 2. Mosaic proliferation blockade in the left limb cartilage slightly reduces bone growth but not left–right limb symmetry. (A) Absolute
length of the indicated left bones of ePit-p21 (n = 4–7 depending on the bone) and ePit-Col-p21 (n = 11–15) E17.5 embryos. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Cell-nonautonomous compensation by spared neighbours in response to
mosaic blockade of chondrocyte proliferation The compensatory response observed in the limbs of ePit-Col-p21 mice could be due to both
bone-intrinsic cellular mechanisms and the aforementioned apparent extrinsic regulation. We 5 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 (B, C) Skeletal
preparations (panel B) and quantification of the left/right ratio (panel C, mean ± SD) of the calcified region of the indicated bones at E17.5 (n =
4–7 ePit-p21 and n = 11–15 ePit-Col-p21 mice, depending on the bone) and P0 (n = 5–9, and 6–9). Dashed lines in panel B mark the ends of the
mineralized region in control bones. In panel A and C, data were analysed by 2-way ANOVA with Genotype and Bone identity as variables. p-
Values are shown below the graphs. For variables with significantly different measurements, Sidak’s post hoc test p-values are shown in the
graph. For panel A and C, see S3 Data. E, embryonic day; P, postnatal day. https://doi.org/10.1371/journal.pbio.2005086.g002 first tested for any organ-intrinsic responses and started by examining proliferation of the
spared chondrocytes in the hindlimbs. Indeed, the left/right ratio of 5-ethynyl-2’-deoxyuridine
(EdU) incorporation by p21−chondrocytes was higher in experimental animals as compared
with controls at E17.5 and P0 but not E15.5 (Fig 3A and S2H Fig), revealing cell-nonautono-
mous compensatory proliferation of p21−cells in the presence of p21+ neighbours. Because
p21−cells did not differ in size from those of control mice (S2I Fig), the hyperproliferation of
these cells at E17.5 likely contributes to the lack of a left-specific growth reduction in ePit-Col-
p21 embryos. In fact, overall EdU incorporation in left and right ePit-Col-p21 PZs (without dis-
tinguishing between p21+ and p21−cells), while tending to be slightly reduced, was not signifi-
cantly different, indicating that the compensatory proliferation phenomenon is quite effective first tested for any organ-intrinsic responses and started by examining proliferation of the
spared chondrocytes in the hindlimbs. Indeed, the left/right ratio of 5-ethynyl-2’-deoxyuridine
(EdU) incorporation by p21−chondrocytes was higher in experimental animals as compared
with controls at E17.5 and P0 but not E15.5 (Fig 3A and S2H Fig), revealing cell-nonautono-
mous compensatory proliferation of p21−cells in the presence of p21+ neighbours. Because
p21−cells did not differ in size from those of control mice (S2I Fig), the hyperproliferation of
these cells at E17.5 likely contributes to the lack of a left-specific growth reduction in ePit-Col-
p21 embryos. https://doi.org/10.1371/journal.pbio.2005086.g003 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 In fact, overall EdU incorporation in left and right ePit-Col-p21 PZs (without dis-
tinguishing between p21+ and p21−cells), while tending to be slightly reduced, was not signifi-
cantly different, indicating that the compensatory proliferation phenomenon is quite effective PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 6 / 28 Long-bone catch-up growth by local and systemic mechanisms Fig 3. Cell-nonautonomous compensation by spared neighbours in response to mosaic blockade of chondrocyte
proliferation. (A) % of p21+ or p21−chondrocytes that have EdU+ nuclei in the PZ in the left and right proximal tibia
of E15.5, E17.5, and P0 ePit-p21 (Control, n = 4, 6, and 4) and ePit-Col-p21 (Exp, n = 3, 5, and 8) embryos. p21−cells
from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables (p-values below
graphs). For each significant variable, p-values for Sidak’s multiple comparisons post hoc test are shown in the graph. (B) % of EdU+ chondrocytes in the PZ of left and right proximal tibias of E17.5 ePit-Col-p21 embryos, without
distinguishing by p21 expression. Comparison by paired 2-tailed t test. (C–D) In situ hybridisation of p21 (panel C,
arrowheads denote ectopic expression) and quantification of tdT and p21 (panel D) on sections of left ePit-Col-p21
tibial PZs at E15.5, E17.5, and P0. n = 3, 5, and 8 for p21; 3 at each stage for tdT. The % of p21+ cells was compared by
1-way ANOVA (p < 0.0001). p-Values for Tukey’s multiple comparisons post hoc test are shown. The % of tdT+ cells
(a proxy for rtTA activity) was compared by unpaired 2-tailed Mann-Whitney test. For panel A, B, and D, see S3 Data. E, embryonic day; EdU, 5-ethynyl-2’-deoxyuridine; Exp, Experimental; PZ, proliferative zone; rtTA, reverse
tetracycline transactivator; tdT, tdTomato. https://doi.org/10.1371/journal.pbio.2005086.g003
Long bone catch up growth by local and systemic mechanisms Fig 3. Cell-nonautonomous compensation by spared neighbours in response to mosaic blockade of chon
proliferation. (A) % of p21+ or p21−chondrocytes that have EdU+ nuclei in the PZ in the left and right proxi
of E15.5, E17.5, and P0 ePit-p21 (Control, n = 4, 6, and 4) and ePit-Col-p21 (Exp, n = 3, 5, and 8) embryos. p2
from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables (p-values
graphs). Long-bone catch-up growth by local and systemic mechanisms (Fig 3B). Moreover, the proliferative disadvantage of p21+ versus p21−chondrocytes in the left
limb of ePit-Col-p21 mice resulted in dilution of p21+ chondrocytes from 45%–50% of PZ
chondrocytes at E15.5 and E17.5 to approximately 20% at P0 (Fig 3C and 3D; n = 3, 5, and 8,
respectively), and this depletion was not due to inactivation of rtTA activity (Fig 3D; n = 3 at
E17.5; n = 3 at P0). Compensatory proliferation involves local cell interactions As a means to examine whether compensatory proliferation was only dependent on local cell–
cell interactions, we cultured left and right E15.5 ePit-Col-p21 tibiae (together in the same well)
for 2 d with Dox, in the absence of surrounding mesenchyme (Fig 4A). We found that the dis-
tal tibia showed chondrocyte proliferation throughout all section levels, with very few or no
senescent cells (S3A Fig, n = 3), whereas EdU incorporation was not detected in the inner core
of the proximal tibia (bulkier than the distal one), probably because of insufficient nutrient dif-
fusion. We therefore focused our analysis on the distal epiphysis. Similar to our findings in
vivo, EdU incorporation in p21−chondrocytes was significantly higher in the left as compared
to the right cultured cartilages (Fig 4B and 4C), suggesting that compensatory proliferation is a
bone-intrinsic phenomenon or at least does not require constant interaction with the sur-
rounding mesenchymal tissues. As a control, we tested whether right cartilage proliferation
was impaired due to the bone being cultured with the injured left tibia, since this would cause
the impression of compensatory proliferation taking place in the left bone. We therefore cul-
tured left and right tibiae from each embryo in different wells (S3B Fig) and found that EdU
incorporation in distal right tibiae was not significantly different between bones cultured
together (n = 4) or separately (n = 6) from the left bones. For each significant variable, p-values for Sidak’s multiple comparisons post hoc test are shown in th
(B) % of EdU+ chondrocytes in the PZ of left and right proximal tibias of E17.5 ePit-Col-p21 embryos, withou
distinguishing by p21 expression. Comparison by paired 2-tailed t test. (C–D) In situ hybridisation of p21 (pa
arrowheads denote ectopic expression) and quantification of tdT and p21 (panel D) on sections of left ePit-Co
tibial PZs at E15.5, E17.5, and P0. n = 3, 5, and 8 for p21; 3 at each stage for tdT. The % of p21+ cells was comp
1-way ANOVA (p < 0.0001). p-Values for Tukey’s multiple comparisons post hoc test are shown. The % of td
(a proxy for rtTA activity) was compared by unpaired 2-tailed Mann-Whitney test. For panel A, B, and D, see
E, embryonic day; EdU, 5-ethynyl-2’-deoxyuridine; Exp, Experimental; PZ, proliferative zone; rtTA, reverse
tetracycline transactivator; tdT, tdTomato. https://doi.org/10.1371/journal.pbio.2005086.g003 Fig 3. Cell-nonautonomous compensation by spared neighbours in response to mosaic blockade of chondrocyte
proliferation. (A) % of p21+ or p21−chondrocytes that have EdU+ nuclei in the PZ in the left and right proximal tibia
of E15.5, E17.5, and P0 ePit-p21 (Control, n = 4, 6, and 4) and ePit-Col-p21 (Exp, n = 3, 5, and 8) embryos. p21−cells
from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables (p-values below
graphs). For each significant variable, p-values for Sidak’s multiple comparisons post hoc test are shown in the graph. (B) % of EdU+ chondrocytes in the PZ of left and right proximal tibias of E17.5 ePit-Col-p21 embryos, without
distinguishing by p21 expression. Comparison by paired 2-tailed t test. (C–D) In situ hybridisation of p21 (panel C,
arrowheads denote ectopic expression) and quantification of tdT and p21 (panel D) on sections of left ePit-Col-p21
tibial PZs at E15.5, E17.5, and P0. n = 3, 5, and 8 for p21; 3 at each stage for tdT. The % of p21+ cells was compared by
1-way ANOVA (p < 0.0001). p-Values for Tukey’s multiple comparisons post hoc test are shown. The % of tdT+ cells
(a proxy for rtTA activity) was compared by unpaired 2-tailed Mann-Whitney test. For panel A, B, and D, see S3 Data. E, embryonic day; EdU, 5-ethynyl-2’-deoxyuridine; Exp, Experimental; PZ, proliferative zone; rtTA, reverse
tetracycline transactivator; tdT, tdTomato. 7 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Compensatory proliferation requires a minimum threshold of p21+
chondrocytes We next addressed whether the proportion of p21+ chondrocytes in the growing cartilage
influences the extent of compensatory proliferation. Given that forelimb bones show a lower
proportion of p21+ chondrocytes than hindlimb bones within each embryo analysed (S1K Fig;
55.4% ± 11.2% in tibia versus 32.8% ± 7.1% in humerus; n = 3; p = 0.0267 for paired t test), we
first tested whether compensatory proliferation was triggered in the proximal humerus. We
found that although there was a trend towards increased proliferation in left p21−chondro-
cytes, the difference was not significant (Fig 4D; n = 3), suggesting that compensatory prolifer-
ation requires a minimum insult threshold to be triggered. Because intrinsic differences
between forelimb and hindlimb bones might exist in regards to the compensatory proliferation
response, we also tested the threshold hypothesis using only hindlimb bones. In order to
induce p21 expression in fewer chondrocytes than in ePit-Col-p21 mice, we made use of a
newly generated Col2a1-tTA line (see Materials and methods) in place of the Col2a1-rtTA
transgene, such that p21 expression was achieved from approximately E12.5 onwards in the
absence of Dox (Pit-tTA-p21 model) (S4A Fig). tTA expression in this line is less extensive
than rtTA in the Col2a1-rtTA line, as p21 misexpression was detected in fewer left tibial chon-
drocytes than in ePit-Col-p21 left tibia (30%–40% at E15.5, 15%–35% at E17.5, 10%–20% at P0;
S4A Fig). Consistent with our prediction of a threshold being needed, compensatory prolifera-
tion was not detected in the Pit-tTA-p21 model (S4B and S4C Fig). To further investigate
whether a minimum insult threshold is required to trigger increased proliferation, we calcu-
lated the correlation coefficient between the percentage of p21+ chondrocytes and the extent of
proliferation in the PZ from left and right ePit-Col-p21 (in vivo and ex vivo) and Pit-tTA-p21
tibiae, at E17.5 (or E15.5 plus 2 d ex vivo). Segmental linear regression analysis revealed that 8 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Fig 4. Compensatory proliferation is bone-intrinsic and takes place when chondrocyte density is lower than normal. (A) Summary of the ex vivo tibial
culture experiment. The boxed regions correspond to the epiphyses shown in panel B. (B) Immunohistochemistry for the indicated molecules. (C) EdU
quantification on distal PZ sections obtained from E15.5 ePit-Col-p21 tibiae cultured for 2 d. p-Value for 2-tailed paired t test comparing left and right
proliferative ratios of p21−chondrocytes is shown (n = 4). The distal cartilage was quantified because the proximal one (bulkier) shows proliferation only in
the periphery. (D) % of p21+ or p21−chondrocytes that have EdU+ nuclei in the PZ in the left and right proximal humerus of E17.5 ePit-p21 (Control, n = 3)
and ePit-Col-p21 (Exp, n = 3) embryos. p21−cells from Control and Exp mice were compared by 2-way ANOVA with Side and Genotype as variables (p-
values below graphs). (E) Correlation analysis between the extent of EdU incorporation in p21−cells and the amount of p21+ nuclei in left and right PZ of
ePit-Col-p21 (n = 5 in vivo and n = 4 ex vivo) and Pit-tTA-p21 tibial cartilage (n = 4) at E17.5. The inflection point revealed by unconstrained segmental
regression was rounded up and used as a dividing threshold for the 2 correlation analyses (colour-coded). Pearson correlation coefficients and 2-tailed p-
values are shown. (F) Comparison of chondrocyte density in the PZ of left and right ePit-p21 and ePit-Col-p21 proximal tibial cartilage at E15.5 (n = 4 and
n = 3), E17.5 (n = 5 and n = 5) and P0 (n = 4 and 7) and analysed by 2-way ANOVA for Genotype and Side (p-values shown in the embedded tables). When
p < 0.05 for these variables, colour-coded p-values for Sidak’s post hoc tests are shown. (G) EdU incorporation in p21−chondrocytes of left and right PZ from
E17.5 ePit-p21 and ePit-Col-p21 embryos (n = 5 each), plotted against cell density in the PZ. Note the sharp change in proliferation beyond 9,000 cells/mm2. For (C–G), see also S3 Data. Dox, doxycycline; E, embryonic day; EdU, 5-ethynyl-2’-deoxyuridine; Exp, Experimental; P, postnatal day; PZ, proliferative
zone. Compensatory proliferation is possibly related to epiphyseal cell density We next asked whether additional cellular changes occur in the left limbs of ePit-Col-p21 mice
that could contribute to the growth compensation and potentially correlate with the number
of insulted chondrocytes. Because an alteration in cell density can influence organ size, we
tested whether cell density was changed in the PZ of ePit-Col-p21 mice. That was indeed the
case, and we found a temporal association between the occurrence of compensatory prolifera-
tion in the ePit-Col-p21 model (i.e., at E17.5 and P0 but not E15.5) and statistically significant
reduction of cell density in the left PZ as compared to the right (Fig 4F). Notably, left and right
PZ cell densities were not significantly different at any stage in ePit-p21 mice (Fig 4F, n = 12). Moreover, in line with the threshold hypothesis, we found that, at E17.5, there was a certain
value of cell density below which EdU incorporation sharply increased in p21−chondrocytes
(Fig 4G, n = 20 bones). https://doi.org/10.1371/journal.pbio.2005086.g004 the extent of EdU incorporation by p21−chondrocytes did not correlate with the proportion
of p21+ neighbours when this proportion was below 35%, but beyond this threshold, there was
a linear correlation between both parameters (Fig 4E; n = 26 bones). These results suggest that
compensatory proliferation is due to a signal produced in proportion to the number of
arrested chondrocytes, that the signal needs to reach a certain threshold to be effective, and
that it remains active until at least P0 despite the dilution of p21+ chondrocytes. Long-bone catch-up growth by local and systemic mechanisms Long-bone catch-up growth by local and systemic mechanisms
S Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018
9 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 9 / 28 https://doi.org/10.1371/journal.pbio.2005086.g004 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction. (A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treated with Dox
(
d
C l
)
d (
d
) C
i
b
A O A
i h G Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth re
(A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treate
(n = 4 ePit-p21 and n = 11 ePit-Col-p21) or untreated (n = 5 and n = 6). Comparison by 2-way ANOVA wit
and Bone identity as variables. p-Values for Genotypes are shown below graphs; p-values for Sidak’s post ho
shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) o Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction. (A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treated with Dox
(n = 4 ePit-p21 and n = 11 ePit-Col-p21) or untreated (n = 5 and n = 6). Comparison by 2-way ANOVA with Genotype
and Bone identity as variables. p-Values for Genotypes are shown below graphs; p-values for Sidak’s post hoc test
shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) of ePit-Col- Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction. Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction. (A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treated with Dox
(n = 4 ePit-p21 and n = 11 ePit-Col-p21) or untreated (n = 5 and n = 6). Comparison by 2-way ANOVA with Genotype
and Bone identity as variables. p-Values for Genotypes are shown below graphs; p-values for Sidak’s post hoc test
shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) of ePit-Col- Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Mosaic local proliferation blockade in chondrocytes of the left limb results
in a systemic growth reduction Given our initial finding that left and right limb bones exhibit reduced growth in ePit-Col-p21
embryos as compared to ePit-p21 littermates (Fig 2), we next investigated whether there was a
systemic response to the p21 insult in the left limbs. Because the growth phenotype is quite
mild (an approximately 10% reduction), we first confirmed the finding by measuring micro-
computerized tomography (μCT)-generated 3D reconstructions instead of flat micrographs
(S5A and S5B Fig; n = 7 for ePit-p21 and n = 13 for ePit-Col-p21 embryos). We found a very
good correlation between both types of measurements (S5C Fig, n = 80 bones) and therefore
used flat micrographs for all measurements in the study. We first tested whether the growth
reduction affected the whole body. We found that, in addition to a decrease in right bone
length, body weight of E17.5 and P0 ePit-Col-p21 mice—but not E15.5 or E16.5 embryos—was
approximately 10% lower than in ePit-p21 littermates (Fig 5A–5C, S5D Fig). Furthermore, the
bone-length and weight effects required Dox treatment and therefore p21 expression (Fig 5A–
5C). As control experiments, we confirmed that there was no leakiness of the intersectional PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 10 / 28 Long-bone catch-up growth by local and systemic mechanisms p21 and Col2a1-rtTA; Igs7TRE-tdT/+ embryos (eCol-tdT, expressing tdT in all cartilage elements), compared to their
controls (ePit-p21 and ePit-tdT) by unpaired t tests. p-Values corrected for multiple comparisons (Holm-Sidak
method) are shown. For panel B, n = 22 ePit-p21, n = 26 ePit-Col-p21, n = 25 ePit-tdT, and n = 5 eCol-tdT). For panel C,
n = 4, 11, 24, and 5. (D) Model of the systemic growth response after local chondrocyte arrest triggers a stress signal. (E–F) qRT-PCR (panel E) and in situ hybridisation (panel F) for the indicated transcripts in the proliferative plus pre-
hypertrophic zone from ePit-Col-p21 embryos. Panel E shows one of 2 independent experiments with 3 distinct
biological replicates each (total n = 6). The –ΔCt (relative to Gapdh) for each stress-related transcript was compared by
a paired t test (left versus right). In panel F, n = 2 E15.5, n = 4 E16.5, and n = 6 E17.5 embryos (arrowheads denote Il6
expression). For panel A–C and E, see also S3 Data. Dox, doxycycline; E, embryonic day; qRT-PCR, quantitative real-
time polymerase chain reaction; tdT, tdTomato. https://doi org/10 1371/journal pbio 2005086 g005 p21 and Col2a1-rtTA; Igs7TRE-tdT/+ embryos (eCol-tdT, expressing tdT in all cartilage elements), compared to their
controls (ePit-p21 and ePit-tdT) by unpaired t tests. p-Values corrected for multiple comparisons (Holm-Sidak
method) are shown. For panel B, n = 22 ePit-p21, n = 26 ePit-Col-p21, n = 25 ePit-tdT, and n = 5 eCol-tdT). For panel C,
n = 4, 11, 24, and 5. (D) Model of the systemic growth response after local chondrocyte arrest triggers a stress signal. (E–F) qRT-PCR (panel E) and in situ hybridisation (panel F) for the indicated transcripts in the proliferative plus pre-
hypertrophic zone from ePit-Col-p21 embryos. Panel E shows one of 2 independent experiments with 3 distinct
biological replicates each (total n = 6). The –ΔCt (relative to Gapdh) for each stress-related transcript was compared by
a paired t test (left versus right). In panel F, n = 2 E15.5, n = 4 E16.5, and n = 6 E17.5 embryos (arrowheads denote Il6
expression). For panel A–C and E, see also S3 Data. Dox, doxycycline; E, embryonic day; qRT-PCR, quantitative real-
time polymerase chain reaction; tdT, tdTomato. htt
//d i
/10 1371/j
l bi 2005086 005 https://doi.org/10.1371/journal.pbio.2005086.g005 misexpression strategy (S5E Fig) that could account for the systemic growth reduction and
that misexpression of tdT in all chondrocytes did not cause a systemic growth reduction (Fig
5B and 5C). Our results thus revealed a whole-body response to an insult in a specific tissue in
mice, similar to what has been described in Drosophila larvae [2–5]. In order to characterize the cartilage response, we performed an RNA-seq experiment to
identify differentially expressed genes (DEGs) between left and right cartilage (PZ plus pre-
hypertrophic region of proximal and distal tibia and femur) of single ePit-Col-p21 embryos at
E17.5 (S6A–S6E Fig, S1 Data and S2 Data). Indeed, overrepresentation analysis of the DEG
(adjusted p-value 0.05) showed enrichment of several pathways related to stress and
immune responses in the left cartilage (S6F Fig). In particular, we found several stress-related
transcripts that shared a similar left–right pattern of expression within each embryo (S6G Fig)
and verified their enrichment in the left cartilage by quantitative real-time polymerase chain
reaction (qRT-PCR) (Fig 5E) or in situ hybridisation (Fig 5F). Relaxin1, the closest homologue
to dilp8, the recently identified [3,27] alarm gene in fly, was not expressed at significant levels
in either limb (S6E Fig), suggesting that the mechanism that links the local insult with a sys-
temic response has diverged during evolution. With regards to the relationship between the
extent of insult and the induction of a systemic response, Pit-tTA-p21 mice did not trigger a
systemic growth defect at E17.5 or P0 (S4D and S4E Fig, summary in Fig 6A), suggesting that
the systemic growth reduction, like compensatory chondrocyte proliferation, is only triggered
when a certain insult threshold is surpassed in the targeted cartilage. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 (A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treated with Dox
(n = 4 ePit-p21 and n = 11 ePit-Col-p21) or untreated (n = 5 and n = 6). Comparison by 2-way ANOVA with Genotype
and Bone identity as variables. p-Values for Genotypes are shown below graphs; p-values for Sidak’s post hoc test
shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) of ePit-Col- PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 11 / 28 Long-bone catch-up growth by local and systemic mechanisms Fig 6. The systemic effect involves impaired placental function and its rescue leads to altered limb–body proportions. (A) Summary of the characteristics and
outcomes of the different injury models. (B, B’) Placental weight (panel B) and placenta/body weight ratio (panel B’) of ePit-Col-p21 embryos (n = 19), normalized to the
average of ePit-p21 littermates (n = 17) at E17.5 and compared by 2-tailed unpaired Mann-Whitney test. (C) qRT-PCR for Igf2, Igf1r, and Igf2r (with Tbp as reference
gene) in the placenta of E17.5 ePit-Col-p21 and ePit-p21 embryos, normalized to the average value of control littermates (n = 4 Control and n = 3 Exp, except Igf2r, for
which 6 of each were analysed). Two-way ANOVA with Gene and Genotype as variables was used. p-Values for Genotype (right) and for Sidak’s post hoc tests (on
graph) are shown. (D) Pregnant females were injected 3 times each day with Leu27-IGF2 in an attempt to improve placental efficiency. (E–F) Characterization of femur
length at E17.5 in Leu27-IGF2-treated versus untreated ePit-Col-p21-embryos (panel E) and between control and experimental embryos within treated litters (panel F); n
= 11 untreated; n = 9 treated ePit-Col-p21 embryos; n = 6 treated ePit-p21 ones. Unpaired 2-tailed Mann-Whitney test was used. (G–H) Same as panel E and F, for body
and placental weight, n = 6 treated ePit-p21 embryos; n = 19 untreated and 9 treated ePit-Col-p21 embryos. Two-way ANOVA with Conceptus part and Treatment as
variables was used in panel G. p-Values for Treatment (bottom) and for Sidak’s post hoc tests (top) are shown. Unpaired 2-tailed Mann-Whitney test was used in panel
H. (I–J) Same as panel E and F, for placenta/body weight ratio, normalized to the average control littermate (n = 6 treated ePit-p21 embryos; n = 19 untreated and 9
treated ePit-Col-p21 embryos). Unpaired 2-tailed Mann-Whitney test was used. (K) Femur length/body weight ratio of untreated or Leu27-IGF2-treated E17.5 ePit-p21
and ePit-Col-p21 embryos, normalized to the average control littermate (n = 5 untreated and n = 6 treated Control; n = 6 untreated and n = 8 treated Exp embryos). For
each treatment, comparisons by unpaired Mann-Whitney test are shown. (L) Left/right ratio of femur length for E17.5 ePit-p21 and ePit-Col-p21 embryos from Leu27-
IGF2-treated (n = 6 Control and n = 8 Exp) and -untreated litters (n = 7 Control and n = 15 Exp). p-Values (2-way ANOVA) for Treatment and Genotype are shown. For panel B through L, see also S3 Data. E, embryonic day; Exp, Experimental; IGF2, insulin-like growth factor 2; qRT-PCR, quantitative real-time polymerase chain
reaction. Fig 6. The systemic effect involves impaired placental function and its rescue leads to altered limb–body proportions. (A) Summary of the characteristics and
outcomes of the different injury models. (B, B’) Placental weight (panel B) and placenta/body weight ratio (panel B’) of ePit-Col-p21 embryos (n = 19), normalized to the
average of ePit-p21 littermates (n = 17) at E17.5 and compared by 2-tailed unpaired Mann-Whitney test. (C) qRT-PCR for Igf2, Igf1r, and Igf2r (with Tbp as reference
gene) in the placenta of E17.5 ePit-Col-p21 and ePit-p21 embryos, normalized to the average value of control littermates (n = 4 Control and n = 3 Exp, except Igf2r, for
which 6 of each were analysed). Two-way ANOVA with Gene and Genotype as variables was used. p-Values for Genotype (right) and for Sidak’s post hoc tests (on
graph) are shown. (D) Pregnant females were injected 3 times each day with Leu27-IGF2 in an attempt to improve placental efficiency. (E–F) Characterization of femur
length at E17.5 in Leu27-IGF2-treated versus untreated ePit-Col-p21-embryos (panel E) and between control and experimental embryos within treated litters (panel F); n
= 11 untreated; n = 9 treated ePit-Col-p21 embryos; n = 6 treated ePit-p21 ones. Unpaired 2-tailed Mann-Whitney test was used. (G–H) Same as panel E and F, for body
and placental weight, n = 6 treated ePit-p21 embryos; n = 19 untreated and 9 treated ePit-Col-p21 embryos. Two-way ANOVA with Conceptus part and Treatment as
variables was used in panel G. p-Values for Treatment (bottom) and for Sidak’s post hoc tests (top) are shown. Unpaired 2-tailed Mann-Whitney test was used in panel
H. (I–J) Same as panel E and F, for placenta/body weight ratio, normalized to the average control littermate (n = 6 treated ePit-p21 embryos; n = 19 untreated and 9
treated ePit-Col-p21 embryos). Unpaired 2-tailed Mann-Whitney test was used. (K) Femur length/body weight ratio of untreated or Leu27-IGF2-treated E17.5 ePit-p21
and ePit-Col-p21 embryos, normalized to the average control littermate (n = 5 untreated and n = 6 treated Control; n = 6 untreated and n = 8 treated Exp embryos). For
each treatment, comparisons by unpaired Mann-Whitney test are shown. The systemic growth reduction of ePit-Col-p21 embryos involves impaired
placental function and when it is rescued, limb–body proportions are
altered We reasoned that the most likely foetal organ to control systemic growth by responding to a
circulating alarm signal is the placenta because in rodents it produces higher IGF levels than
any other organ [28] and is considered the main organ controlling foetal growth [29], whereas
hepatic IGFs regulate systemic growth mainly after weaning [18]. Placental weight was not
diminished in ePit-Col-p21 embryos (n = 19) as compared to ePit-p21 controls (n = 17), such
that the placenta/body weight ratio was increased (Fig 6B). This result suggests that placental
efficiency is reduced in response to the left-cartilage p21 insult. To determine the status of pla-
cental IGF signalling, we tested the expression of several pathway members by qRT-PCR and
found that Igf2 levels were increased in the placentas of ePit-Col-p21 embryos as compared to
ePit-p21 controls, whereas the levels of Igf1r did not vary significantly (Fig 6C; n = 3 experi-
mental and n = 4 control). Increased expression of IGF2 by the placenta has been seen as part
of a placental stress response triggered by prenatal insults such as alcohol exposure, which is
also associated with placental functional impairment, increased placental/body weight ratio,
and foetal growth restriction [30,31]. Therefore, one possible interpretation of our results is
that the left limb cartilage stress response is relayed to the placenta, which then indirectly PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 12 / 28 https://doi.org/10.1371/journal.pbio.2005086.g006 Because the body size was “over-rescued” as compared to the bones, the femur/body weight
ratio of rescued ePit-Col-p21 embryos was diminished compared to ePit-p21 littermates or
untreated litters (Fig 6K, n = 5 untreated and n = 6 treated ePit-p21 embryos; n = 6
untreated and n = 8 treated ePit-Col-p21). Taken together, these results suggest that, in the
presence of local growth defects, a systemic growth reduction is necessary to maintain
limb–body proportions. Unexpectedly, rescue of the systemic effect did not result in left–
right asymmetry in ePit-Col-p21 embryos (Fig 6L), indicating that a specific decrease in
growth of the unmanipulated limb, which is independent of placental function, contributes
to the maintenance of left–right symmetry upon a unilateral insult. 3. Because the body size was “over-rescued” as compared to the bones, the femur/body weight
ratio of rescued ePit-Col-p21 embryos was diminished compared to ePit-p21 littermates or
untreated litters (Fig 6K, n = 5 untreated and n = 6 treated ePit-p21 embryos; n = 6
untreated and n = 8 treated ePit-Col-p21). Taken together, these results suggest that, in the
presence of local growth defects, a systemic growth reduction is necessary to maintain
limb–body proportions. Unexpectedly, rescue of the systemic effect did not result in left–
right asymmetry in ePit-Col-p21 embryos (Fig 6L), indicating that a specific decrease in
growth of the unmanipulated limb, which is independent of placental function, contributes
to the maintenance of left–right symmetry upon a unilateral insult. (L) Left/right ratio of femur length for E17.5 ePit-p21 and ePit-Col-p21 embryos from Leu27-
IGF2-treated (n = 6 Control and n = 8 Exp) and -untreated litters (n = 7 Control and n = 15 Exp). p-Values (2-way ANOVA) for Treatment and Genotype are shown. For panel B through L, see also S3 Data. E, embryonic day; Exp, Experimental; IGF2, insulin-like growth factor 2; qRT-PCR, quantitative real-time polymerase chain
reaction. 13 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms impacts on foetal growth. Perhaps explaining why increased IGF2 expression does not corre-
late with enhanced embryo growth, we found an increase in the level of Igf2r (Fig 6C; n = 6
experimental and n = 6 control), which encodes a decoy receptor that can decrease local IGF2
availability [32]. Furthermore, inhibition of IGF2R has been shown to boost placental effi-
ciency [33]. As a means to test whether the systemic growth reduction in ePit-Col-p21 embryos was due
to impaired IGF signalling within the placenta, we injected pregnant dams (from E15.25 to
E17.25, 3 times per day, Fig 6D) with an IGF2 analogue (Leu27-IGF2) that does not cross the pla-
cental barrier, can only bind IGF2R (and not IGF1R), and thus was shown to increase placental
efficiency [33]. Body and placental weight and femur length of ePit-Col-p21 embryos were com-
pared between litters that were either treated or not treated with Leu27-IGF2 to determine the
degree to which body/organ size was rescued, and they were also compared with ePit-p21 embryos
within treated litters to determine whether Leu27-IGF2 differentially affected experimental and
control embryo growth. Boosting placental function led to the following results: 1. Femur length of treated ePit-Col-p21 embryos was significantly increased compared to
untreated experimental embryos (n = 9 and n = 11, respectively), and within treated litters,
femur length was not significantly different between ePit-Col-p21 and ePit-p21 littermates
(n = 6), demonstrating preferential rescue of the mutant embryos (Fig 6E and 6F). 2. Whereas placental weight remained unchanged following Leu27-IGF2 administration, body
weight of treated ePit-Col-p21 embryos was significantly increased compared to untreated
experimental embryos (n = 9 and n = 19), and within-litter comparison revealed that body
weight was not only rescued but also slightly increased beyond that of ePit-p21 littermates
(n = 6), suggesting that ePit-Col-p21 embryos are especially sensitized to the systemic
growth factors produced by the placenta (Fig 6G and 6H). Consequently, the placenta/body
ratio was notably reduced in treated versus untreated ePit-Col-p21 embryos, to levels even
lower than in treated ePit-p21 littermates (Fig 6I and 6J). 3. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Compensatory proliferation We have shown that a few days into mosaic inhibition of proliferation affecting >35% of chon-
drocytes of the left limb, spared chondrocytes undergo increased proliferation, such that the
overall proliferative rate in the left cartilage almost matches that of the right limb. We propose
the following order of events, based on correlative data from our study: 1. In response to a reduction in the number of chondrocytes produced, extracellular matrix
production is increased (and thus cell density is reduced). As a consequence, the amount of
cartilage scaffold being laid down is not reduced, and thus endochondral ossification can
proceed at an almost normal rate. This proposed response fits with stereological studies
that showed that, in growth plates with low proliferative rates, it is mainly the production of
extracellular matrix that contributes to bone growth [34]. In this regard, the small decrease
in chondrocyte density seen in Pit-tTA-p21 mice could explain why these mice do not show
increased left–right asymmetry despite lacking compensatory proliferation and systemic
growth reduction (S4F and S4G Fig). 2. When the insult is extensive enough for cell density to drop below a certain threshold, com-
pensatory proliferation is triggered in spared chondrocytes, to an extent proportional to the
number of affected chondrocytes. Because we found that the extent of compensatory prolif-
eration does not linearly correlate with cell density—but it does with the proportion of p21+
chondrocytes—we posit that density plays a permissive rather than an instructive role and
that stress signals emanating from p21+ chondrocytes are needed as well. These stress sig-
nals could be diffusible molecules or cell–cell interactions, and they could act either in short
range, affecting only cells in their proximity, or in long range, via a self-propagated travel-
ling wave [35] and leading to a whole-organ (i.e., community) effect (S7A and S7B Fig). Moreover, although our ex vivo experiments suggest that compensatory proliferation is a
bone-intrinsic process, we cannot rule out that tissues such as the cartilage–bone interface
or the perichondrium (also present in the cultures) play a role in modulating compensatory
proliferation. To distinguish between all these possibilities, new insult models that allow
triggering of focal insult domains of variable size and positions will be required. 2. When the insult is extensive enough for cell density to drop below a certain threshold, com-
pensatory proliferation is triggered in spared chondrocytes, to an extent proportional to the
number of affected chondrocytes. Compensatory proliferation Because we found that the extent of compensatory prolif-
eration does not linearly correlate with cell density—but it does with the proportion of p21+
chondrocytes—we posit that density plays a permissive rather than an instructive role and
that stress signals emanating from p21+ chondrocytes are needed as well. These stress sig-
nals could be diffusible molecules or cell–cell interactions, and they could act either in short
range, affecting only cells in their proximity, or in long range, via a self-propagated travel-
ling wave [35] and leading to a whole-organ (i.e., community) effect (S7A and S7B Fig). Moreover, although our ex vivo experiments suggest that compensatory proliferation is a
bone-intrinsic process, we cannot rule out that tissues such as the cartilage–bone interface
or the perichondrium (also present in the cultures) play a role in modulating compensatory
proliferation. To distinguish between all these possibilities, new insult models that allow
triggering of focal insult domains of variable size and positions will be required. 3. Once p21+ chondrocytes have been replaced by p21−cells and the threshold cell density has
recovered, compensatory proliferation stops, such that overgrowth is not generated. Long-bone catch-up growth by local and systemic mechanisms autonomous [9,11,20]. Therefore, our results introduce a new conceptual framework for inter-
preting studies of perturbed long-bone growth. Furthermore, the experimental approach we
devised for the study of growth regulation in mice makes a strong case for using unilateral pertur-
bation approaches when studying bilateral organs. Although a local response such as compensa-
tory proliferation or reduced cell density could have been unveiled with a mosaic bilateral injury,
a subtle body-weight effect would likely be ascribed to the reduced size of all limbs and not to
inter-organ communication. Indeed, the hint that prompted us to explore inter-organ communi-
cation was the observed reduction in the unmanipulated limb between experimental and control
mice. Below, we discuss the potential mechanisms and evolutionary conservation of local and sys-
temic responses to developmental injury. A holistic view of compensatory responses triggered by developmental
insults In summary, our results show that when the embryonic long bones experience mosaic inhibition
of chondrocyte proliferation, an adaptive growth response can be triggered that involves cell-non-
autonomous local mechanisms and systemic changes during the time frame of the insult, such
that body proportions are maintained. We refer to this new type of catch-up growth that happens
during an on-going insult as ‘adaptive growth’ (S7 Fig). Our finding that a local compensatory
response occurs during the insult and involves cell-nonautonomous mechanisms is distinct from
previous models that proposed that compensation occurs after the insult is lifted and is cell- PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 14 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Left–right limb crosstalk An unexpected result of our study is that when placental function is boosted in ePit-Col-p21 con-
cepti through maternal Leu27-IGF2 treatment, long-bone growth is not enhanced to the same ex-
tent as body weight, resulting in a reduction in the ratio of bone length to body weight (Fig 6). Given that the right cartilage templates are not experiencing the same p21 insult as the left ones, the
dampened response of the right skeletal elements to the systemic rescue suggests that the insult in
the left cartilage influences growth of the right limb through some sort of left–right crosstalk. A sim-
ilar crosstalk has been previously proposed in studies of amphibian limb regeneration, in which it
was shown that amputation of the contralateral limb at the same rostrocaudal level as the originally
amputated limb reduced the regenerative rate of the latter, whereas ipsilateral or contralateral am-
putation at a different rostrocaudal level did not [38]. Moreover, a study of tibial fracture repair in
young rats showed that the healing environment of a fractured bone triggers the release of growth-
promoting signals in the growth plate of the fractured bone and that the same signalling is induced
in the contralateral growth plate [39]. As previously proposed [38], the most obvious candidate sys-
tem to mediate crosstalk between the left and right limbs is the nervous system. While the exact
mechanism remains to be determined, a recent study showed that peripheral sympathetic nerves
might inhibit bone growth in response to sustained social stress [40]. Regardless of the mechanism,
these results suggest that the observed systemic growth reduction in ePit-Col-p21 embryos is a com-
bination of 2 effects: reduced growth efficiency of the contralateral bones in response to the left-spe-
cific insult, and impairment of placental function that affects the rest of the body. Systemic growth reduction Our results reveal a mild but consistent systemic growth reduction (approximately 10%) in
response to a local insult in the cartilage. We propose that the stress response generated in the
left cartilage is somehow communicated to the placenta, which in turn systemically reduces 15 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms growth (S7 Fig). We suggest the following 2 mechanisms that could account for the injured
cartilage–placenta communication: 1. Stress signals are produced locally—either by p21+ chondrocytes, p21−chondrocytes, the
perichondrium, or cells at the cartilage–bone interface—are released into circulation, and,
when they surpass a threshold, impact on placental efficiency (S7C Fig). It was recently
shown that a subpopulation of natural killer (NK) cells that are transiently abundant in the
decidual region of the placenta can promote placental function and foetal growth [36]. If
the function of these cells was impaired by the circulating stress signals in ePit-Col-p21
embryos, this could negatively impact on placental function and explain the systemic
growth reduction. 2. There is a size-monitoring system that detects impaired long-bone growth and leads to a
systemic growth reduction that allows the impaired organ to keep up with the rest of the
body. Such a mechanism would likely require a central integrator where size-for-age infor-
mation is stored and compared to actual organ size, but as of yet, there is almost no evi-
dence for such a system (see discussion in [13]). With regards to potential growth correction treatments, it would be important to determine
whether all ePit-Col-p21 organs are equally reduced or whether the musculoskeletal system
(which is especially dependent on IGF signalling) is primarily affected. Resolution of the latter
question is currently difficult because the embryos are too small for individual organs to be
weighed reliably. Volumetric analyses using mesoscopic techniques such as optical projection
tomography [37] on embryos expressing fluorescent reporters in the tissue of interest will be
necessary to achieve the necessary level of resolution. Ethics statement All animal studies were performed under an approved Institutional Animal Care and Use
Committee mouse protocol (#07-01-001) according to MSKCC institutional guidelines. Study design To correct for interlitter variability when studying the effect of p21 misexpression on systemic
growth, we normalized each measurement from an experimental animal (ePit-Col-p21 or Pit-
tTA-p21) to the average measurement for its control littermates (ePit-p21 or Pit-p21). This is
important because the absolute measurements vary significantly between litters, in part
because they differ in exact developmental stage, number of embryos, and age of the mother
[36]. For paired measurements, the use of left/right ratios allowed for intra-individual normali-
zation. For each experiment, the minimum sample size was estimated using an online tool
(http://powerandsamplesize.com/Calculators), based on the average SD observed in pilot
experiments, to achieve an effect size of 0.03 in the left/right bone length ratio, 0.5 in the left/
right ratio of EdU incorporation, or 10% in normalized systemic measurements, with a power
of 0.8 and a 95% CI. In Fig 5B and 5C, 2 embryos (one from the ePit-Col-p21 and one from the
eCol-tdT populations) were abnormally small, possibly dead, and were excluded from the anal-
ysis. For comparison of qualitative expression, a minimum of 2 specimens per stage and 5
across several stages were used. The investigator measuring bone length was blinded to the
treatment/genotype of the specimens. No blinding was done for other measurements. No ran-
domization was used for animal processing. Evolutionary conservation of compensatory mechanisms Collectively, our results reveal that the processes leading to coordination of growth within and
between organs to achieve normal proportions upon developmental insults are conserved 16 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms across metazoans. However, the magnitude of the contributions of local and systemic mecha-
nisms likely varies across phyla because the extent of the systemic growth reduction observed
in mice seems to be less extreme than in Drosophila, with the caveat that different insults or tis-
sues could elicit distinct responses. The exact underlying mechanisms also vary because we did
not observe up-regulation of the dilp8 homologue Relaxin1 in the insulted cartilage. Different
molecular mechanisms aside, the compensatory response in vertebrates shares some features
with the response in insects, such as our finding that the injured tissue is able to catch up
despite being exposed to an environment that stunts growth of the rest of the body. One expla-
nation for this result is that local compensatory proliferation overrides a systemic effect. We
further speculate that if the same ‘alarm’ signal were to trigger both the intrinsic and systemic
mechanisms following injury, this would provide an evolutionarily advantageous strategy to
achieve robust coordination of organ growth. While many unknowns remain in the field of organ growth and repair, further exploration
of the mechanisms revealed by this study will open exciting new avenues for basic and transla-
tional research and lead to an understanding of human growth disorders. Long-bone catch-up growth by local and systemic mechanisms measurements were compared within experimental animals only, paired 2-tailed t test was
used. For all ANOVA, alpha = 0.05. All relevant parameters for the statistical tests can be
found in S1 Table. When parametric tests were used, data normality was confirmed by Sha-
piro-Wilk test and equality of variance by F-test. Prism7 software (Graphpad) was used for
most analyses. Most graphs show individual values and mean ± SD, unless otherwise
indicated. Dox treatment Dox hyclate (Sigma) was added to the drinking water at a final concentration of 1 mg/ml, with
1% sucrose to increase palatability. Animals To generate the Igs7TRE-LtSL-p21 mouse line, the NruI-STOP-loxP-tdTomato-SnaBI fragment in
the Ai62(TITL-tdT) Flp-in replacement vector [23] was replaced by a custom NruI-tdTomato-
STOP-loxP-MluI-HpaI-SnaBI cassette, to generate an empty DRAGON vector. A PCR-ampli-
fied Kozak-Cdkn1a cassette was subsequently cloned into the MluI and SnaBI sites to generate
the DRAGON-p21 vector. This vector was then used for recombinase-mediated cassette
exchange into Igs7-targeted G4 ES cells [23]. Two successfully targeted clones were injected
into C2J blastocysts to generate chimeras, obtaining 27 chimeric males (out of 30 born) with
75% to 100% chimerism. Two males from each clone were crossed to BL6 albino mice (Charles
River, Wilmington, MA) to assess germline transmission and to establish the new mouse lines. To generate the Col2a1-tTA line, a Kozak-tTA fragment was PCR-amplified from plasmid
pEnt L1L3 tTA-3 (Addgene plasmid #27105, gift of Edward Hsiao) and cloned into a vector
containing the regulatory region of mouse Col2a1 obtained from plasmid p3000i3020Col2a1
[41]. Backbone-free vector DNA was injected into FVB zygotes to generate transgenic lines. Four out of 11 founders transmitted the Col2a1-tTA allele. The progeny of one of those
(founder number 92) expressed the tTA faithfully in the highest percentage of chondrocytes
and was bred with Pitx2-Cre animals to generate breeders for the experiments. Col2a1-tTA mice were maintained on an outbred Swiss Webster background and geno-
typed using primers Col2a1-F (CCAGGGTTTCCTTGATGATG) and tTA-R (GCTACTTGAT
GCTCCTGATCCTCC) and a standard PCR program with 55˚C annealing temperature. The
Pitx2-Cre [25] (kind gift of Dr. H. Hamada), Col2a1-rtTA [26] (kind gift of Dr. K. Posey), Ai9
(R26CAGGS-LSL-tdTomato) [42], and Ai62 (Igs7TRE-LSL-tdTomato) [23] mouse lines were maintained
on an outbred Swiss Webster background and genotyped as previously described. Igs7TRE-LtSL-p21
animals were genotyped like Ai62 mice. Pitx2-Cre/Cre; Col2a1-(r)tTA/+ females and males homo-
zygous for the conditional misexpression allele were crossed to generate experimental and control
animals. Noon of the day of vaginal plug detection was considered E0.5. The equivalent of E19.5
is referred to as P0. Col2a1-tTA mice were maintained on an outbred Swiss Webster background and geno-
typed using primers Col2a1-F (CCAGGGTTTCCTTGATGATG) and tTA-R (GCTACTTGAT
GCTCCTGATCCTCC) and a standard PCR program with 55˚C annealing temperature. The
Pitx2-Cre [25] (kind gift of Dr. H. Hamada), Col2a1-rtTA [26] (kind gift of Dr. K. Posey), Ai9
(R26CAGGS-LSL-tdTomato) [42], and Ai62 (Igs7TRE-LSL-tdTomato) [23] mouse lines were maintained
TRE LtSL 21 Statistics When data were available for control and experimental, a normalized measurement (left/right
ratio or percentage of average control mice) was calculated for both. Between different time
points, the normalized measurements were compared by multiple unpaired t test with Holm-
Sidak correction for multiple comparisons. Within the same time point, comparisons were
done by an unpaired Mann-Whitney test (1 variable and 2 conditions), by 1-way ANOVA (1
variable and 3 conditions), or by 2-way ANOVA (2 variables and 2 or more conditions) fol-
lowing a matched (paired) design when possible (indicated when not). When left and right PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 17 / 28 17 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Sample processing for histology Mouse embryos were euthanized by hypothermia in cold PBS. Mouse pups were euthanized
by decapitation after hypothermia-induced analgesia. Knees (or isolated full tibiae and femora)
were dissected out, skinned, and fixed by immersion in 4% paraformaldehyde (PFA; Electron
Microscopy Sciences) in PBS for 2 d at 4˚C. After several washes with PBS, the tissue was then
cryoprotected, first by brief incubation with a solution of 15% sucrose and then 30% sucrose in
PBS for at least 4 h at 4˚C, and then embedded in Cryomatrix (Thermo) using dry-ice-cold iso-
pentane (Sigma). The knees were oriented sagittally and facing each other, with the tibiae on
the bottom of the block (i.e., closest to the blade when sectioning). Serial 7-micron sections
were collected with a Leica Cryostat on Superfrost slides, allowed to dry for at least 30 min,
and stored at −80˚C until used. For all histological techniques, frozen slides were allowed to
reach room temperature in a closed box, and Cryomatrix was washed away for 15 min with
warm PBS (37˚C). Long-bone catch-up growth by local and systemic mechanisms Note that after 2 d, we consistently observed growth of 19% to 23% in control limbs as com-
pared to the original length. This is less than in vivo (approximately 87% growth between
E15.5 and E17.5), and the main difference seemed to be at the level of the proximal cartilage,
which does not proliferate, likely due to insufficient diffusion of nutrients because it is larger
than the distal cartilage. We therefore focused our analysis on the distal cartilage, which at
these stages is expected to contribute one-third of total growth [44], i.e., approximately 29%,
quite similar to the observed growth. Micro-CT scans and measurements Whole femora and tibiae were scanned using a Scanco μCT 35 (Scanco Medical, Bru¨ttisellen,
Switzerland) system. Six-μm voxel size, 45 KVp, 0.36-degree rotation step (180-degree angular
range), and a 400-ms exposure per view were used for the scans, which were performed in air. Scanco μCT software (HP, DECwindows Motif 1.6) was used for 3D reconstruction and view-
ing of images. After 3D reconstruction, ‘Distance 3D’ tool was used for measuring the length
of the ossified region. Three measurements were taken and the average derived for each bone. The observer was blinded to the genotype of the mouse. Skeletal preparations and measurements Staining of cartilage and bone was performed as described [45]. Bone length was measured on
digital micrographs using the Line tool in Adobe Photoshop. Unless otherwise indicated, only
the mineralized region was measured. Leu27-IGF2 injections Human Leu27-IGF2 (GroPep, Australia) was prepared at 500 ng/μl in sterile 0.01 N HCl solu-
tion and kept at 4˚C in between injections. From E15.25 to E17.25, the pregnant dam was sub-
cutaneously injected every 8 h, for a total dose of 1 μg/g of body weight per day. Tibial culture A previously described protocol [43] was slightly adapted to culture embryonic long bones. Briefly, E15.5 tibiae were obtained from the embryos of Dox-treated pregnant females, dissected
free of as much soft tissues as possible in ice-cold PBS, and then cultured (at 37˚C, 5% CO2) in
24-well plates with serum-free DMEM (Gibco) containing 0.2% Bovine Serum Albumin (BSA),
0.5 mM L-glutamine, 40 U/ml penicillin/streptomycin (Gibco), 0.05 mg/ml ascorbic acid
(Sigma), and 1 mM betaglycerophosphate (Sigma). The medium additionally contained 1 ng/μl
Dox to maintain transgene expression. After 2 d, the bones were incubated with 10 μM EdU for
90 min, then fixed in PFA and processed for histological analysis. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 18 / 28 Long-bone catch-up growth by local and systemic mechanisms PBSTx with DAPI) was performed for 1 h at room temperature. After PBSTx washes, the slides
were mounted with Fluoro-Gel (Electron Microscopy Sciences). For TUNEL staining, endoge-
nous biotin was blocked after antigen retrieval using the Avidin/Biotin blocking kit (Vector
#SP-2001), and TdT enzyme and Biotin-16-dUTP (Sigma #3333566001 and #11093070910)
were subsequently used following manufacturer instructions. Biotin-tagged DNA nicks were
revealed with Alexa488- or Alexa647-conjugated streptavidin (Molecular Probes, 1/1000) dur-
ing the incubation with the secondary antibody. Antibodies (host species, vendor, catalogue number, dilution) included tdT (rabbit poly-
clonal, Rockland #600-401-379, 1/500), p21 (rabbit polyclonal, Santa Cruz Biotechnology #sc-
471, 1/300), p19Arf (rat monoclonal, clone 12-A1-1, Novus Biologicals #NB200-169, 1/100),
and p16-INK4A (rabbit polyclonal, Proteintech #10883-1-AP, 1/300). Imaging For in vivo experiments, sagittal sections of the knees were imaged, focusing on the region
between both menisci and analysing at least 2, and typically 4, sections per limb. For cultured
distal tibiae, frontal sections were used because they allow for better identification of the differ-
ent epiphyseal regions. The transition between round (resting) and flat (columnar) nuclei,
roughly describing an arch between the upper point of the wedges formed by the groove of
Ranvier, was chosen as the start of the PZ, while the transition towards bigger, more spaced
nuclei (pre-hypertrophic) was chosen as the end of the PZ. The point where pericellular matrix
is sharply reduced around enlarging chondrocytes was considered as the start of the HZ, while
the end of the HZ was marked as the distal end of the last intact chondrocyte. Bright-field and
fluorescence images were taken on a Zeiss inverted microscope (Observer.Z1) using Axiovi-
sion software (Zeiss). Mosaic pictures were automatically reconstructed from individual 10×
(bright-field) or 20× (fluorescence) tiles. In situ hybridisation The protocol described in [46] was followed. For embryos and newborns, samples were not
decalcified. Except for Col2a1, Col10a1, and Ihh (provided by Dr. Licia Selleri), the templates for
most riboprobes were generated by PCR from embryonic cDNA, using primers containing the
SP6 or T7 RNA polymerase promoters. Sequence of the primers is available upon request. After
purification of the PCR product (Qiagen PCR purification kit), DIG-labelled probes were tran-
scribed following manufacturer instructions (Roche), treated with DNAase for 30 min, and
purified by LiCl-mediated precipitation in alcoholic solvent. Probes were kept at −80˚C in 50%
formamide (Fluka). For immunohistochemistry after in situ hybridisation, sections were incu-
bated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15 min at 90˚C, allowed
to cool down, washed with PBSTx, and incubated with 1% H2O2 in PBSTx for 1 h to block
endogenous peroxidases. After BSA blocking and primary antibody incubation, endogenous
biotin was blocked using Avidin/Biotin Blocking kit (Vector #SP-2001), and then the slides
were incubated with a biotinylated secondary antibody. A brown precipitate was obtained using
a peroxidase-coupled streptavidin-biotin complex (Vectastain Elite ABC Kit, Vector #PK-6100)
and DAB substrate (Vector #SK-4100), following manufacturer instructions. Immunohistochemistry and TUNEL Sections were incubated in citrate buffer (10 mM citric acid, 0.05% Tween 20 [pH 6.0]) for 15
min at 90˚C, allowed to cool down, washed with PBSTx (PBS containing 0.1% Triton X-100),
blocked with 5% BSA in PBSTx for 30 min at room temperature, and incubated with the pri-
mary antibody over night at 4˚C (see list of antibodies below). After PBSTx washes, incubation
with Alexa647- and/or Alexa555-conjugated secondary antibodies (Molecular Probes; 1/500 in PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 19 / 28 EdU incorporation Five mg/ml EdU in PBS was injected (50μg/g body weight, s.c for pups, i.p. for adults and preg-
nant females) 1.5 h before euthanizing the mice. EdU was detected using the Click-iT
Alexa488 Imaging Kit (Invitrogen, C-10337) once the immunohistochemistry and/or TUNEL
staining were finished on the same slides. The fraction of nuclei that were positive for EdU, 20 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Cell size analysis The PZ was cropped from 20×-imaged sections of left and right ePit-Col-p21 proximal tibial
cartilage. tdT+ chondrocytes were segmented and counted, and their individual area was mea-
sured using Cell Profiler. Long-bone catch-up growth by local and systemic mechanisms Table 1. Sequence of the oligonucleotides used for qRT-PCR. Primer name
Sequence 5’ ! 3’
qPCR Cdkn1a F
CCTGGTGATGTCCGACCTG
qPCR Cdkn1a R
CCATGAGCGCATCGCAATC
qPCR Arc F
AAGTGCCGAGCTGAGATGC
qPCR Arc R
CGACCTGTGCAACCCTTTC
qPCR Il17ra F
AGTGTTTCCTCTACCCAGCAC
qPCR Il17ra R
GAAAACCGCCACCGCTTAC
qPCR Gapdh F
CCAATGTGTCCGTCGTGGATCT
qPCR Gapdh R
GTTGAAGTCGCAGGAGACAACC
qPCR Igf2 F
GTGCTGCATCGCTGCTTAC
qPCR Igf2 R
ACGTCCCTCTCGGACTTGG
qPCR Igf1r F
GTGGGGGCTCGTGTTTCTC
qPCR Igf1r R
GATCACCGTGCAGTTTTCCA
qPCR Igf2r F
TGAATGGTGATCCTTGCCCTC
qPCR Igf2r R
CCGGTAGCTGTTGGTCTGTC
qPCR Tbp F
GGGAGAATCATGGACCAGAA
qPCR Tbp R
GATGGGAATTCCAGGAGTCA
Abbreviation: qRT-PCR, quantitative real-time polymerase chain reaction. p21, or tdT in the PZ of the cartilage was determined using ImageJ or CellProfiler, followed by
manual curation. Cell density analysis The PZ was cropped from 20×-imaged sections of left and right experimental and control
proximal tibial cartilage, stained for DAPI, p21, and EdU. The area of the region of interest
(PZ) was measured in pixels using the Histogram tool in Adobe Photoshop and converted into
mm2 using the resolution and scale information. The DAPI channel was segmented and quan-
tified using Cell Profiler. Cell density was calculated as the number of chondrocytes per area
unit. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 https://doi.org/10.1371/journal.pbio.2005086.t001 S2 Data. Data for S6 Fig. List of DEGs between left and right ePit-Col-p21 cartilage at
E17.5. The DESeq2 tool (adjusted p 0.05) was used to obtain the list.
(TXT) S2 Data. Data for S6 Fig. List of DEGs between left and right ePit-Col-p21 cartilage at
E17.5. The DESeq2 tool (adjusted p 0.05) was used to obtain the list. (TXT) S3 Data. Data for Figs 1–6 and S1–S6 Figs. Individual values of the measurements presented
throughout the study. (XLSX) qRT-PCR cDNA was synthesized from purified RNA using iScript reverse transcriptase (RT) as
described by the manufacturer (Bio-Rad). Each target was amplified in triplicate to obtain an
average per sample, using SYBR Green (Applied Biosystems) on a StepOnePlus real-time PCR
system (Applied Biosystems). Primer sequences are shown in Table 1. Negative controls (no
template) and no-RT cDNA controls were included for each primer/sample combination. Rel-
ative expression on each sample was calculated by the 2−ΔCT method, with Gapdh (for carti-
lage) or Tbp (for placenta) as a reference. Long-bone catch-up growth by local and systemic mechanisms RNA-seq High-quality RNA was deep sequenced (50 million paired-end reads) by the New York
Genome Center. Aligned reads were analysed using DESeq2 tool in R. A paired design was
used, such that left and right comparison was performed for each specimen, which minimized
the effect of sequencing batch and interspecimen variability. DEGs were obtained using a
threshold of adjusted p-value 0.05. NMF library tools were used to generate heatmaps. Enrichment analysis was performed using DAVID [47] and WebGestalt [48]. Supporting information S1 Data. Data for S2 Fig and S6 Fig. RNA-seq data from left and right ePit-Col-p21 growth
plates. Left (L) and right (R) proliferative and pre-hypertrophic zones from 3 different E17.5
ePit-Col-p21 embryos were dissected and sequenced. Growth plates from femur and tibia were
pooled. Normalized counts are shown. The original numbering (#386–388) was changed to
1–3. (TXT) PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 RNA isolation and analysis The distal left or right femoral and proximal tibial cartilage from E17.5 Pit-Col-p21 em-
bryos was dissected in cold PBS, the condyles and hypertrophic zones removed using a micro-
knife, and the perichondrium removed by a combination of collagenase type II treatment
(Worthington, 2 mg/ml in DMEM, 2 min at room temperature) and mechanical dissection. Left and right cartilage fragments from each embryo (number 1, 2, and 3) were kept in sepa-
rated tubes and flash-frozen in liquid nitrogen. RNA was extracted using Trizol (Invitrogen)
and a mechanical tissue disruptor. 21 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 doxycycline; E, embryonic day; PZ, proliferative zone; tdT, tdTomato.
(TIF) doxycycline; E, embryonic day; PZ, proliferative zone; tdT, tdTomato. (TIF) doxycycline; E, embryonic day; PZ, proliferative zone; tdT, tdTomato. (TIF) S2 Fig. Histological, molecular, and cellular characterization of the effects of p21 misex-
pression. (A–C) The expression of chondrocyte maturation markers Cdkn1c, Col10a1, and Ihh
is not ectopically triggered by p21 misexpression (panel A, B), but their expression is qualita-
tively and quantitatively diminished in the left cartilage (panel C, normalized counts and
adjusted p-value from the RNA-seq experiment of S6 Fig are shown). For panel C, see S1 Data
as well. (D–E) Misexpression of p21 does not lead to cell senescence in the experimental carti-
lage at E17.5 (panel D, monitored by p16 and p19 expression, n = 3), nor to ectopic cell death
at E15.5 or E17.5 (panel E, arrows indicate TUNEL+ cells, n = 5). (F) Hematoxylin–eosin
staining of E15.5 femora and E17.5 proximal tibiae from ePit-Col-p21 embryos. (G) Compari-
son of the length of the left and right proliferative and hypertrophic zones (PZ and HZ) of the
femora from ePit-Col-p21 (n = 4) and ePit-p21 embryos (n = 3) at E15.5 (2-way ANOVA with
Genotype and Side as variables was used, and p-values are shown). (H) Left/right ratios of
EdU+ incorporation in the PZ of ePit-p21 and ePit-Col-p21 embryos at E15.5 (n = 4 and n = 3),
E17.5 (n = 5 and n = 5), and P0 (n = 4 and n = 8). Comparison by 2-way ANOVA for Genotype
and Stage (p-values below graphs). p-Values for Sidak’s post hoc test are shown in the graphs. (I) Cell size of tdT+ (i.e., p21−) chondrocytes was characterized for ePit-Col-p21 embryos at
E17.5 (n = 10, see Materials and methods). Representative pictures of left and right PZ are
shown. No significant difference between left and right distribution was found (p-value for
2-tailed unpaired Mann-Whitney test for ranks is shown). For panel G–I, see S3 Data. E,
embryonic day; HZ, hypertrophic zone; PZ, proliferative zone; RNA-seq, RNA sequencing. (TIF) S3 Fig. Characterization of the tibial culture system. (A) After 2 d of culture, the distal tibial
cartilage does not show signs of senescence, as shown by lack of p16 immunostaining (n = 3). (B) Right tibiae show the same extent of proliferation regardless of whether they are cultured
together (n = 4) or separated (n = 6) from the contralateral tibia. See also S3 Data. (TIF) S3 Fig. S1 Table. Parameters of the statistical tests used in this study.
(XLSX) S1 Fig. Characterization of the left limb-specific intersectional approach to induce tran-
sient growth defects. (A–F’) Pitx2-Cre females were crossed with Ai9 males to characterize the
specificity of Cre-mediated labelling. Seven-μm sections from left and right hindlimbs are
shown at 2 different stages: E12.5 (A–D) and E18.5 (E–F’), n = 4 for each stage. Boxed regions
in panel E and panel F are shown in E’, (E”, and F’. Most of the red signal on right limbs corre-
sponds to autofluorescent blood cells. (G–H’) Dynamics of tdT and CDKN1A (p21) activation
in ePit-Col-p21 embryos, 1 d (G, G’, n = 2) and 2 d (H, H’, n = 3) after Dox administration to
the pregnant female. Boxed regions in panel G and H are shown in G’ and H’. Note that activa-
tion of the transgene starts to be detectable 1 d post Dox administration, but it is not complete
until 2 d post Dox. Asterisks indicate autofluorescent cells. Of note, the Pitx2-Cre allele is con-
sistently left-predominant only when inherited from the female. (I–J”) Same as above, but
E17.5 elbow sections are shown. (K) Intra-individual comparison of the proportion of p21+
nuclei in the left proximal humerus versus left proximal tibia PZ (n = 3). See also S3 Data. p-
Value for 2-tailed paired t test is shown. Cre, recombinase from P1 bacteriophage; Dox, PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 22 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Long-bone catch-up growth by local and systemic mechanisms mice. Comparisons by 2-way ANOVA with Genotype and Bone identity as variables; p-values
are shown. (G) Cell density in left and right PZ of E17.5 Pit-p21 (n = 4) and Pit-tTA-p21 (n =
4) mice. Comparisons were done by 2-way ANOVA with Genotype and Side as variables; p-
values are shown below the graph (p-values for Sidak’s test are shown colour-coded in the
graph). For panel A–G, see also S3 Data. Dox, doxycycline; E, embryonic day; EdU, 5-ethynyl-
2’-deoxyuridine; Exp, experimental; PZ, proliferative zone; tTA, tetracycline transactivator. (TIF) S5 Fig. Micro-CT measurements confirm that transient and local p21 misexpression trig-
gers a systemic growth reduction, which is progressive and not due to leakiness in other
organs. (A–B) Representative 3D reconstruction (panel A) and normalized measurements
(panel B) of E17.5 bones scanned by μCT (n = 7 ePit-p21, n = 13 ePit-Col-p21). (C) Correlation
analysis between the μCT measurements and the measurements done on micrographs of the
same bones (n = 80). Spearman’s correlation coefficient (and 95% CI) is shown. (D) Left panel:
weight of E15.5 and E16.5 ePit-p21 (n = 10 and n = 5) and ePit-Col-p21 (n = 11 and n = 6)
embryos, normalized to the average control littermate and compared by 2-tailed unpaired
Mann-Whitney test. Right panel: comparison of right bone length at P0. n = 4 ePit-p21 and
n = 4 ePit-Col-p21 pups. Comparison by 2-way ANOVA with Bone and Genotype as variables. p-Values for Sidak’s post hoc test are shown on the graph. (E) Analysis of tdT expression in
E17.5 Pitx2-Cre/+; Col2a1-rtTA/+; Igs7TRE-LSL-tdT/+ embryos (Pit-Col-tdT model, Dox at E12.5)
does not reveal spurious activation outside the left cartilage templates (n = 2). The embryos
were bisected sagittally to facilitate sectioning. For panel B–D, see S3 Data. Dox, doxycycline;
E, embryonic day; LFL, left forelimb; RFL, right forelimb; tdT, tdTomato. (TIF) S6 Fig. Transcriptomic comparison of left and right ePit-Col-p21 cartilage. (A) Schematic
of the experimental approach. After dissection and perichondrium removal, left and right car-
tilage elements were deprived of condyles and hypertrophic zone and were flash frozen. Left
and right samples from each embryo were kept separated, and RNA was extracted for deep
sequencing. (B) Unsupervised hierarchical clustering of 6 samples (left and right cartilage
from 3 embryos). Note that each sample is closest to its contralateral one. doxycycline; E, embryonic day; PZ, proliferative zone; tdT, tdTomato.
(TIF) Characterization of the tibial culture system. (A) After 2 d of culture, the distal tibial
cartilage does not show signs of senescence, as shown by lack of p16 immunostaining (n = 3). (B) Right tibiae show the same extent of proliferation regardless of whether they are cultured
together (n = 4) or separated (n = 6) from the contralateral tibia. See also S3 Data. (TIF) S4 Fig. Compensatory proliferation and systemic growth reduction are not detected by
birth when p21 is expressed in less than 35% of chondrocytes. (A) Left: schematic of the new
Col2a1-tTA allele. See ref. [41] for details on the Col2a1 regulatory region used. In the absence
of Dox, the tTA is activated around E12.5 (detected by a germline-recombined reporter Ai62
allele) [23]. Right: percentage of p21+ chondrocytes in the PZ of left proximal tibia of Pit-tTA-
p21 embryos unexposed to Dox, at E15.5, E17.5, and P0 (n = 3, 4, and 3). Comparison by
1-way ANOVA (p = 0.0368), followed by Tukey’s post hoc tests (shown). (B) Left/Right ratio
of EdU incorporation in PZ chondrocytes of Pit-tTA and Pit-tTA-p21 mice at E15.5 (n = 3
each), E17.5 (n = 4 each), and P0 (n = 3 each). Comparison by 2-way ANOVA for Genotype
and Stage (p-values below graphs). (C) Percentage of p21+ or p21−chondrocytes that have
EdU+ nuclei in the PZ in the left and right tibias of E17.5 ePit-p21 (Control) and ePit-Col-p21
(Exp) embryos. p21−cells from Control and Exp mice were compared by 2-way ANOVA with
Side and Genotype as variables (p-values below graphs). n as in panel B. (D) Length of P0 Pit-
p21 (n = 6–10 depending on the bone) and Pit-tTA-p21 (n = 3–7) right bones, normalized to
the average value of control littermates. Comparisons were done by 2-way ANOVA with
Genotype and Bone identity as variables; p-values are shown. (E) Weight of pooled E17.5
and E18.5 Pit-p21 (n = 9) and Pit-tTA-p21 (n = 11) mice, normalized to the average value of
control littermates and compared by unpaired 2-tailed Mann-Whitney test. (F) Left/right
length ratio for femur and tibia from newborn Pit-p21 (n = 10) and Pit-tTA-p21 (n = 3–8) PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 23 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Project administration: Alexandra L. Joyner. Project administration: Alexandra L. Joyner. Resources: Alberto Rosello´-Dı´ez, Linda Madisen, Hongkui Zeng. Supervision: Alexandra L. Joyner. Validation: Alberto Rosello´-Dı´ez. Visualization: Alberto Rosello´-Dı´ez, Se´bastien Bastide. Writing – original draft: Alberto Rosello´-Dı´ez. Writing – review & editing: Alexandra L. Joyner. Acknowledgments We thank the Joyner lab for scientific discussions, D. Stephen for technical support, N.S. Bayin, J.M. Gonza´lez-Rosa and A. Wojcinski for comments on the manuscript, the Mouse
Genetics core at MSKCC for generating chimeras and new transgenic lines, the μCT core at
the Hospital for Special Surgery for μCT scans and measurements, B. de Crombrugghe for the
p3000i3020Col2a1vector and H. Hamada and K. Posey for the Pitx2-Cre and Col2a1-rtTA
mouse lines, respectively. Long-bone catch-up growth by local and systemic mechanisms placental function, causing a systemic reduction in growth (stunting). (TIF) placental function, causing a systemic reduction in growth (stunting). (TIF) (C–D) MA plot
(panel C) and clustered heatmap (panel D) of the 285 DEGs (red dots in panel C) obtained by
a paired DESeq2 design with adjusted p 0.05. (E) Normalized counts for Cdkn1a (p21) and
Rln1 (Relaxin1, the closest vertebrate homologue to dilp8) are shown for each sample. Note
that Rln1 is virtually absent from control and experimental cartilage. See also S1 Data and S2
Data. (F) Overrepresented pathways obtained from the 285 DEGs (FDR < 0.05). Note the
presence of immune response pathways. (G) Normalized counts for the transcripts following a
similar left–right pattern as Cdkn1a. The 4 examples shown are involved in cellular stress re-
sponse [49–52]. For panel C, E, and G, see S1 Data and S2 Data. DEG, differentially expressed
gene; FDR, false discovery rate; MA plot, log ratio (M) versus mean average (A) plot. (TIF) S7 Fig. Potential mechanisms leading to adaptive growth after unilateral mosaic growth
inhibition in long-bone chondrocytes. (A–B) Two alternative mechanisms that could under-
lie compensatory proliferation in response to a stress signal, classified based on whether they
work at the whole growth plate level (panel A, community effect mediated by a self-propagated
travelling wave) or just by proximity to the stress signal (panel B). Coloured outlines identify
chondrocytes producing the stress molecule. Note that in panel A, the self-propagating signal
could be the same as the original stress molecule. t1–t3 refer to subsequent times of the travel-
ling wave. (C) Potential relay of the stress signal into circulation, which in turn impacts on PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 24 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Author Contributions Conceptualization: Alberto Rosello´-Dı´ez, Alexandra L. Joyner. Conceptualization: Alberto Rosello´-Dı´ez, Alexandra L. Joyner. Data curation: Alberto Rosello´-Dı´ez. Formal analysis: Alberto Rosello´-Dı´ez, Se´bastien Bastide. Funding acquisition: Alberto Rosello´-Dı´ez, Alexandra L. Joyner. Investigation: Alberto Rosello´-Dı´ez, Se´bastien Bastide. Methodology: Alberto Rosello´-Dı´ez, Linda Madisen. Project administration: Alexandra L Joyner Funding acquisition: Alberto Rosello´-Dı´ez, Alexandra L. Joyner. Project administration: Alexandra L. Joyner. Long-bone catch-up growth by local and systemic mechanisms 8. Tanner JM (1963) Regulation of Growth in Size in Mammals. Nature 199: 845–850. PMID: 14079891 9. Gafni RI, Weise M, Robrecht DT, Meyers JL, Barnes KM, et al. (2001) Catch-up growth is associated
with delayed senescence of the growth plate in rabbits. Pediatr Res 50: 618–623. https://doi.org/10. 1203/00006450-200111000-00014 PMID: 11641457 10. Prader A, Tanner JM, von Harnack G (1963) Catch-up growth following illness or starvation. An exam-
ple of developmental canalization in man. J Pediatr 62: 646–659. PMID: 13985887 11. Baron J, Klein KO, Colli MJ, Yanovski JA, Novosad JA, et al. (1994) Catch-up growth after glucocorti-
coid excess: a mechanism intrinsic to the growth plate. Endocrinology 135: 1367–1371. https://doi.org/
10.1210/endo.135.4.7925098 PMID: 7925098 12. Kronenberg HM (2003) Developmental regulation of the growth plate. Nature 423: 332–336. https://doi. org/10.1038/nature01657 PMID: 12748651 13. Rosello-Diez A, Joyner AL (2015) Regulation of Long Bone Growth in Vertebrates; It Is Time to Catch
Up. Endocr Rev 36: 646–680. https://doi.org/10.1210/er.2015-1048 PMID: 26485225 14. Kronenberg HM (2007) The role of the perichondrium in fetal bone development. Ann N Y Acad Sci
1116: 59–64. https://doi.org/10.1196/annals.1402.059 PMID: 18083921 15. Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, et al. (2010) Osteoblast precursors, but not
mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev
Cell 19: 329–344. https://doi.org/10.1016/j.devcel.2010.07.010 PMID: 20708594 16. Yang L, Tsang KY, Tang HC, Chan D, Cheah KS (2014) Hypertrophic chondrocytes can become osteo-
blasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci U S A 111: 12097–12102. https://doi.org/10.1073/pnas.1302703111 PMID: 25092332 17. Zhou X, von der Mark K, Henry S, Norton W, Adams H, et al. (2014) Chondrocytes transdifferentiate
into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in
mice. PLoS Genet 10: e1004820. https://doi.org/10.1371/journal.pgen.1004820 PMID: 25474590 18. Stratikopoulos E, Szabolcs M, Dragatsis I, Klinakis A, Efstratiadis A (2008) The hormonal action of
IGF1 in postnatal mouse growth. Proc Natl Acad Sci U S A 105: 19378–19383. https://doi.org/10.1073/
pnas.0809223105 PMID: 19033454 19. Rosello-Diez A, Stephen D, Joyner AL (2017) Altered paracrine signaling from the injured knee joint
impairs postnatal long bone growth. Elife 6. 20. Lui JLC, Nilsson O, Baron J (2011) Growth Plate Senescence and Catch-Up Growth. Cartilage and
Bone Development and Its Disorders 21: 23–29. 21. Lui JC, Forcinito P, Chang M, Chen W, Barnes KM, et al. PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 References 1. Schubiger G (1971) Regeneration, duplication and transdetermination in fragments of the leg disc of
Drosophila melanogaster. Dev Biol 26: 277–295. PMID: 5003476 2. Hussey RG, Thompson WR, Calhoun ET (1927) The Influence of X-Rays on the Development of Dro-
sophila Larvae. Science 66: 65–66. https://doi.org/10.1126/science.66.1698.65 PMID: 17735569 3. Colombani J, Andersen DS, Leopold P (2012) Secreted peptide Dilp8 coordinates Drosophila tissue
growth with developmental timing. Science 336: 582–585. https://doi.org/10.1126/science.1216689
PMID: 22556251 4. Jaszczak JS, Wolpe JB, Dao AQ, Halme A (2015) Nitric Oxide Synthase Regulates Growth Coordina-
tion During Drosophila melanogaster Imaginal Disc Regeneration. Genetics 200: 1219–1228. https://
doi.org/10.1534/genetics.115.178053 PMID: 26081194 5. Vallejo DM, Juarez-Carreno S, Bolivar J, Morante J, Dominguez M (2015) A brain circuit that synchro-
nizes growth and maturation revealed through Dilp8 binding to Lgr3. Science 350: aac6767. https://doi. org/10.1126/science.aac6767 PMID: 26429885 6. Mollereau B, Perez-Garijo A, Bergmann A, Miura M, Gerlitz O, et al. (2013) Compensatory proliferation
and apoptosis-induced proliferation: a need for clarification. Cell Death Differ 20: 181. https://doi.org/
10.1038/cdd.2012.82 PMID: 22722336 7. Diaz-Garcia S, Ahmed S, Baonza A (2016) Analysis of the Function of Apoptosis during Imaginal Wing
Disc Regeneration in Drosophila melanogaster. PLoS ONE 11: e0165554. https://doi.org/10.1371/
journal.pone.0165554 PMID: 27893747 25 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms 30. Joya X, Salat-Batlle J, Velezmoro-Jauregui G, Clave S, Garcia-Algar O, et al. (2015) Prenatal ethanol
exposure and placental hCG and IGF2 expression. Placenta 36: 854–862. https://doi.org/10.1016/j. placenta.2015.05.011 PMID: 26031386 31. Gardebjer EM, Cuffe JS, Pantaleon M, Wlodek ME, Moritz KM (2014) Periconceptional alcohol con-
sumption causes fetal growth restriction and increases glycogen accumulation in the late gestation rat
placenta. Placenta 35: 50–57. https://doi.org/10.1016/j.placenta.2013.10.008 PMID: 24239160 32. Ludwig T, Eggenschwiler J, Fisher P, D’Ercole AJ, Davenport ML, et al. (1996) Mouse mutants lacking
the type 2 IGF receptor (IGF2R) are rescued from perinatal lethality in Igf2 and Igf1r null backgrounds. Dev Biol 177: 517–535. https://doi.org/10.1006/dbio.1996.0182 PMID: 8806828 33. Charnock JC, Dilworth MR, Aplin JD, Sibley CP, Westwood M, et al. (2016) The impact of a human
IGF-II analog ([Leu27]IGF-II) on fetal growth in a mouse model of fetal growth restriction. Am J Physiol
Endocrinol Metab 310: E24–31. https://doi.org/10.1152/ajpendo.00379.2015 PMID: 26530156 34. Farnum CE, Wilsman NJ (2002) Chondrocyte Kinetics in the Growth Plate. In: Shapiro IM, Boyan B,
Anderson HC, editors. The growth plate. Amsterdam; Washington, DC: IOS Press. pp. x, 265 p. 35. Deneke VE, Di Talia S (2018) Chemical waves in cell and developmental biology. J Cell Biol. 36. Fu B, Zhou Y, Ni X, Tong X, Xu X, et al. (2017) Natural Killer Cells Promote Fetal Development through
the Secretion of Growth-Promoting Factors. Immunity 47: 1100–1113 e1106. https://doi.org/10.1016/j. immuni.2017.11.018 PMID: 29262349 37. Sharpe J, Ahlgren U, Perry P, Hill B, Ross A, et al. (2002) Optical projection tomography as a tool for 3D
microscopy and gene expression studies. Science 296: 541–545. https://doi.org/10.1126/science. 1068206 PMID: 11964482 38. Tweedle C (1971) Transneuronal effects on amphibian limb regeneration. J Exp Zool 177: 13–29. https://doi.org/10.1002/jez.1401770104 PMID: 5569236 39. Fischerauer EE, Manninger M, Seles M, Janezic G, Pichler K, et al. (2013) BMP-6 and BMPR-1a are
up-regulated in the growth plate of the fractured tibia. J Orthop Res 31: 357–363. https://doi.org/10. 1002/jor.22238 PMID: 23097200 40. Foertsch S, Haffner-Luntzer M, Kroner J, Gross F, Kaiser K, et al. (2017) Chronic psychosocial stress
disturbs long-bone growth in adolescent mice. Dis Model Mech 10: 1399–1409. https://doi.org/10. 1242/dmm.030916 PMID: 28982680 41. Zhou G, Garofalo S, Mukhopadhyay K, Lefebvre V, Smith CN, et al. (1995) A 182 bp fragment of the
mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice. (2010) Coordinated postnatal down-regulation
of multiple growth-promoting genes: evidence for a genetic program limiting organ growth. FASEB J
24: 3083–3092. https://doi.org/10.1096/fj.09-152835 PMID: 20371622 22. Wit J, Boersma B (2002) Catch-up growth: definition, mechanisms, and models. J Pediatr Endocrinol
Metab 15 Suppl 5: 1229–1241. 23. Madisen L, Garner AR, Shimaoka D, Chuong AS, Klapoetke NC, et al. (2015) Transgenic Mice for Inter-
sectional Targeting of Neural Sensors and Effectors with High Specificity and Performance. Neuron 85:
942–958. https://doi.org/10.1016/j.neuron.2015.02.022 PMID: 25741722 24. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is
a potent inhibitor of G1 cyclin-dependent kinases. Cell 75: 805–816. PMID: 8242751 25. Shiratori H, Yashiro K, Shen MM, Hamada H (2006) Conserved regulation and role of Pitx2 in situs-spe-
cific morphogenesis of visceral organs. Development 133: 3015–3025. https://doi.org/10.1242/dev. 02470 PMID: 16835440 26. Posey KL, Veerisetty AC, Liu P, Wang HR, Poindexter BJ, et al. (2009) An inducible cartilage oligomeric
matrix protein mouse model recapitulates human pseudoachondroplasia phenotype. Am J Pathol 175:
1555–1563. https://doi.org/10.2353/ajpath.2009.090184 PMID: 19762713 27. Garelli A, Gontijo AM, Miguela V, Caparros E, Dominguez M (2012) Imaginal discs secrete insulin-like
peptide 8 to mediate plasticity of growth and maturation. Science 336: 579–582. https://doi.org/10. 1126/science.1216735 PMID: 22556250 28. White V, Jawerbaum A, Mazzucco MB, Gauster M, Desoye G, et al. (2015) Diabetes-associated
changes in the fetal insulin/insulin-like growth factor system are organ specific in rats. Pediatr Res 77:
48–55. https://doi.org/10.1038/pr.2014.139 PMID: 25268143 29. Sferruzzi-Perri AN, Sandovici I, Constancia M, Fowden AL (2017) Placental phenotype and the insulin-
like growth factors: resource allocation to fetal growth. J Physiol 595: 5057–5093. https://doi.org/10. 1113/JP273330 PMID: 28337745 26 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 51.
Willenberg HS, Path G, Vogeli TA, Scherbaum WA, Bornstein SR (2002) Role of interleukin-6 in stress
response in normal and tumorous adrenal cells and during chronic inflammation. Ann N Y Acad Sci
966: 304–314. PMID: 12114287 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 52.
Kim SR, Kim HJ, Kim DI, Lee KB, Park HJ, et al. (2015) Blockade of Interplay between IL-17A and
Endoplasmic Reticulum Stress Attenuates LPS-Induced Lung Injury. Theranostics 5: 1343–1362.
https://doi.org/10.7150/thno.11685 PMID: 26516372 J
Cell Sci 108 (Pt 12): 3677–3684. 42. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, et al. (2010) A robust and high-throughput
Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13: 133–140. https://doi.org/10.1038/nn.2467 PMID: 20023653 43. Agoston H, Khan S, James CG, Gillespie JR, Serra R, et al. (2007) C-type natriuretic peptide regulates
endochondral bone growth through p38 MAP kinase-dependent and -independent pathways. BMC Dev
Biol 7: 18. https://doi.org/10.1186/1471-213X-7-18 PMID: 17374144 44. Stern T, Aviram R, Rot C, Galili T, Sharir A, et al. (2015) Isometric Scaling in Developing Long Bones Is
Achieved by an Optimal Epiphyseal Growth Balance. PLoS Biol 13: e1002212. https://doi.org/10.1371/
journal.pbio.1002212 PMID: 26241802 45. Rigueur D, Lyons KM (2014) Whole-mount skeletal staining. Methods Mol Biol 1130: 113–121. https://
doi.org/10.1007/978-1-62703-989-5_9 PMID: 24482169 46. Nomura S, Hirota S (2003) In Situ Hybridization of Bone and Cartilage. In: An YH, Martin KL, editors. Handbook of Histology Methods for Bone and Cartilage. Totowa, NJ: Humana Press. pp. 321–327. 47. Huang da W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists
using DAVID bioinformatics resources. Nat Protoc 4: 44–57. https://doi.org/10.1038/nprot.2008.211
PMID: 19131956 48. Zhang B, Kirov S, Snoddy J (2005) WebGestalt: an integrated system for exploring gene sets in various
biological contexts. Nucleic Acids Res 33: W741–748. https://doi.org/10.1093/nar/gki475 PMID:
15980575 49. Zhang F, Kumano M, Beraldi E, Fazli L, Du C, et al. (2014) Clusterin facilitates stress-induced lipidation
of LC3 and autophagosome biogenesis to enhance cancer cell survival. Nat Commun 5: 5775. https://
doi.org/10.1038/ncomms6775 PMID: 25503391 50. Ons S, Marti O, Armario A (2004) Stress-induced activation of the immediate early gene Arc (activity-
regulated cytoskeleton-associated protein) is restricted to telencephalic areas in the rat brain: relation-
ship to c-fos mRNA. J Neurochem 89: 1111–1118. https://doi.org/10.1111/j.1471-4159.2004.02396.x
PMID: 15147503 27 / 28 PLOS Biology | https://doi.org/10.1371/journal.pbio.2005086
June 26, 2018 Long-bone catch-up growth by local and systemic mechanisms Long-bone catch-up growth by local and systemic mechanisms 28 / 28
|
https://openalex.org/W4281715005
|
https://www.researchsquare.com/article/rs-1513250/latest.pdf
|
English
| null |
Climate Change, Food Choices, and the Theory of Behavioral Choice
|
Research Square (Research Square)
| 2,022
|
cc-by
| 9,551
|
1. Introduction Climate change is driven in large part by the use of fossil fuels, and this has increased the level of
greenhouse gases to historic levels of over 400 parts per million. In multiple ways, climate change will
affect every person and animal on the planet. Addressing the vast and varied environmental, social, and
economic consequences of climate change is one of the great challenges the world faces in the 21st
century (Swim et al. 2009). An overwhelming majority of scientists agree that global warming is occurring,
and that it is predominantly caused by human activities (Cook et al. 2016; Pachauri and Meyer 2014). Unsurprisingly, therefore, many researchers have focused their efforts on understanding the significant
effect that altering human behavior can have on strategies to reduce the negative effects of climate
change (e.g. Schultz and Kaiser 2012). Despite efforts in many quarters that have led to increased environmental awareness and education (Steg
and Gifford 2005), greenhouse gas emissions still trend upward. One often-overlooked area where human
behavior change can have an important impact on climate change is food consumption (Rees et al. 2018). Food consumption – particularly of animal-based products – accounts for a significant amount of
both global water use and greenhouse gas (GHG) emissions (Reisch et al. 2013). In an analysis of over 60
studies, Aleksandrowicz et al. (2016) found a median decrease in GHG emissions across all sustainable
diets, with the strongest effect observed in the diets that most reduced meat consumption. Furthermore,
reducing meat consumption is a particularly useful area of sustainability to focus on when studying
behavioral change, because it has an especially high potential for reducing GHG emissions on an
individual and household level (Lacroix and Gifford 2020; Stehfest et al. 2009). To help address this
threat, the present study’s goal is to improve understanding of the gap between harboring good
environmental intentions and weak mitigative action within the food-choice domain. Despite efforts in many quarters that have led to increased environmental awareness and education (Steg
and Gifford 2005), greenhouse gas emissions still trend upward. One often-overlooked area where human
behavior change can have an important impact on climate change is food consumption (Rees et al. The disparity between choosing climate-negative behaviors despite awareness of long-term consequence,
is a significant challenge (Joireman et al. 2004). Pro-environmental values do not necessarily translate
into employed, actionable strategies (e.g. Aitken et al. 2016; Gifford 2011). Research Article Posted Date: June 7th, 2022 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/26 Page 1/26 Page 1/26 Page 1/26 Abstract Food choices are an important aspect of climate change mitigation. How best to account for them? The
dietary change intentions of 454 adults in the United States and Canada were predicted from the theory of
planned behavior (TPB) and the recently proposed theory of behavioral choice (TBC), using an online
survey. The TBC accounted for a statistically significant greater proportion of variance than the TPB (83
versus 61 percent) in explaining the respondents’ intentions. The strongest TBC predictor of intention was
felt obligation, followed by social norms, affect, and habitual behavior. Three interactions among the TBC
predictors also contributed, in small but significant ways, in accounting for the food choice intentions. Translational policy implications are discussed. 1. Introduction An important step toward
mitigation is the development of a theory that can provide a sound basis for effective policy. The theory of
planned behavior (TPB; Ajzen 1991, 2011) has been widely used in other areas of psychology and has Page 2/26 Page 2/26 Page 2/26 served as a reasonable framework for addressing pro-environmental behavior (PEB). It was developed as
an enhancement of theory of reasoned action (TRA), adding perceived behavioral control as a direct, and
indirect, mediator of behavior. This helped to make the TPB appropriate for studying PEB and, indeed, it
has been shown to be a stronger predictor of pro-environmental behavior achievement than the original
TRA (Rossi and Armstrong 1999). served as a reasonable framework for addressing pro-environmental behavior (PEB). It was developed as
an enhancement of theory of reasoned action (TRA), adding perceived behavioral control as a direct, and
indirect, mediator of behavior. This helped to make the TPB appropriate for studying PEB and, indeed, it
has been shown to be a stronger predictor of pro-environmental behavior achievement than the original
TRA (Rossi and Armstrong 1999). In the TPB, intentions are said to be influenced by three factors: attitude toward the behavior, subjective
norms, and perceived control over one’s behavior, each of which is further divided into sub-categories
(Ajzen 1991). Successful attempts to expand the TPB have been made, for example, to help account for transportation
choices (e.g. Heath and Gifford 2002). However, given the ubiquitous gulf between intentions and
behavior, the time has come specifically deal with the intention = behavior gap. To do so, the recently
proposed theory of behavioral choice (TBC; Gifford Lacroix and Chen 2018) suggests accounting for the
gap by adding structural and psychological barriers as potential explanations of the intention-behavior
chasm, as well as three added antecedent predictors (Figure 2). Some of the intention-behavior gap can be explained by structural barriers – those that individuals truly
are unable to overcome themselves, such as a physical disability, low income, or the non-existence of local
public transportation. However, many individuals are objectively capable of making pro-environmental
changes to their behavior, but do not, or not as much as they can, because of what might be called
psychological barriers, justifications, or rationalizations (Gifford 2011). Understanding the psychological
barriers that prevent people from achieving their pro-environmental intentions is necessary to develop the
interventions needed to effect meaningful and successful behavioral change. 2.1 Participants Using the Turk Prime platform, 664 participants from the United States and Canada completed the online
survey. Some were removed from the sample for a number of reasons: 50 failed the validated questions
and 160 “speeders” completed the survey unreasonably quickly. Thus, the final sample size included 454
validated participants. Their mean age was 42.63 years (SD = 12.56 years), and the sample included 191
males (42 %) and 262 (57.7 %) females. A few participants (n = 8, 1.8 %) chose not to disclose their level
of education, 110 participants had a high school diploma (24.2 %), 104 had a college diploma (22.9 %),
163 had a bachelor’s degree (25.9 %), and the rest had a Master’s degree (n = 59 or 13%) or a doctorate
degree (n = 9 or 2 %). Their political ideology was slightly left leaning on average (M = 2.76on a 5-point
scale from “strong left” to “strong right”). 1. Introduction In order to better explain the remaining gap between pro-environmental intentions and actually completed
actions, a taxonomy of psychological barriers called the “dragons of inaction” was proposed (Gifford
2011). Subsequent empirical research has resulted in the development of the Dragons of Inaction
Psychological Barrier (DIPB) scale, comprised of five scales that incorporate about two dozen of the
dragon species: Change Unnecessary, Conflicting Goals and Aspirations, Interpersonal Relations, Lacking
Knowledge, and Tokenism (Lacroix Gifford Chen 2019). More recently, to incorporate these barriers into a model, the theory of behavioral choice (TBC; see Figure
2; Gifford Lacroix Chen 2018) was proposed. The TBC shares some elements with the theory of planned
behavior (TPB) but adds the following elements: values (as an enhancement to the TPB’s attitude toward
the behavior), affect, habit, and felt obligation (as predictors of intention), psychological barriers and
structural barriers (as moderators of the success or failure of intentions), and reported behavior (as
distinct from observed behavior). The new predictors emerged from a preliminary qualitative study in
which 65 respondents were asked to freely suggest reasons why they would consider making an
important new choice in their lives. The TBC aims to improve the prediction of behavior by adding these
constructs, with the aim of striking a productive balance between comprehensiveness and parsimony. Page 3/26 Page 3/26 Page 3/26 The TBC strives to answer the often-asked question of why, if individuals profess to care about the
environment, they do not more actively change their behavior to reflect that concern. Developing a clearer
understanding of the influences that encourage or discourage pro-environmental intentions, including the
barriers that prevent those intentions from coming to fruition, is important for achieving success in
changing behavior. The primary purpose of this study was to evaluate the relative strength of the well-established TPB versus
the newly-developed TBC as competing models for predicting pro-environmental intention and behavior. The domain for their comparison of the models in this study is one’s choice to choose a climate-positive
food diet (or not). To be specific, the main hypothesis was that the TBC model will fit the data better than
the TPB model. Another aim of the study was to investigate whether the interaction effects among the
predictors would improve the predictive power. Predictor interactions have recently been examined for TPB
(Kothe Mullan 2015; La Barbera and Ajzen 2020; La Barbera and Ajzen 2021; Steinmetz Davidov and
Schmidt 2011). 2.2 Materials The three predictors of intention in the TPB model were measured on a 7-point “strongly disagree” to
“strongly agree” scale: attitude toward the behavior, using three items (e.g. “Making this change would be
good for the environment”); social norms, using four items (e.g. “Most people who are important to me
think that I should eat a more plant-based diet”); and perceived behavioral control, using four items (e.g. “If
I really want to, I can change my diet to include fewer animal products”). The six predictors of the TBC model were assessed as follows: Attitude Plus: This predictor aims to improve the TPB’s attitude predictor by adding questions about
values. Therefore, it was named “Attitude Plus” to differentiate between these two scales.Eight items were Page 4/26 used to measure attitude plus, the five items from the TBC attitude scale plus, on a scale of 1 ("strongly
disagree") to 7 ("strongly agree"): “My values require me to change my consumption towards more plant-
based foods,” “I believe this dietary change is the right thing to do,” and “Eating fewer animal products is
one of my principles.” used to measure attitude plus, the five items from the TBC attitude scale plus, on a scale of 1 ("strongly
disagree") to 7 ("strongly agree"): “My values require me to change my consumption towards more plant-
based foods,” “I believe this dietary change is the right thing to do,” and “Eating fewer animal products is
one of my principles.” Social Norms: As in the TPB, four items were used: "Most people who are important to me think that I
should eat a more plant-based diet." Respondents were asked to rate these items on scales
indicating strongly disagree (= 1) to strongly agree (= 7). Perceived Behavioral Control: As in the TPB, four items were used to measure perceived behavioral
control: “If I really want to, I can change my diet to include fewer animal products?”, and “Whether or not I
change my eating behavior is up to me.”On a scale of 1 ("strongly disagree") to 7 ("strongly
agree"), respondents were asked to rate these items. 2.2 Materials Habitual Behavior: For assessing habitual behavior, a TBC addition, four items were used: "I have been
eating animal products for such a long time that I'm not sure how I will make this change," and "I am too
much of a creature of habit to actually reduce my consumption of animal products."A 7-point response-
option scaleof 1 to 7 from strongly disagree (= 1) to strongly agree (= 7) was asked to measure these
items. Felt Obligation: Four items were used in this TBC addition. Respondents were asked to rate the following
statements about the obligation they may perceive on a 7-point scale, from 1 = strongly disagree, to 7 =
strongly agree: “I feel obligated to change my eating behavior for the duration of this study", and "For this
study, I ought to change my dietary choices.” Affect: Five items were used in this third TBC addition, to assess emotions about the behavior. Affect: Five items were used in this third TBC addition, to assess emotions about the behavior. Respondents were asked to rate the following statements about their desire to change their behavior on a
7-point scale, with 1 = strongly disagree, and 7 = strongly agree such as: “I would feel delighted to eat a
more plant-based diet.”, and “I am excited to make this dietary change.” Affect: Five items were used in this third TBC addition, to assess emotions about the behavior. Respondents were asked to rate the following statements about their desire to change their behavior on a
7-point scale, with 1 = strongly disagree, and 7 = strongly agree such as: “I would feel delighted to eat a
more plant-based diet.”, and “I am excited to make this dietary change.” Intention was measured using six items (e.g. “Moving toward a plant-based diet over the next two weeks is
something that I intend to do”). Respondents were asked to rate these items on a scale from 1 = strongly
disagree, to 7 = strongly agree. Without positing specific hypotheses, we were also interested in examining whether demographic
variables such as age, gender, level of education, income, and political position might be related to
behavioral intention. The coding of these variables is shown in Table 4. 3. Results 3.1 Means, Standard Deviations, and Reliabilities 3.1 Means, Standard Deviations, and Reliabilities Page 5/26 On average, responses to the intention items were in mid- to high- levels of their 7-point scale (m = 4.43, sd
= 1.51). Among the predictors of intention, respondents reported high levels of the 7-point
scale for attitude (m = 5.07, sd = 1.32) andperceived behavioral control (m = 5.73, sd = .93). The
participants reported mid- to high-levels of attitude plus values (m = 4.70, sd = 1.28), social norms (m =
4.29, sd = 1.29), affect (m = 4.27, sd = 1.45) and obligation (m = 4.32, sd = 1.54). However, participants
reported mid-levels of the 7-point scale for habitual behavior (m = 3.54, sd = 1.47). The internal On average, responses to the intention items were in mid- to high- levels of their 7-point scale (m = 4.43, sd
= 1.51). Among the predictors of intention, respondents reported high levels of the 7-point
scale for attitude (m = 5.07, sd = 1.32) andperceived behavioral control (m = 5.73, sd = .93). The
participants reported mid- to high-levels of attitude plus values (m = 4.70, sd = 1.28), social norms (m =
4.29, sd = 1.29), affect (m = 4.27, sd = 1.45) and obligation (m = 4.32, sd = 1.54). However, participants
reported mid-levels of the 7-point scale for habitual behavior (m = 3.54, sd = 1.47). The internal consistency reliability of the predictor scales was assessed using Cronbach's alpha. They ranged from α =
.76 to .91 (see Table 5). The social norm and perceived behavioral control factors in TBC were identical to
those of the TPB Table 1 Predictors of behavioral intention in the TPB model Page 6/26 Attitude
Mean
SD
Response range
1. I believe eating a more plant-based diet would be…
5.41
1.47
Extremely bad (1)
to Extremely good
(2)
2. I want to make this dietary change because the alternative,
eating more animal products, is worse. 4.60
1.67
Strongly disagree
(1) to
Strongly agree (7)
3. Making this change would be good for the environment. 5.20
1.51
4. On a scale of Bad to Good, to make this behavior change
would be…
5.37
1.46
5. I will move to a plant-based diet simply because it seems like
a good idea. 4.65
1.65
Social Norms
1. 3. Results I will be reducing my consumption of animal products
because others strongly encouraged me to. 4.46
2.01
2. Most people who are important to me think that I should eat
a more plant-based diet. 4.09
1.65
3. I am going to make this change because people around me
would criticize me if I did not try. 4.20
2.24
4. I have been strongly influenced by other people to eat fewer
animal products. 4.41
1.85
Perceived Behavioral Control
1. If I really want to, I can change my diet to include fewer
animal products. 5.71
1.20
2. I am confident that I will be able to eat fewer animal
products. 5.43
1.34
3. I can choose to change my dietary behavior, at least for this
study. 5.46
1.45
4. Whether or not I change my eating behavior is up to me. 6.34
.83
Table 2
Predictors of behavioral intention in the TBC model Page 7/26 Mean
SD
Response range Items Attitude Plus Attitude Plus Attitude Plus
1. I believe that making this dietary change would be…
5.41
1.47
Extremely useless (1)
to Extremely useful
(7)
2. I want to make this dietary change because the
alternative, eating more animal products, is worse. 4.60
1.67
Extremely bad (1) to
Extremely (7) good
3. On a scale of Bad to Good, to make this behavior change
would be…
5.37
1.46
4. Making this change would be good for the environment. 5.20
1.51
Strongly disagree (1)
to
Strongly agree (7)
5. Eating fewer animal products is one of my principles. 3.85
1.69
6. My values require me to change my consumption
towards more plant-based foods. 3.68
1.80
7. I believe this dietary change is the right thing to do. 4.88
1.62
8. I will move to a plant-based diet simply because it seems
like a good idea. 4.65
1.65
Social Norms
1. I will be reducing my consumption of animal products
because others strongly encouraged me to. 4.46
2.01
Strongly disagree (1)
to
Strongly agree (7)
2. Most people who are important to me think that I should
eat a more plant-based diet. 4.09
1.65
3. I am going to make this change because people around
me would criticize me if I did not try. 4.20
2.24
4. I have been strongly influenced by other people to eat
fewer animal products. 4.41
1.85
Perceived Behavioral Control
1. If I really want to, I can change my diet to include fewer
animal products. 5.71
1.20
Strongly disagree (1)
to
Strongly agree (7)
2. I am confident that I will be able to eat fewer animal
products. 5.43
1.34
3. I can choose to change my dietary behavior, at least for
this study. 5.46
1.45
4. Whether or not I change my eating behavior is up to me. 6.34
.83
Habitual Behavior
1. I have been eating animal products for such a long time
that I'm not sure how I will make this change
3.66
1.76 Strongly disagree (1)
to
Strongly agree (7)
2. I am set in my ways, so it's difficult to consider changing
my dietary habits. 3.32
1.62
3. I have to admit that I am so used to my current eating
pattern that this could be a difficult change to make. 3.86
1.72
4. I am too much of a creature of habit to actually reduce
my consumption of animal products. Attitude Plus 3.43
1.68 Felt Obligation
Strongly disagree (1)
to
Strongly agree (7)
1. I feel obligated to change my eating behavior for the
duration of this study. 3.86
1.87
2. I really must change my eating behavior, at least for this
study
4.26
1.85
3. For this study, I ought to change my dietary choices
4.75
1.67
4. Really, I don’t believe that I should change my eating
choices during this study (reversed)
4.43
1.18
Affect
1. I would feel delighted to eat a more plant-based diet
4.25
1.79
Strongly disagree (1)
to
Strongly agree (7)
2. I really don’t think I would enjoy changing my current
eating choices (Reversed)
3.94
1.69
3. I am excited to make this dietary change. 4.23
1.76
4. Attempting to make this change will feel good. 4.81
1.65
5. Changing my diet towards a plant-based diet would be... 5.16
1.63
Extremely
unappetizing (1) to
Extremely appetizing
(7) Felt Obligation Table 3 Intention items Table 4 Demographic variables Table 4 Demographic variables Table 4 Demographic variables Page 9/26 Mean
SD
Response range
1. Moving toward a plant-based diet over the next two weeks
is something that I intend to do. 4.08
1.97
Strongly disagree
(1) to Strongly
agree (7)
2. To see how it goes, my intention is to try to eat a more
plant-based diet for the length of this study. 4.48
1.86
3. I definitely plan to eat more plant-based foods. 4.66
1.76
4. I don’t expect that I will be changing my dietary
choices (reversed)
4.18
1.85
5. My aim, honestly, is to switch toward a more plant-oriented
diet. 4.39
1.78
6. I just can’t see myself trying to change my food
choices (reversed)
4.84
1.83
Response range
Age
-
Gender
Male (1), Female (2), Other (3)
Level of
education
1 = high school graduate, 2 = diploma/technical training, 3 = bachelor degree, 4 =
master's or equivalent degree, 5 = PhD, 6 = other
Income
-
Political
stands
1 = Strong left, 2 = Somewhat left, 3 = Moderate, 4 = Somewhat right, 5 = Strong right
Table 5 Descriptive statistics (N = 454) Mean
SD
Response range
1. Moving toward a plant-based diet over the next two weeks
is something that I intend to do. 4.08
1.97
Strongly disagree
(1) to Strongly
agree (7)
2. Attitude Plus R
d
t
h
h d b
ti
t f
l
ti
h
d
l
d
h bit th t th
f
d it h
d t M
SD
α
TPB
Attitude (1-7)
5.04
1.30
.89
Social norms (1-7)
4.29
1.55
.81
Perceived behavioral control (1-7)
5.73
.93
.76
TBC
Attitude plus values (1-7)
4.70
1.28
.92
Social norms (1-7)
4.29
1.55
.81
Perceived behavioral control (1-7)
5.73
.93
.76
Habitual behavior (1-7)
3.56
1.47
.89
Obligation (1-7)
4.32
1.54
.88
Affect (1-7)
4.27
1.45
.89
Intention (1-7)
4.43
1.51
.91
Recent dietary behavior
12.75
5.75
-
3.2. Relations among the
Predictors and Intention
All the predictors were
significantly related to intent Attitude (1-7) g
y
(see Table 6). A significant positive relation was observed between attitude and intention (r = .72, p <
.001). Social norm was significantly negatively related to intention (r = -.46, p < .001). Respondents who
perceived greater control over their dietary behaviors, tended to report stronger intentions to take
precautionary actions in the future (r = .51, p < .001). Participants with positive attitude and values
towards changing their diet to more plant-based diets expressed stronger intentions (r = .75, p < .001). (see Table 6). A significant positive relation was observed between attitude and intention (r = .72, p <
.001). Social norm was significantly negatively related to intention (r = -.46, p < .001). Respondents who
perceived greater control over their dietary behaviors, tended to report stronger intentions to take
precautionary actions in the future (r = .51, p < .001). Participants with positive attitude and values
towards changing their diet to more plant-based diets expressed stronger intentions (r = .75, p < .001). Respondents who had been eating meat for a long time have developed a habit that they found it hard to
break; they expressed weaker intentions to change their behavior (r = -.52, p < .001). The more that
respondents believed that they were obligated to change their behavior, the stronger their intentions to take
action in the near future (r = .82, p < .001). Those with greater more positive affect about towards
changing their behavior intended more to change toward a plant-based diet (r = .80, p < .001). Finally,
respondents who had recently consumed more animal-based food reported weaker intention to adopt
plant-based diets. Attitude Plus To see how it goes, my intention is to try to eat a more
plant-based diet for the length of this study. 4.48
1.86
3. I definitely plan to eat more plant-based foods. 4.66
1.76
4. I don’t expect that I will be changing my dietary
choices (reversed)
4.18
1.85
5. My aim, honestly, is to switch toward a more plant-oriented
diet. 4.39
1.78
6. I just can’t see myself trying to change my food
choices (reversed)
4.84
1.83
Response range
Age
-
Gender
Male (1), Female (2), Other (3)
Level of
education
1 = high school graduate, 2 = diploma/technical training, 3 = bachelor degree, 4 =
master's or equivalent degree, 5 = PhD, 6 = other
Income
-
Political
stands
1 = Strong left, 2 = Somewhat left, 3 = Moderate, 4 = Somewhat right, 5 = Strong right Table 5 Descriptive statistics (N = 454) Page 10/26 M
SD
α
TPB
Attitude (1-7)
5.04
1.30
.89
Social norms (1-7)
4.29
1.55
.81
Perceived behavioral control (1-7)
5.73
.93
.76
TBC
Attitude plus values (1-7)
4.70
1.28
.92
Social norms (1-7)
4.29
1.55
.81
Perceived behavioral control (1-7)
5.73
.93
.76
Habitual behavior (1-7)
3.56
1.47
.89
Obligation (1-7)
4.32
1.54
.88
Affect (1-7)
4.27
1.45
.89
Intention (1-7)
4.43
1.51
.91
Recent dietary behavior
12.75
5.75
-
3.2. Relations among the
Predictors and Intention
All the predictors were
significantly related to intention
(see Table 6). A significant positive relation was observed between attitude and intention (r = .72, p <
.001). Social norm was significantly negatively related to intention (r = -.46, p < .001). Respondents who
perceived greater control over their dietary behaviors, tended to report stronger intentions to take
precautionary actions in the future (r = .51, p < .001). Participants with positive attitude and values
towards changing their diet to more plant-based diets expressed stronger intentions (r = .75, p < .001). Attitude Plus Page 11/26
Table 6 Correlations among the predictors and intention
Attitude
Social
norms
Perceived
behavioral
control
Attitude
plus
Habitual
behavior
Obligation
Affect
Recent
dietary
behavior
Intention
.72**
-.46**
.51**
.75**
-.52**
.82**
.80**
-.10*
**Significant at the .01 level *Significant at the .05 level. Table 6 Correlations among the predictors and intention **Significant at the .01 level *Significant at the .05 level. Page 11/26
**Significant at the .01 level *Significant at the .05 level. Page 11/26
**Significant at the .01 level *Significant at the .05 level. The main hypothesis was that the TBC model predictors would account for more variance in intention
than would the predictors of TPB model. Linear regression using the Enter method in three steps was
employed to test this assumption. In the first step, recent dietary behavior was entered as a control
variable; a change from eating little (or much) meat recently could importantly affect one’s intention to
change to a plant-based diet. For example, those who were already eating little meat would have less
“room” (intention) to change. In the second step, the TPB predictors were added as a second block, and in
the third step the TBC predictors were entered as a final block. The results were that the TBC accounted for a significantly greater proportion of the variance in
intention [F (6, 455) = 255.45, p < .001, R2
Adjusted = .82] than that of the TBC [F (3, 449) = 202.31, p <
.001, R2
Adjusted = .61]. Thus, the main hypothesis was supported (see Table 7). Among TPB predictors, attitude was the most important predictor of intention [β = .56, p < .001]. The
second-most important predictor was social norms; it had a negative relation to intention [β = -.30, p <
.001]. The least important predictor was perceived behavioral control [β = .18, p < .001], which was
positively correlated with intention. In the TBC model, the most important predictor was felt obligation [β = .41, p < .001]. The more
participants felt obligated to change their behavior towards a plant-based diet, the stronger intention they
reported. Affect was the second-most important predictor [β = .22, p < .001]; it was positively related to
intention. The third-most important predictor was social norms [β = -.16, p < .001]; its correlation was
negative. Attitude Plus Participants tended to deny that adoption of a more plant-based diet would occur under the
influence of their social circle [β = -.16, p < .001]. The fourth-most important variable was habit [β = -.11, p
< .001]. Those who felt trapped in their behavior pattern of consuming meat-based diets were less likely to
change their behavior towards a plant-based diet. Finally, the least-important predictor was perceived
behavioral control [β = .05, p < .001]. Participants who reported being in greater control of their dietary
behaviors reported stronger intentions to change. Interestingly, attitude (TPB) and attitude plus (TBC) were
not significantly related to intention (p > .05; see Table 7). In the TBC model, the most important predictor was felt obligation [β = .41, p < .001]. The more
participants felt obligated to change their behavior towards a plant-based diet, the stronger intention they
reported. Affect was the second-most important predictor [β = .22, p < .001]; it was positively related to
intention. The third-most important predictor was social norms [β = -.16, p < .001]; its correlation was
negative. Participants tended to deny that adoption of a more plant-based diet would occur under the
influence of their social circle [β = -.16, p < .001]. The fourth-most important variable was habit [β = -.11, p
< .001]. Those who felt trapped in their behavior pattern of consuming meat-based diets were less likely to
change their behavior towards a plant-based diet. Finally, the least-important predictor was perceived
behavioral control [β = .05, p < .001]. Participants who reported being in greater control of their dietary
behaviors reported stronger intentions to change. Interestingly, attitude (TPB) and attitude plus (TBC) were
not significantly related to intention (p > .05; see Table 7). Table 7 The prediction of intention Page 12/26 Page 12/26 Page 12/26 Model
Standardized Coefficients
β
t
p
1
Constant
27.68
.00
Recent dietary behavior
-.10
-2.16
.03
2
(Constant)
2.14
.03
Recent dietary behavior
-.02
-.81
.41
Attitude
.56
17.44
.00
Social norms
-.30
-10.34
.00
Perceived behavioral control
.18
5.70
.00
3
(Constant)
4.43
.00
Recent dietary behavior
-.01
-.60
.54
Attitude
.03
.42
.67
Social norm
-.16
-7.18
.00
Perceived behavioral control
.05
2.19
.02
Habitual
-.11
-4.81
.00
Obligation
.41
13.21
.00
Affect
.22
5.91
.00
Attitude plus values
.14
1.75
.08
ole of Interactions among the Predictors 3.3. The Role of Interactions among the Predictors 3.3. The Role of Interactions among the Predictors All possible interactions of the predictors were then computed and their effects on intention were
examined independently with structural equation modeling. Social norms, in conjunction with (each of)
perceived behavioral control, felt obligation, and affect, significantly predicted intention. Affect, interacting
with felt obligation, significantly predicted intention. Finally, the interaction of perceived behavioral control
and habitual behavior predicted intention. Therefore, these five interactions were included in the model-comparison analyses reported earlier. Recent
dietary behavior, as a control variable, was again entered as a first block. The three TPB predictors were
entered in the second block, followed by the six TBC predictors were entered as the third block. Finally, the
five aforementioned TBC interactions were entered in the fourth block. The resulting model was significant
and it accounted for an adjusted 83 percent of the variance in intention [F (13, 440) = 168.69, p < .001,
R2
Adjusted = .83].The variance accounted for by the interactions was small (added R2 = .01, but this was
statistically significant [F (5, 440) = 6.16, p < .001]. R2
Adjusted = .83].The variance accounted for by the interactions was small (added R2 = .01, but this was
statistically significant [F (5, 440) = 6.16, p < .001]. Page 13/26 Page 13/26 This new analysis shifted the overall results slightly. Attitude plus significantly predicted intention once
the interactions had been entered into the analysis, but attitude (TPB) and social norms were no longer
significant. Once they were entered into the full analysis, three of the five interactions (habitual behavior ×
perceived behavioral control, affect × felt obligation, and social norms × perceived behavioral control)
significantly predicted intention. Felt obligation [β = .52, p < .001] remained the most important predictor of food choice intentions. However, perceived behavioral control, which had been the least important predictor in the earlier analysis,
now became the second-most important predictor of intention [β =.41, p < .001]. The third-most important
predictor of intention [β =.36, p < .001] was affect. Next, participants who were more in the habit of
consuming plant-based food reported stronger intentions in the future [β = .25, p < .001]. Attitude plus was
also a significant predictor [β = .16, p < .001]; it was positively related to intention (see Table 8). 3.3. The Role of Interactions among the Predictors Table 8 The prediction of intention including interaction terms Page 14/26 Model
Standardized Coefficients
β
t
p
1
(Constant)
27.68
<.01
Recent dietary behavior
-.10
-2.16
.03
2
(Constant)
2.14
.03
Recent dietary behavior
-.02
-.81
.41
Attitude
.56
17.44
<.01
Social norms
-.30
-10.34
<.01
Perceived behavioral control
.18
5.70
<.01
3
(Constant)
4.43
<.01
Recent dietary behavior
-.01
-.60
.54
Attitude
.03
.42
.67
Social norms
-.16
-7.18
<.01
Perceived behavioral control
.05
2.19
.02
Attitude plus
.14
1.75
.08
Habitual behavior
-.11
-4.81
<.01
Felt obligation
.41
13.21
<.01
Affect
.22
5.91
<.01
4
(Constant)
-2.47
.01
Recent dietary behavior
.00
-.01
.99
Attitude
.02
.38
.70
Social norms
.12
.96
.33
Perceived behavioral control
.41
4.71
<.01
Attitude plus
.16
1.93
.05
Habitual behavior
.25
2.48
.01
Felt obligation
.52
4.70
<.01
Affect
.36
3.22
<.01
Habitual behavior × Perceived behavioral control
-.36
-3.80
<.01 Social norms × Perceived behavioral control
-.42
-2.76
<.01
Affect × Felt obligation
-.37
-3.27
<.01
Social norms × Affect
.04
.45
.65
3.3.1. Inside the Significant Interactions Simple slopes analyses were conducted to examine the nature of the three significant TBC interactions. All
were based on creating three groups: respondents with scores lower than one standard deviation from the
mean, those with scores between minus one and plus one standard deviations from the mean, and those
with scores higher than one standard deviation from the mean. The first interaction, between perceived behavioral control and habitual behavior, is shown in . Intention
weakened for all three groups as respondents believed less that they could not change their habits. Second, those who believed that they had greatest control expressed the strongest intentions to adopt
plant-based diets. Third, those who believed they had the least control reduced their intention to change
their behavior the least; this is the heart of the interaction. Second, those who believed that they had greatest control expressed the strongest intentions to adopt
plant-based diets. Third, those who believed they had the least control reduced their intention to change
their behavior the least; this is the heart of the interaction. The second interaction was between affect and felt obligation. First, as respondents felt more delighted
and excited about the idea of changing their behaviors, their intention to take those steps became
stronger. Second, those with the strongest sense of obligation reported stronger intentions to change their
behavior. 3.3. The Role of Interactions among the Predictors However, this tendency was slightly flatter among respondents who reported the strongest sense
of obligation; this is the heart of the interaction (see Figure 4). The third significant interaction involved social norms and perceived behavioral control. First, intention declined for all groups with stronger perceived norms. This is consistent with the earlier
finding of a strong negative correlation between social norms and intention. Second, intention was
strongest for those who perceived greater perceived behavioral control. However, intention declined faster
for those who perceived greater perceived behavioral control than for others; that is the heart of this
interaction (see Figure 5). 4. Discussion This study’s results show that the theory of behavioral choice (TBC) predicts a significantly greater
proportion of variance in the intention to adopt plant-based behaviors than does the theory of planned
behavior, as hypothesized. The 21 percent increase is partly caused by the TBC’s additional predictor
variables including felt obligation, affect, and habitual behavior. The findings also confirm the small but
significant value of considering interactions between TBC components. Several previous studies have considered adding constructs in order to enhance the TPB model (e.g. Conner and Abraham 2001; Conner and Armitage 1998; Gifford Lacroix and Chen, 2018; Richard van der
Pligt and Vries 1996; Zhang et al. 2020). Most focused on extending the original model by including one
or two additional predictors. The theory of behavioral choice is a more comprehensive framework for
successfully gathering the most important predictors. In particular, felt obligation played an essential role in determining individuals' food-choice intentions. Previous studies have also reported a positive and significant relation between a sense of obligation to
engage in pro‐environmental intentions or behavior (Doran and Larsen 2016; Han Lee and Kim 2018;
Harland Staats and Wilke 1999; Onwezen et al. 2013; Stern 2000). In accordance with the TPB model and other studies’ findings (Croker et al. 2009; Huang et al. 2020;
Zhang et al. 2020; Ong et al. 2021; Rees et al. 2018; Richard, van der Pligt and Vries 1996) social norms
also play a key role in influencing on the intention of individuals to prepare, at least in our first analysis
(that did not include predictor interactions). Social norms and intention were negatively related. Perhaps
this was because participants wished to deny that they were under the influence of others in their social
circles. Many of us would like to think that our decisions are our own. Alternatively, some authors attribute
the absence of a connection between intention and social influence to lack of awareness (Croker et al. 2009) As for affect, many studies find it to be one of most important factors in predicting intention (Ajzen 2011;
Rapaport and Orbell 2000; Richard van der Pligt and Vries, 1996; Wolff et al. 2011). Although breaking one’s habits is often considered the most difficult part of behavior change to overcome,
many climate-relevant behaviors are associated with habit. 3.4. Demographic Variables The respondents’ intentions to adopt plant-based diets was examined for the five demographic variables. Females (r = .24, p < .01) and those with more left political positions (r = -.13, p < .01) expressed stronger
intentions to change their behavior towards a plant-based diet in the future. The other demographic
variables were not significantly related and intention (ps > .05; see Table 8). Table 8 Correlations between demographic variables and intention Table 8 Correlations between demographic variables and intention Page 16/26 Age
Gender
Academic standing
Income
Political ideology
Intention
.05
.24**
.04
.04
-.13**
Note: ** Significant at the .01 level; * Significant at the .05 level. Note: ** Significant at the .01 level; * Significant at the .05 level. 4. Discussion The results of this study confirmed the
importance of breaking habits for increasing one’s pro-environmental intentions, which is consistent with
other studies (Gifford Lacroix and Chen, 2018; Rees et al. 2018; Verplanken and Whitmarsh 2021). Page 17/26 Page 17/26 In the present study’s second analysis, the variance accounted for by the interactions between constructs
within the TBC was small but significant. Previous studies have estimated interaction effects in the TPB. These mostly focused on the moderator impact of perceived behavioral control (e.g. La Barbera and Ajzen
2020; La Barbera and Ajzen 2021; Steinmetz Davidov and Schmidt 2011). The present investigated the
interaction effects of the TBC intention constructs, which is consistent with Kothe and Mullan’s (2015)
conclusion that considering interactions between constructs is essential for a complete understanding of
outcomes. Although the TBC predictor attitude plus was not significantly related to intention in the first analysis, it
was when the interaction effects were added to the model. Therefore, the inclusion of values and
emotions to the TPB’s attitude construct was confirmed in this work. To the best of our knowledge this
construct, attitude plus, was used first in the present study, although the idea of adding principles to the
TPB model was introduced by Gifford, Lacroix and Chen (2020). Perceived behavioral control moved from being the least to the second-most important predictor of
intention when the interaction effects were added to the TBC. That greater PBC tends to weaken the
relative importance of social norm is consistent with the results of other studies (La Barbera and Ajzen
2020). Three demographic variables (age, income, and educational level) were not significantly related to
intention, butfemales and those with stronger left political positions did express stronger intentions than
males and those with stronger right political stands. Thus, efforts to shift toward encouraging more plant-
based dietary choices might best be focused on men and those who hold stronger right political positions. Which sorts of messages appeal to these groups might well be discovered by reviewing the literature on
masculinity and conservatism. This study focused on dietary intentions. Of course, future studies based on the TBC should enhance this
research by including food behaviors. And, of course, other important climate-related intentions and
behaviors should be investigated. Also, future work should continue the search for the value of examining
the import of predictor interactions. 4. Discussion As is very often the case, we note that participants’ reports of their
recent dietary behavior relied on their memories, which may not be perfect. Policymakers who are engaged in designing pro-environment messages should give greater consideration
to the strongest influences on intention. For instance, we found that having a sense of obligation was the
most important factor predicting the intention to adopt plant-based behavior in the near future. Adopting
plant-based food was also said by the participants to be negatively influenced by their social circle. Social
norms influence individuals’ dietary decisions and they should be taken into account by awareness
campaigns and policymakers. Furthermore, anticipating positive effect, such as enjoying engaging in pro-environmental behavior or
having a good feeling about it strongly motivates individuals to undertake action in the future. Breaking a
habit is the most difficult challenge that individuals encounter and can act as a barrier through the Page 18/26 effectiveness of interventions. Therefore, providing information for people and educating them how to
overcome it is vital. In addition, based on the results of interaction effects, policy makers need also
emphasize on perceived control over dietary decisions and attitude plus values toward behavior. However,
they should be aware that the importance of social norm weakens with greater PBC. effectiveness of interventions. Therefore, providing information for people and educating them how to
overcome it is vital. In addition, based on the results of interaction effects, policy makers need also
emphasize on perceived control over dietary decisions and attitude plus values toward behavior. However,
they should be aware that the importance of social norm weakens with greater PBC. In conclusion, the present work suggests that adding three important constructs to the theory of planned
behavior (felt obligation, perceived behavioral control, affect, habit, and attitude plus), as part of the new
theory of behavioral choice, is an important advance. Declarations Competing interests The authors declare no competing interests Funding We are grateful for the support from the Social Sciences and Humanities Research Council of
Canada, grant number 435-2020-1295. Author contributions Robert Gifford: conceptualization; writing; revising; editing Karine Lacroix: data
gathering; writing Zahra Asgarizadeh: data analysis; methodology; visualization; writing; editing Emily
Ashford Anderson: data gathering; writing Madison Milne-Ives: writing Peter Sugrue: data
analysis; methodology Author contributions Robert Gifford: conceptualization; writing; revising; editing Karine Lacroix: data
gathering; writing Zahra Asgarizadeh: data analysis; methodology; visualization; writing; editing Emily
Ashford Anderson: data gathering; writing Madison Milne-Ives: writing Peter Sugrue: data
analysis; methodology Data Availability The data generated during and analyzed during the current study are available from the
corresponding author upon reasonable request. Data Availability The data generated during and analyzed during the current study are available from the
corresponding author upon reasonable request. Consent to Publish All authors gave their consent to publish and our university encourages us to publish
scientific articles, so the University’s consent is assumed. Consent to Publish All authors gave their consent to publish and our university encourages us to publish
scientific articles, so the University’s consent is assumed. Ethics approval Ethics approval was granted by University's Research Ethics Board. References 1. Aitken N M, Pelletier L G, Baxter D E (2016) Doing the difficult stuff: Influence of self-determined
motivation toward the environment on transportation pro-environmental behavior. Eco psychology, 8,
153-162. doi:10.1089/eco.2015.0079 2. Ajzen I (1991) The theory of planned behavior. Organizational behavior and human decision
processes, 50, 179-211. 3. Ajzen I (2011) The theory of planned behavior: Reactions and reflections. Psychology & Health, 26,
1113-1127. http://dx.doi.org/10.1080/08870446.2011.613995 4. Aleksandrowicz L, Green R. Joy, E J M, Smith P, Haines A (2016) The impacts of dietary change on
greenhouse gas emissions, land use, water use, and health: A systematic review. PloS One, 11, 1–16. https://doi.org/10.1371/journal.pone.0165797 Page 19/26 5. Conner M, Abraham C (2001) Conscientiousness and the theory of planned behavior: Toward a more
complete model of the antecedents of intentions and behavior. Journal of Applied Social Psychology,
27, 1547–61. doi:10.1177/01461672012711014. 6. Conner M, Armitage C J (1998) Extending the theory of planned behavior: A review and avenues for
further research. Journal of Applied Social Psychology, 28, 1429–64. doi:10.1111/j.1559-
1816.1998.tb01685.x. 6. Conner M, Armitage C J (1998) Extending the theory of planned behavior: A review and avenues for
further research. Journal of Applied Social Psychology, 28, 1429–64. doi:10.1111/j.1559-
1816.1998.tb01685.x. 7. Cook J, Oreskes N, Doran P T et al (2016) Consensus on consensus: a synthesis of consensus
estimates on human-caused global warming. Environmental Research Letters, 11. 7. Cook J, Oreskes N, Doran P T et al (2016) Consensus on consensus: a synthesis of consensus
estimates on human-caused global warming. Environmental Research Letters, 11. 8. Croker H, Whitaker K L, Cooke L, Wardle J (2009) Do social norms affect intended food choice? Preventive Medicine, 49, 190-3. 8. Croker H, Whitaker K L, Cooke L, Wardle J (2009) Do social norms affect intended food choice? Preventive Medicine, 49, 190-3. 9. Doran R, Larsen S (2016) The relative importance of social and personal norms in explaining
intentions to choose eco‐friendly travel options. International Journal of Tourism Research, 18, 159–
166. 9. Doran R, Larsen S (2016) The relative importance of social and personal norms in explaining
intentions to choose eco‐friendly travel options. International Journal of Tourism Research, 18, 159–
166. 10. Gifford R (2011) The dragons of inaction: Psychological barriers that limit climate change mitigation
and adaptation. American Psychologist, 66, 290–302. https://doi.org/10.1037/a0023566. 10. Gifford R (2011) The dragons of inaction: Psychological barriers that limit climate change mitigation
and adaptation. American Psychologist, 66, 290–302. https://doi.org/10.1037/a0023566. 11. References Gifford R, Lacroix K, Chen A (2018) Understanding responses to climate change: Psychological
barriers to mitigation and a new theory of behavioral choice. In Clayton, S. & Manning, C. (Eds.). Psychology and climate change: Human perceptions, impacts, and responses (pp. 161-184). New
York: Academic. 12. Han H, Lee M J, Kim W (2018) Promoting towel reuse behavior in guests: A water conservation
management and environmental policy in the hotel industry. Business Strategy and the Environment ,
27, 1302–1312. 13. Harland P, Staats H, Wilke H A M (1999) Explaining proenvironmental behavior by personal norms and
the theory of planned behavior. Journal of Applied Social Psychology, 29, 2505–2528. 13. Harland P, Staats H, Wilke H A M (1999) Explaining proenvironmental behavior by personal norms and
the theory of planned behavior. Journal of Applied Social Psychology, 29, 2505–2528. 14. Heath Y, Gifford R (2002) Extending the theory of planned behavior: Predicting the use of public
transportation. Journal of Applied Social Psychology, 32, 2154. 14. Heath Y, Gifford R (2002) Extending the theory of planned behavior: Predicting the use of public
transportation. Journal of Applied Social Psychology, 32, 2154. 15. Huang J, Wang J, Pang T W Y, Chan M K Y, Leung S, Chen X, Leung C, Zheng Z J, Wong M C S (2020)
Does theory of planned behavior play a role in predicting uptake of colorectal cancer screening? A
cross-sectional study in Hong Kong. BMJ Open, 10. https://doi.org/10.1136/bmjopen-2020-037619. 15. Huang J, Wang J, Pang T W Y, Chan M K Y, Leung S, Chen X, Leung C, Zheng Z J, Wong M C S (2020)
Does theory of planned behavior play a role in predicting uptake of colorectal cancer screening? A
cross-sectional study in Hong Kong. BMJ Open, 10. https://doi.org/10.1136/bmjopen-2020-037619. 16. Kothe E J, Mullan B A (2015) Interaction effects in the theory of planned behavior: Predicting fruit
and vegetable consumption in three prospective cohorts. British Journal of Psychology, 20, 549-62. doi: 10.1111/bjhp.12115. Epub 2014 Sep 11. PMID: 25209256. 16. Kothe E J, Mullan B A (2015) Interaction effects in the theory of planned behavior: Predicting fruit
and vegetable consumption in three prospective cohorts. British Journal of Psychology, 20, 549-62. doi: 10.1111/bjhp.12115. Epub 2014 Sep 11. PMID: 25209256. 17. La Barbera F, Ajzen I (2020) Control interactions in the theory of planned behavior: rethinking the role
of subjective norm. Europe's Journal of Psychology, 16, 401–417. https://doi.org/10.5964/ejop.v16i3.2056. 17. References 23. Pachauri R K, L A Meyer, L A (Eds.). Climate Change 2014: Synthesis Report. Contribution of Working
Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland, 151 pp. 24. Palmer C L, Burwitz L, Smith N C, Borrie A (2000) Enhancing fitness training adherence of elite netball
players: An evaluation of Maddux's revised theory of planned behavior. Journal of Sports Sciences,
18, 627 - 641. 25. Rapaport P, Orbell S (2000) Augmenting the theory of planned behavior: motivation to provide
practical assistance and emotional support to parents. Psychology and Health, 15, 309–24. doi:
10.1080/08870440008401995. 26. Rees J H, Bamberg S, Jäger A, Victor L, Bergmeyer M, Friese M (2018) Breaking the habit: On the
highly habitualized nature of meat consumption and implementation intentions as one effective way
of reducing it. Basic and Applied Social Psychology, 40, 136–147. https://doi.org/10.1080/01973533.2018.1449111 27. Reisch L, Eberle U, Lorek S (2013) Sustainable food consumption: An overview of contemporary
issues and policies. Sustainability: Science, Practice and Policy, 9, 7-25. DOI:
10.1080/15487733.2013.11908111 27. Reisch L, Eberle U, Lorek S (2013) Sustainable food consumption: An overview of contemporary
issues and policies. Sustainability: Science, Practice and Policy, 9, 7-25. DOI:
10.1080/15487733.2013.11908111 28. Richard R, van der Pligt J de Vries N (1996) Anticipated affect and behavioral choice. Basic & Applied
Social Psychology, 18, 111–129. https://doi-
org.ezproxy.library.uvic.ca/10.1207/s15324834basp1802_1. 28. Richard R, van der Pligt J de Vries N (1996) Anticipated affect and behavioral choice. Basic & Applied
Social Psychology, 18, 111–129. https://doi-
org.ezproxy.library.uvic.ca/10.1207/s15324834basp1802_1. 29. Rossi A N, Armstrong J B (1999) Theory of reasoned action vs. theory of planned behavior: Testing
the suitability and sufficiency of a popular behavior model using hunting intentions. Human
Dimensions of Wildlife, 4, 40-56. doi:10.1080/10871209909359156 29. Rossi A N, Armstrong J B (1999) Theory of reasoned action vs. theory of planned behavior: Testing
the suitability and sufficiency of a popular behavior model using hunting intentions. Human
Dimensions of Wildlife, 4, 40-56. doi:10.1080/10871209909359156 30. Schultz P W, Kaiser F G (2012) Promoting pro-environmental behavior. In S. Clayton (Ed.), The Oxford
handbook of environmental and conservation psychology (pp. 556–580). Oxford. 30. Schultz P W, Kaiser F G (2012) Promoting pro-environmental behavior. In S. Clayton (Ed.), The Oxford
handbook of environmental and conservation psychology (pp. 556–580). Oxford. 31. Steg L, Gifford R (2005) Sustainable transportation and quality of life. Journal of Transport
Geography, 13, 59-69. doi:10.1016/j.jtrangeo.2004.11.003 31. References La Barbera F, Ajzen I (2020) Control interactions in the theory of planned behavior: rethinking the role
of subjective norm. Europe's Journal of Psychology, 16, 401–417. https://doi.org/10.5964/ejop.v16i3.2056. 18. La Barbera F, Ajzen I (2021) Moderating role of perceived behavioral control in the theory of planned
behavior: A preregistered study. Journal of Theoretical Social Psychology, 5, DOI: 10.1002/jts5.83 18. La Barbera F, Ajzen I (2021) Moderating role of perceived behavioral control in the theory of planned
behavior: A preregistered study. Journal of Theoretical Social Psychology, 5, DOI: 10.1002/jts5.83 18. La Barbera F, Ajzen I (2021) Moderating role of perceived behavioral control in the theory of planned
behavior: A preregistered study. Journal of Theoretical Social Psychology, 5, DOI: 10.1002/jts5.83 Page 20/26 Page 20/26 19. Lacroix K, Gifford R (2020) Targeting interventions to distinct meat-eating groups reduces meat
consumption. Food Quality and Preference, 86. https://doi.org/10.1016/j.foodqual.2020.103997 20. Lacroix K, Gifford R, Chen A (2019) Developing and validating the Dragons of Inaction Psychological
Barriers (DIPB) scale. Journal of Environmental Psychology, 63, 9–18. https://doi.org/10.1016/j.jenvp.2019.03.001. 21. Ong AK, Prasetyo Y T, Lagura, F C, Ramos R N, Sigua K M, Villas J A, Young M N, Diaz J F, Persada S F,
Redi A A (2021) Factors affecting intention to prepare for mitigation of “the big one” earthquake in the
Philippines: Integrating protection motivation theory and extended theory of planned behavior. International Journal of Disaster Risk Reduction, 63, 102467. 21. Ong AK, Prasetyo Y T, Lagura, F C, Ramos R N, Sigua K M, Villas J A, Young M N, Diaz J F, Persada S F,
Redi A A (2021) Factors affecting intention to prepare for mitigation of “the big one” earthquake in the
Philippines: Integrating protection motivation theory and extended theory of planned behavior. International Journal of Disaster Risk Reduction, 63, 102467. 22. Onwezen M C, Antonides G, Bartels J (2013) The norm activation model: An exploration of the
functions of anticipated pride and guilt in pro‐environmental behavior. Journal of Economic
Psychology, 39, 141–153. 22. Onwezen M C, Antonides G, Bartels J (2013) The norm activation model: An exploration of the
functions of anticipated pride and guilt in pro‐environmental behavior. Journal of Economic
Psychology, 39, 141–153. 23. Pachauri R K, L A Meyer, L A (Eds.). Climate Change 2014: Synthesis Report. Contribution of Working
Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change. IPCC, Geneva, Switzerland, 151 pp. References Steg L, Gifford R (2005) Sustainable transportation and quality of life. Journal of Transport
Geography, 13, 59-69. doi:10.1016/j.jtrangeo.2004.11.003 Page 21/26 Page 21/26 32. Stehfest E, Bouwman L, van Vuuren D P et al (2009) Climate benefits of changing diet. Climatic
Change, 95, 83–102. https://doi.org/10.1007/s10584-008-9534-6 33. Steinmetz H, Davidov E, Schmidt P (2011) Three approaches to estimate latent interaction effects:
Intention and perceived behavioral control in the theory of planned behavior. Methodological
Innovations Online, 6, 95–110. https://doi.org/10.4256/mio.2010.0030 34. Stern P C (2000) Toward a coherent theory of environmentally significant behavior. Journal of Social
Issues, 56, 407–42 34. Stern P C (2000) Toward a coherent theory of environmentally significant behavior. Journal of Social
Issues, 56, 407–42 35. Swim J, Howard G, Clayton S, Reser J P, Doherty T J, Stern P C, Gifford R, Weber E U (2009)
Psychology and global climate change: Addressing a multi-faceted phenomenon and set of
challenges (pp. 1–108). American Psychological Association. http://www.apa.org/science/about/publications/climate-change.aspx 35. Swim J, Howard G, Clayton S, Reser J P, Doherty T J, Stern P C, Gifford R, Weber E U (2009)
Psychology and global climate change: Addressing a multi-faceted phenomenon and set of
challenges (pp. 1–108). American Psychological Association. http://www.apa.org/science/about/publications/climate-change.aspx 36. Verplanken B, Whitmarsh L (2021) Habit and climate change. Current Opinion in Behavioral Sciences,
42, 42-46. https://doi.org/10.1016/j.cobeha.2021.02.020. 36. Verplanken B, Whitmarsh L (2021) Habit and climate change. Current Opinion in Behavioral Sciences,
42, 42-46. https://doi.org/10.1016/j.cobeha.2021.02.020. 37. Wolff K, Nordin K, Brun W, Berglund G, Kvale G (2011) Affective and cognitive attitudes, uncertainty
avoidance and intention to obtain genetic testing: An extension of the theory of planned behavior. Psychology Health, 26, 1143–55. doi:10.1080/08870441003763253. 37. Wolff K, Nordin K, Brun W, Berglund G, Kvale G (2011) Affective and cognitive attitudes, uncertainty
avoidance and intention to obtain genetic testing: An extension of the theory of planned behavior. Psychology Health, 26, 1143–55. doi:10.1080/08870441003763253. 38. Zhang Y, Yang H, Cheng P, Luqman A (2020) Predicting consumers’ intention to consume poultry
during an H7N9 emergency: An extension of the theory of planned behavior model. Human and
Ecological Risk Assessment: An International Journal, 26, 190-211, DOI:
10.1080/10807039.2018.1503931. 38. Zhang Y, Yang H, Cheng P, Luqman A (2020) Predicting consumers’ intention to consume poultry
during an H7N9 emergency: An extension of the theory of planned behavior model. Human and
Ecological Risk Assessment: An International Journal, 26, 190-211, DOI:
10.1080/10807039.2018.1503931. Figures Figures Page 22/26 Figure 1
The theory of planned behavior
Figure 2 Page 23/26
Figure 1
The theory of planned behavior
Figure 2 Figure 1 Figure 1 The theory of planned behavior The theory of planned behavior Figure 2 Figure 2 Page 23/26 The theory of behavioral choice The theory of behavioral choice
igure 3
ntention and the habitual behavior x perceived behavioral control interaction gure 3
ntention and the habitual behavior x perceived behavioral control interaction Figure 3 Figure 3 Intention and the habitual behavior x perceived behavioral control interaction ntention and the habitual behavior x perceived behavioral control interaction Intention and the habitual behavior x perceived behavioral control interaction Page 24/26 Page 24/26 Figure 4
Intention and the interaction between affect and felt obligation Figure 4 Figure 4 Intention and the interaction between affect and felt obligation Page 25/26 gure 5
ntention and the interaction between social norms and perceived behavioral control Figure 5 Intention and the interaction between social norms and perceived behavioral control Intention and the interaction between social norms and perceived behavioral control Page 26/26
|
https://openalex.org/W4321486470
|
https://www.biorxiv.org/content/biorxiv/early/2023/06/18/2023.02.22.529480.full.pdf
|
English
| null |
Image-based Flow Simulation of Platelet Aggregates under Different Shear Rates
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,023
|
cc-by
| 15,741
|
.
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023.
;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Image-based Flow Simulation of Platelet Aggregates under
Different Shear Rates Yue Hao1*, G´abor Z´avodszky1,2, Claudia Tersteeg3, Mojtaba Barzegari4, Alfons G. Hoekstra1 Yue Hao1*, G´abor Z´avodszky1,2, Claudia Tersteeg3, Mojtaba Barzegari4, Alfons G. Hoekstra1 1 Computational Science Lab, Informatics Institute, Faculty of Science, University of
Amsterdam, The Netherlands
2 Department of Hydrodynamic Systems, Budapest University of Technology and
Economics, Budapest, Hungary
3 Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak
Kortrijk, Kortrijk, Belgium
4 Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven,
Belgium 1 Computational Science Lab, Informatics Institute, Faculty of Science, University of
Amsterdam, The Netherlands
2 Department of Hydrodynamic Systems, Budapest University of Technology and
Economics, Budapest, Hungary
3 Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Campus Kulak
Kortrijk, Kortrijk, Belgium
4 Biomechanics Section, Department of Mechanical Engineering, KU Leuven, Leuven,
Belgium * y.hao@uva.nl * y.hao@uva.nl Abstract Hemodynamics is crucial for the activation and aggregation of platelets in response to
flow-induced shear. In this paper, a novel image-based computational model simulating
blood flow through and around platelet aggregates is presented. The microstructure of
aggregates was captured by two different modalities of microscopy images of in vitro
whole blood perfusion experiments in microfluidic chambers coated with collagen. One
set of images captured the geometry of the aggregate outline, while the other employed
platelet labelling to infer the internal density. The platelet aggregates were modelled as
a porous medium, the permeability of which was calculated with the Kozeny-Carman
equation. The computational model was subsequently applied to study hemodynamics
inside and around the platelet aggregates. The blood flow velocity, shear stress and
kinetic force exerted on the aggregates were investigated and compared under 800 s−1,
1600 s−1 and 4000 s−1 wall shear rates. The advection-diffusion balance of agonist
transport inside the platelet aggregates was also evaluated by local P´eclet number. The
findings show that the transport of agonists is not only affected by the shear rate but
also significantly influenced by the microstructure of the aggregates. Moreover, large
kinetic forces were found at the transition zone from shell to core of the aggregates,
which could contribute to identifying the boundary between the shell and the core. The
shear rate and the rate of elongation flow were investigated as well. The results imply
that the emerging shapes of aggregates are highly correlated to the shear rate and the
rate of elongation. The framework provides a way to incorporate the internal
microstructure of the aggregates into the computational model and yields a better
understanding of the hemodynamics and physiology of platelet aggregates, hence laying
the foundation for predicting aggregation and deformation under different flow
conditions. .
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023.
;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Introduction Platelet aggregation is the initial step of thrombus formation, that begins with platelet
adhesion to the sites of vascular injury [1–3]. It is a multistep dynamic process involving
distinct receptors and adhesive ligands such as GPIbα and αIIbβ3 receptors, von
Willebrand factor (VWF), fibrinogen and fibronectin [4]. Those receptor-ligand
interactions regulate tethering, platelet-platelet cohesion and stabilization of the formed
aggregates. Blood flow also plays a critical role in the process as distinct shear ranges
lead to unique ways to form aggregates [4,5]. Under low shear conditions, fibrinogen
and integrin αIIbβ3 are known to be the predominant factors of platelet aggregation
while VWF and fibronectin have progressively increasing contribution to the
aggregation with the increase of the flow shear rate [6]. To better understand the impact of the hemodynamics on the formation and
mechanics of blood clots, several computational models were developed [7–12]. Since the
porosity of the forming aggregate influences its formation [13–16], a growing number of
computational studies considered the thrombus as a porous medium. Tomaiuolo et
al. [17] established a two-dimensional computational model, in which the thrombus
consists of two homogeneous porous parts. One with highly activated, densely packed
platelets was named core and the other with lower levels of activation was named shell. Different permeabilities were assigned for the core and shell, respectively. They have
shown that the solute transport behaviour changes with changes of the platelet packing
density. Xu et al. [18] developed a similar novel two-dimensional multi-phase [
]
computational model to characterise the interplay between the main components of the
clot. They showed that the shear force acting on the clot surface is affected by the
internal structure of clots. Also, their results indicated that the porosity of the clots
decreased as the flow shear rate increased. The growing interest in the porous structure of thrombus or platelet aggregate has
prompted measurements of their permeability [19,20]. Experimental studies have
demonstrated several ways to measure the permeability of clots [20–24]. However, owing
to the significant differences in the setup, the composition of the flow and the
heterogeneous nature of the formation, the experimental results of permeability usually
correspond to unique conditions and are difficult to reuse in different scenarios. At
present, the Kozeny-Carman formula [25,26] is one of the most widely applied methods
to estimate permeability. Author summary The initial step in the formation of an arterial thrombus is the rapid aggregation of the
tiny blood particles called platelets. This process significantly influences the formation 1/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint and structure of the resulting thrombi. The mechanical properties of the aggregates
depend on their microstructure, which in turn is dictated by their interaction with the
flow during formation. However, due to currently existing technological limitations, it is
not possible to measure these interactions in sufficient detail experimentally. In this
paper, an image-based computational model is proposed based on two different
modalities of experimental images, that can complement the experiments and give
detailed information on hemodynamics during the aggregation. The image sets are
captured from whole blood perfused microfluidic chambers coated with collagen. One
modality of images captured the shape of the aggregate outline with high contrast,
while the other employed platelet labeling to infer the internal density. The platelet
aggregates are considered as porous media in the simulations, informed by the images. This framework incorporates the internal microstructure of the aggregates into the
computational model and yields a better understanding of the hemodynamics and
physiology of platelet aggregates, hence laying the foundation for predicting aggregation
and deformation under different flow conditions. Introduction In [32], the porous geometry of the clot was reconstructed from
the experimental images. This setting is closer to reality compared to the impermeable
solid and provides the prediction of the transport of inert solutes in the aggregates. In this paper, an image-based method to capture the internal microstructure is
proposed. Based on this method, a three-dimensional computational model is developed,
that can simulate the blood flows through the formed platelet aggregates in a
microchannel accurately. In this model, the platelet aggregates are considered as porous
media and the microstructure of platelet aggregates is based on in vitro experiments of
whole blood perfusion in microfluidic chambers coated with collagen. Two sets of
images were used to capture the geometries and internal structures of the aggregates. The images without platelet labeling captured the geometry of the aggregate outline,
while the images with platelet labeling are employed to infer the internal density of the
aggregates and estimate the permeability. The computational model was subsequently
applied to study the hemodynamics inside and around the platelet aggregates. The
blood flow velocity, shear stress, kinetic forces of blood flow exerted on aggregates, flow
elongational rate and advection-diffusion balance of agonist transport inside the platelet
aggregates were investigated and compared under 800 s−1, 1600 s−1 and 4000 s−1 wall
shear rates (WSRs). The proposed model incorporating the internal microstructure of
the aggregates provides an accurate estimate of the hemodynamics of platelet
aggregates, and thus lays the foundation for predicting further aggregation and
deformation under different flow conditions. Introduction It approximates the permeability based on the volume fraction
and geometric properties of the solid components that form the porous
substance [14,27]. 2/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Instead of considering the clot geometry as a simplified object such as half ellipse in
3
the model, the geometry of the clot can be obtained from in vivo or in vitro imaging
3
data, contributing to further studies on the microstructure of platelet aggregates [28]. 3
For example, in [29], a two-dimensional model was developed to calculate the shear
3
stress of the near-thrombus region using the fluorescent labeling images captured in mice. 4
They quantitatively measured the thrombus dynamics in the early stages of hemostasis
4
based on the images, but did not consider the internal structure of the thrombus. 4
Taylor et al. [30] and Pinar et al. [31] examined the dynamics of the blood flow around
4
the thrombus based on the in vitro experiments, but both of them considered thrombi
4
as impermeable solids. In [32], the porous geometry of the clot was reconstructed from
4
the experimental images. This setting is closer to reality compared to the impermeable
4
solid and provides the prediction of the transport of inert solutes in the aggregates. 4 Instead of considering the clot geometry as a simplified object such as half ellipse in
36
the model, the geometry of the clot can be obtained from in vivo or in vitro imaging
37
data, contributing to further studies on the microstructure of platelet aggregates [28]. 38
For example, in [29], a two-dimensional model was developed to calculate the shear
39
stress of the near-thrombus region using the fluorescent labeling images captured in mice. 40
They quantitatively measured the thrombus dynamics in the early stages of hemostasis
41
based on the images, but did not consider the internal structure of the thrombus. 42 Taylor et al. [30] and Pinar et al. [31] examined the dynamics of the blood flow around
the thrombus based on the in vitro experiments, but both of them considered thrombi
as impermeable solids. In vitro experiments The three-dimensional structure of
85
platelet aggregates was reconstructed by stacking all the images over the z-direction
86
later. Two types of images modality were captured for the platelet aggregates. One set
87
of images captured the shape of the aggregate outline with non-labelled platelets (DIC
88
images), while the other employed platelet labelling to infer the internal density
89
(fluorescent images). 90 ensure after which a stable clot was formed in vitro. The three-dimensional structure of
85
platelet aggregates was reconstructed by stacking all the images over the z-direction
86
later. Two types of images modality were captured for the platelet aggregates. One set
87
of images captured the shape of the aggregate outline with non-labelled platelets (DIC
88
images), while the other employed platelet labelling to infer the internal density
89
(fluorescent images). 90 Fig 1. The schematic of the experiment set-up. The width and the height of the
chamber is 1 mm and 100 µm, respectively. The dimension of the recorded
experimental domain is 125 µm × 100 µm × 100 µm. Fig 1. The schematic of the experiment set-up. The width and the height of the
chamber is 1 mm and 100 µm, respectively. The dimension of the recorded
experimental domain is 125 µm × 100 µm × 100 µm. In vitro experiments All the experiments were performed at the Laboratory for Thrombosis Research at KU
Leuven. A schematic diagram of the in vitro experiments of platelet aggregates is shown
in Fig 1. Fresh human blood was drawn into a citrated tube (3.8% sodium citrate,
citrate:blood = 1:9) and mixed with MitoTracker Deep Red (Invitrogen, USA). Citrate
chelates extracellular calcium, and without calcium coagulation cannot occur, hence
fibrin cannot be formed. A temperature control (Thermo Fisher Scientific, US) was
used to ensure the blood was at a human-body temperature. The chip µ−Slide VI 0.1
(Ibidi, Germany) was positioned under the microscope and connected to the test tube
with blood and the pump via tubing. Before the experiments, the complete surface of
the perfusion chamber was coated using 100 µg/ml Horm-collagen (Takeda, Austria)
and stood overnight, resulting in a uniform distribution of collagen fibrils over the
complete perfused area. The pump (Harvard Apparatus, US), which was on the other
side of the chip, pulled the blood through the chamber with a preset WSR. After five
minutes of blood perfusion, for every one micrometer in the z-direction, the images of
cross-sections of the channel were captured under an Axio Observer Z1 inverted
fluorescence microscope (Zeiss, Germany) using differential interference contrast (DIC)
and fluorescence microscopy at 100x magnification. The perfusion time was chosen to 3/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint ensure after which a stable clot was formed in vitro. Image data processing CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 2. Flow diagram for the image-based modelling methodology
implemented in this work. Experiment: microscopy images of platelet aggregates
with labelled and non-labelled platelets caught by differential interference contrast
microscopy with 100x magnification. Extracted information: reconstructed platelet
aggregate geometry and permeability distribution over the aggregate. Computational
modelling: the simulated domain for blood flow. Fig 2. Flow diagram for the image-based modelling methodology Fig 2. Flow diagram for the image-based modelling methodology
implemented in this work. Experiment: microscopy images of platelet aggregates
with labelled and non-labelled platelets caught by differential interference contrast
microscopy with 100x magnification. Extracted information: reconstructed platelet
aggregate geometry and permeability distribution over the aggregate. Computational
modelling: the simulated domain for blood flow. implemented in this work. Experiment: microscopy images of platelet aggregates
with labelled and non-labelled platelets caught by differential interference contrast
microscopy with 100x magnification. Extracted information: reconstructed platelet
aggregate geometry and permeability distribution over the aggregate. Computational
modelling: the simulated domain for blood flow. Fig 3. Fluorescence intensity (arbitrary units) - density relation inside platelet aggregates. (a) 800 s−1 WSR. Slope = 0.0027. Intercept = -0.19. (b) 1600 s−1 WSR. Slope = 0.0014. Intercept = 0.28. (c) 4000 s−1 WSR. Slope = 0.0004. Intercept = -0.23. The unit in each subfigure is different due to the influence of external environment such as exposure level
and labelling time. Fig 3. Fluorescence intensity (arbitrary units) - density relation inside platelet aggregates. (a) 800 s−1 WSR. Slope = 0.0027. Intercept = -0.19. (b) 1600 s−1 WSR. Slope = 0.0014. Intercept = 0.28. (c) 4000 s−1 WSR. Slope = 0.0004. Intercept = -0.23. The unit in each subfigure is different due to the influence of external environment such as exposure level
and labelling time. simulated domain. Image data processing Finally, this volume mesh of the flow domain was then used as the
spatial discretization of the finite element method to simulate the blood flow. Image data processing A schematic workflow of the methodology employed in this work to transfer the
experimental image data to the computational model is provided in Fig 2. The non-labelled images were segmented with 3D Slicer [33] manually and stacked
together over the z direction. As a final step to reconstruct the surface of the platelet
aggregate, interpolation was applied to fill between the image slices. Smoothing was
applied to further improve the surface quality of the platelet aggregate meshes. f
f However, the surface mesh may still contain low quality mesh elements, therefore it was
remeshed with MeshLab [34] using the uniform resampling filter. After proceeding with
the surface mesh, a corresponding volume mesh was created in Gmsh [35] for use in the
numerical simulations. The intensity of the fluorescence in the labelled image data is proportional to the
102
local density of the platelet aggregate, and can be further used to infer the porosity of
103
the aggregates. Therefore density measurements have been made to approximate the
104
local density of aggregates. This was done by manually counting and the details of it
105
are described in S1 File. Fig 3 shows the results of this measurement which provide us
106
with a mapping between the fluorescence intensity and density of the entire aggregates. 107
The permeability of the aggregates was subsequently inferred using the Kozeny-Carman
108
equation. Further details on this are presented in the next subsection. 109 To simulate the blood flow around the platelet aggregates, a rectangular domain was
110
created and uniformly meshed to represent a part of the channel of the chip in the
111
experiments, as shown in Fig 4. The platelet aggregates formed on the collagen coated
112
bottom plate, approximately in the middle of the channel. The intensity data of platelet
113
aggregates were interpolated from the platelet aggregate volume mesh to the volume
114
mesh of the flow domain. This interpolation between two meshes allows us to assign the
115
corresponding fluorescence intensity value of the aggregate to a specific location in the
116 June 18, 2023 4/22 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . Computational blood flow model Blood flow was modelled as an incompressible Newtonian fluid governed by the
120
Navier-Stokes equations, and the influence of the porous clot on the flow was introduced
121
using a Darcy term:
122 ρ
∂u
∂t + u · ∇u
= −∇p + µ∆u −µ
k u,
(1) (1) 5/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 4. Schematic diagram of the simulation domain. (a) Blood perfusion channel and the location of the simulated
domain. (b) Top view of the blood perfusion channel. (c) Domain of the blood flow simulation including the position of the
platelet aggregate. Fig 4. Schematic diagram of the simulation domain. (a) Blood perfusion channel and the location of the simulated
domain. (b) Top view of the blood perfusion channel. (c) Domain of the blood flow simulation including the position of the
platelet aggregate. ∇· u = 0. (2) (2) ∇· u = 0. Here, u is the velocity of the fluid, p is the pressure, ρ is the fluid density, and µ is the
dynamic viscosity. The last term on the right-hand side accounts for the momentum
exchange between the fluid and the solid phases of the porous medium. It is defined by
Darcy’s law [36,37], which describes flow through a porous medium. In this term, k is
the permeability of the platelet aggregates. Permeability is a function of the volume
fraction of pore size and fiber or cell/cell aggregate size [21] and was estimated using
the Kozeny-Carman equation is the velocity of the fluid, p is the pressure, ρ is the fluid density, and µ is the
123
c viscosity. The last term on the right-hand side accounts for the momentum
124 k = Φ2
s
ϵ3D2
p
150 (1 −ϵ)2 . Computational blood flow model (3) (3) Here Φs, ϵ and Dp are the sphericity of the platelets, the porosity of the aggregates and
130
the platelet diameter, respectively. 131 Here Φs, ϵ and Dp are the sphericity of the platelets, the porosity of the aggregates and
130
the platelet diameter, respectively. 131 p
,
p
y
The complete list of parameter values used in the model and their literature sources
132
are shown in Table 1. According to the inlet flow settings in experiments, the Reynolds
133
numbers for 800 s−1, 1600 s−1 and 4000 s−1 WSRs is 0.67, 1.37 and 3.33, respectively. 134
We expect that the flow velocity in the platelet aggregates are significantly smaller than
135
the velocity in the channel due to the porous structure, which fits the Stokes flow
136
assumption of the Kozeny-Carman equation. The time-step size was chosen to satisfy
137
the Courant–Friedrichs–Lewy condition. A mesh convergence study was carried out to
138
select the appropriate mesh resolution for the simulation. Considering the
139
computational cost and the convergence of the simulation, a mesh with 3.0 million
140
tetrahedral elements was chosen. The average element size of the mesh is 0.837 µm3,
141
which is significantly smaller than the average size of a platelet (7.2 −11.7 fL) [38]. As
142
shown in Fig 4, the blood flowed through the domain from left to right (along the
143
positive y direction). Since the blood flow domain was considered as a small part of the
144
channel in the x direction, constant and parabolic velocity profiles were prescribed in
145
the x and z directions respectively at the inlet (for the coordinate system see Fig 4). A
146
constant-pressure condition was prescribed at the outlet. No-slip boundary conditions
147
were imposed at the top and bottom walls. 148 The flow field was solved using the finite element method (FEM) [43] and
149
implemented in FreeFEM [44]. An iterative solver based on flexible generalized minimal
150
residual (FGMRES) method [45] was used and implemented via PETSc [46]. This work
151
was carried out on the Dutch national supercomputer Snelllius (SURF, Netherlands). 152 The flow field was solved using the finite element method (FEM) [43] and
149
implemented in FreeFEM [44]. An iterative solver based on flexible generalized minimal
150
residual (FGMRES) method [45] was used and implemented via PETSc [46]. Results The distribution of the fluorescence intensity obtained from the experimental images
157
under three WSRs is presented in Fig 5 (a)-(c). With the increase of the shear rate, the
158
ratio of fluorescence intensity clustering at the tail of the distribution increases, which
159
means more parts in the aggregates have a higher density. Note that the values of this
160
fluorescence intensity are in arbitrary units and not comparable under different
161
scenarios due to the fact that they are influenced by other factors such as initial
162
labeling concentration and light intensity. By computing the corresponding aggregate
163
density from the fluorescence intensity via regression, the average density and the
164
distribution of this density inside the aggregates are demonstrated in Fig 5(d) and
165
Fig 5(e). Subsequently, Fig 5(f) shows the distribution of the permeability. The denser
166
the aggregate is, the lower the permeability. 167 To further study how fluorescence intensity, porosity and permeability distribute
168
inside the platelet aggregate, the corresponding results on the cross-sections of the
169
aggregate formed under 1600 s−1 WSR are shown in Fig 6. In the core of the aggregate,
170
the intensity is higher than that in the shell. Correspondingly, the platelets in the core
171
are positioned denser, which leads to much lower permeability in this part. This
172
non-homogeneity results in complex blood flow behavior inside the platelet aggregates. 173 Computational blood flow model This work
151
was carried out on the Dutch national supercomputer Snelllius (SURF, Netherlands). 152 (
)
[
]
p
[
]
as carried out on the Dutch national supercomputer Snelllius (SURF, Netherlands). 152 June 18, 2023 6/22 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Table 1. Parameter used in simulations. Notation
Description
Value
Unit
Reference
ρ
density of fluid
1.025 × 10−3
g mm−3
[39]
µ
fluid viscosity
3 × 10−3
g mm−1 s−1
[32,40]
Φs
sphericity of activated platelet
0.71
—
[41]
Dp
platelet diameter
2 × 10−3
mm
[42]
dt
time-step size
10−5
s
—
—
average element size
0.837
µm3
— Table 1. Parameter used in simulations. Each simulation was performed on AMD Rome 7H12 CPU × 2 and parallelized on 128
153
cores. The corresponding code of the simulation is available at
154
https://github.com/UvaCsl/AggregateFlowSimu/releases/tag/v1.0.0. 155 Each simulation was performed on AMD Rome 7H12 CPU × 2 and parallelized on 128
153
cores. The corresponding code of the simulation is available at
154
https://github.com/UvaCsl/AggregateFlowSimu/releases/tag/v1.0.0. 155 https://github.com/UvaCsl/AggregateFlowSimu/releases/tag/v1.0.0. 1 Flow within and around the platelet aggregate The steady flow pattern on a cross-section under 1600 s−1 WSR is shown in Fig 7(a). 175
Although the platelet aggregate is considered as a porous medium, the permeability of
176
the aggregates is extremely small. Consequently, most of the blood flows over the
177
aggregate and only a small amount of the blood permeates the platelet aggregate. 178
Fig 7(b) shows the corresponding blood flow velocity inside the platelet aggregates on
179
cross-sections. Although in the shell with relatively high permeability, the blood flow
180
velocity is significantly lower than the extra-thrombus flow, it is still considerably higher
181
than the velocity in the core with lower permeability. However, in the transition from
182
the shell to the core, the magnitude of the velocity is greater than that of some regions
183
of the shell. After blood enters the core, its velocity becomes extremely low. The
184
minimum velocity in the interior core reaches 10−11 mm/s, which means barely any
185
blood flow. 186 7/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 5. Distribution of fluorescence intensity, distribution of platelet aggregates density and permeability
under 800 s−1, 1600 s−1 and 4000 s−1 WSRs. (a)-(c) Distributions of fluorescence intensity inside the platelet
aggregates. (d) Average volume fraction of platelets of the platelet aggregates. (e) Distribution of volume fraction of platelets
inside the platelet aggregates. (f) Distribution of permeability inside the platelet aggregates in log-scale. Fig 5. Distribution of fluorescence intensity, distribution of platelet aggregates density and permeability
under 800 s−1, 1600 s−1 and 4000 s−1 WSRs. (a)-(c) Distributions of fluorescence intensity inside the platelet
aggregates. (d) Average volume fraction of platelets of the platelet aggregates. (e) Distribution of volume fraction of platelets
inside the platelet aggregates. (f) Distribution of permeability inside the platelet aggregates in log-scale. Fig 6. Flow within and around the platelet aggregate Intensity of the fluorescence, corresponding porosity and the permeability of the platelet aggregate
under 1600 s−1 WSR. (a) Intensity of the fluorescence obtained from the experimental data. (b) Corresponding porosity of
the platelet aggregate. (c) Permeability of the platelet aggregate on the cross-sections of the aggregate obtained from the
Kozeny-Carman equation. The results inside the platelet aggregates formed under 800 s−1 and 4000 s−1 WSRs are
demonstrated in S1 Fig. Fig 6. Intensity of the fluorescence, corresponding porosity and the permeability of the platelet aggregate
under 1600 s−1 WSR. (a) Intensity of the fluorescence obtained from the experimental data. (b) Corresponding porosity of
the platelet aggregate. (c) Permeability of the platelet aggregate on the cross-sections of the aggregate obtained from the
Kozeny-Carman equation. The results inside the platelet aggregates formed under 800 s−1 and 4000 s−1 WSRs are
demonstrated in S1 Fig. Stress analysis of the blood flow Since the platelet aggregate is considered as a porous medium, the momentum of the
188
flow exerts a kinetic force inside the aggregate, which can lead to partial or complete
189
aggregate embolism [32]. These forces indicate the interaction between blood flow and
190
platelet aggregates under the assumption that the deformation of the aggregate is
191
insignificant, and induce stresses in the aggregate structure. However, these are not
192
discussed in the current work. This kinetic force was calculated by Darcy’s law,
193
f = −µ
k u, and the result is shown in Fig 8(a). The highest forces appear on the top
194 8/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 7. Flow field at WSR of 1600 s−1. (a) The velocity field of the blood flow on a
cross-section of the flow domain. The red arrow indicates the direction of blood flow. (b) The velocity field of the blood flow inside the platelet aggregate on the cross-sections. The orange arrow points out the high flow velocity area between the shell and the core. Fig 7. Flow field at WSR of 1600 s−1. (a) The velocity field of the blood flow on a
cross-section of the flow domain. The red arrow indicates the direction of blood flow. (b) The velocity field of the blood flow inside the platelet aggregate on the cross-sections. The orange arrow points out the high flow velocity area between the shell and the core. Stress analysis of the blood flow outer layer of the platelet aggregate, due to the relatively high fluid velocity over these
195
parts. Also, similar to the blood velocity in the transition from the shell to the core,
196
there exists an area with higher kinetic force compared to the force in the shell and core. 197 Fig 8. Stress analysis of the blood flow and the platelet aggregate under
1600 s−1 WSR. (a) The kinetic force exerted on the platelet aggregate. (b) The fluid
shear stress on the surface of the platelet aggregate. The simulation results inside the
platelet aggregates formed under 800 s−1 and 4000 s−1 WSRs are shown in S2 Fig. Fig 8. Stress analysis of the blood flow and the platelet aggregate under
1600 s−1 WSR. (a) The kinetic force exerted on the platelet aggregate. (b) The fluid
shear stress on the surface of the platelet aggregate. The simulation results inside the
platelet aggregates formed under 800 s−1 and 4000 s−1 WSRs are shown in S2 Fig. Fig 8. Stress analysis of the blood flow and the platelet aggregate under
1600 s−1 WSR. (a) The kinetic force exerted on the platelet aggregate. (b) The fluid
shear stress on the surface of the platelet aggregate. The simulation results inside the
platelet aggregates formed under 800 s−1 and 4000 s−1 WSRs are shown in S2 Fig. Furthermore, the local shear stress has a significant influence on platelet activation,
198
aggregation and adhesion [47]. It has been shown in [48] and [49] that high levels of
199
shear stress could initiate platelet aggregation. Fig 8(b) demonstrates the fluid shear
200
stress on the surface of such platelet aggregate under 1600 s−1 WSR condition. These
201
fluid-induced shear stress magnitude σ is estimated by:
202 σ = µ · ˙γ,
(4) σ = µ · ˙γ,
(4)
ude obtained by:
2
˙γ =
√
2 × D : D. (5) (4) where ˙γ is the shear rate magnitude obtained by: ˙γ =
√
2 × D : D. (5) (5) Here, D represents the strain rate tensor which is defined as D = 1
2(∇u + ∇uT ). As
204
expected, shear stress at the top part of the aggregate is several-fold higher than at the
205
bottom. This is because the aggregate protrudes into the center of the channel and is
206
exposed to a higher blood flow velocity. Advection versus diffusion Biochemical processes involving different agonists such as adenosine-5′-diphosphate
210
(ADP), require a certain amount of time to take place. In the advection-dominated
211
region, if such a process takes more time than the characteristic time of agonist
212
advection (i.e. the average time of the agonist to be convected over a distance of
213
platelet), this process is hindered since the flow velocity is high enough to wash away
214
the agonists before any reaction takes place. Therefore, identifying the
215
advection-dominated region is critical for understanding the dynamics of the
216
aggregation process. The advection- and diffusion-dominated regions of inner chemicals
217
were evaluated by P´eclet number, a ratio of advection and diffusion time:
218 PeL = u/L
D/L2 ,
(6) (6) where u denotes the magnitude of local flow velocity, D is the mass diffusion coefficient
219
and L denotes the characteristic length. In our simulation, the characteristic length is
220
defined as the platelet diameter, therefore the advection time in the denominator of PeL
221
indicates the time required to move the distance of a platelet diameter. When the
222
P´eclet number equals one, advection and diffusion contribute to mass transport equally. 223
If PeL > 1, the diffusion time of the chemical is longer than the advection time in such
224
area, which means the motion of the chemical is advection-dominated. Otherwise, it is
225
diffusion-dominated. 226
2 where u denotes the magnitude of local flow velocity, D is the mass diffusion coefficient
219
and L denotes the characteristic length. In our simulation, the characteristic length is
220
defined as the platelet diameter, therefore the advection time in the denominator of PeL
221
indicates the time required to move the distance of a platelet diameter. When the
222
P´eclet number equals one, advection and diffusion contribute to mass transport equally. 223
If PeL > 1, the diffusion time of the chemical is longer than the advection time in such
224
area, which means the motion of the chemical is advection-dominated. Otherwise, it is
225
diffusion-dominated. 226 Three agonists, Calcium (Ca2+), ADP and Factor X, were evaluated in this work
227
(see Table 2). Ca2+ contributes to multiple stages of cellular activation in platelets [50]. 228
It has the lowest molecular weight of these three chemicals but the highest diffusion
229
coefficient. Stress analysis of the blood flow This result agrees with the shear stress
207
distribution reported in [28] and [32]. 208 Here, D represents the strain rate tensor which is defined as D = 1
2(∇u + ∇uT ). As
204
expected, shear stress at the top part of the aggregate is several-fold higher than at the
205
bottom. This is because the aggregate protrudes into the center of the channel and is
206
exposed to a higher blood flow velocity. This result agrees with the shear stress
207
distribution reported in [28] and [32]. 208 9/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Advection versus diffusion The second one is ADP, an agonist that plays an important role in platelet
230
activation. Finally, Factor X, an enzyme in the coagulation cascade that can increase
231
the thrombosis propensity [51]. It has the highest molecular weight and the lowest
232
diffusion coefficient among the three agonists. The results demonstrate that the
233
advection-dominated volume in the platelet aggregate increases with the decrease of the
234
diffusion velocities of agonists. Furthermore, Fig 9 visualises the distribution of the
235
advection- and diffusion-dominated areas under the same flow condition. It can be
236
observed that for the highly diffusive agonists only minuscule parts of the platelet
237
aggregate are advection-dominated, even in the shell region. However for Factor X,
238
advection takes over the transport at almost the entire outer layer of the aggregate. In
239
conclusion, with the decrease of the diffusion coefficient, advection gradually dominates
240
the transport of chemicals in the shell region of the platelet aggregate. This aligns well
241
with previous findings predicting such behaviour within the core-shell structure of
242
platelet aggregates [13,17]. 243 Table 2. Intrathrombus transport simulation for coagulation factors with different molecular weights at WSR
of 1600 s−1. Molecular weight [g mol−1]
Diffusion coefficient [mm2 s−1]
Advection dominated volume [%]
Ca2+
40.08
6.64 × 10−4 [32]
4.67
ADP
472.201
2.57 × 10−4 [52]
11.78
Factor X
59000
5 × 10−5 [53]
31.61 Table 2. Intrathrombus transport simulation for coagulation factors with different molecular weights at WSR
f 1600
−1 Table 2. Intrathrombus transport simulation for coagulation factors with different molecular weights at WSR
of 1600 s−1. Molecular weight [g mol−1]
Diffusion coefficient [mm2 s−1]
Advection dominated volume [%]
Ca2+
40 08
6 64 × 10−4 [32]
4 67 rt simulation for coagulation factors with different molecular weights at WSR Intrathrombus transport simulation for coagulation factors with different molec Effects of increasing WSR Under all three shear rate conditions, the average intrathrombus velocity is at least two
245
orders of magnitude lower than the average extra-thrombus velocity, as shown in Table 3. 246 10/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 9. Advection-diffusion balance. Advection-diffusion balance of (a) Ca2+, (b) ADP and (c) Factor X on cross-sections
inside the platelet aggregate under 1600 s−1 WSR. The upper end of the color scale is set to 1. Therefore, areas with red color
correspond to advection-dominated regions, while colors towards the lower end of the scale denote diffusion-dominated regions. Fig 9. Advection-diffusion balance. Advection-diffusion balance of (a) Ca2+, (b) ADP and (c) Factor X on cross-sections
inside the platelet aggregate under 1600 s−1 WSR. The upper end of the color scale is set to 1. Therefore, areas with red color
correspond to advection-dominated regions, while colors towards the lower end of the scale denote diffusion-dominated regions. Fig 9. Advection-diffusion balance. Advection-diffusion balance of (a) Ca2+, (b) ADP and (c) Factor X on cross-sections
inside the platelet aggregate under 1600 s−1 WSR. The upper end of the color scale is set to 1. Therefore, areas with red color
correspond to advection-dominated regions, while colors towards the lower end of the scale denote diffusion-dominated regions. Table 3. Comparison of simulation results under various WSRs. Effects of increasing WSR Wall shear rate [s−1]
800
1600
4000
Intrathrombus velocity [mm s−1]
0.05 ± 0.09
0.06 ± 0.15
0.13 ± 0.34
Extra-thrombus velocity [mm s−1]
13.78 ± 6.28
27.50 ± 12.79
69.06 ± 32.18
Kinetic force [N] (×10−12)
1.36 ± 2.07
4.07 ± 5.34
11.20 ± 19.34
Average P´eclet number
Ca2+
0.16 ± 0.27
0.18 ± 0.44
0.38 ± 1.02
ADP
0.40 ± 0.69
0.45 ± 1.13
0.99 ± 3.64
Factor X
2.08 ± 3.56
2.33 ± 5.80
5.11 ± 13.59
Advection-dominated
volume [%]
Ca2+
1.97
4.67
10.90
ADP
14.46
11.78
18.82
Factor X
37.82
31.61
33.83
Average intrathrombus velocity, average extra-thrombus velocity, average kinetic force,
average P´eclet number inside the aggregates and the advection-dominated volume of
Ca2+, ADP and Factor X inside the platelet aggregates under three WSRs. Table 3. Comparison of simulation results under various WSRs. Average intrathrombus velocity, average extra-thrombus velocity, average kinetic force,
average P´eclet number inside the aggregates and the advection-dominated volume of
Ca2+, ADP and Factor X inside the platelet aggregates under three WSRs. This is comparable to the previously reported results on the microscale thrombus
247
aggregate simulation [16,54]. Furthermore, the increase in inlet blood velocity leads to
248
higher average intrathrombus velocity and stronger kinetic forces on platelet aggregates. 249 This is comparable to the previously reported results on the microscale thrombus
247
aggregate simulation [16,54]. Furthermore, the increase in inlet blood velocity leads to
248
higher average intrathrombus velocity and stronger kinetic forces on platelet aggregates. 249 p
p
y
p
aggregate simulation [16,54]. Furthermore, the increase in inlet blood velocity leads to
248
higher average intrathrombus velocity and stronger kinetic forces on platelet aggregates. 249
In order to explore how the inlet WSRs affect the solute transport, the average
250
P´eclet number and advection-dominated volume inside the platelet aggregates are also
251
computed. The result indicates that the average P´eclet number inside the aggregates
252
has a positive correlation with WSRs, while the relation between advection-dominated
253
volume and WSRs is not clear. 254
To further understand the influence of the blood flow velocity on the platelet
255
aggregate formation, the shapes of the platelet aggregates formed under three shear
256 In order to explore how the inlet WSRs affect the solute transport, the average
250
P´eclet number and advection-dominated volume inside the platelet aggregates are also
251
computed. Effects of increasing WSR The result indicates that the average P´eclet number inside the aggregates
252
has a positive correlation with WSRs, while the relation between advection-dominated
253
volume and WSRs is not clear. 254 To further understand the influence of the blood flow velocity on the platelet
255
aggregate formation, the shapes of the platelet aggregates formed under three shear
256
rates are shown in Fig 10. It can be observed that the platelet aggregate formed under
257
a high shear rate condition (4000 s−1) has a distinctly different shape. It is the tallest
258
out of the three, while the shape of the aggregate formed under low shear rates is the
259
flattest. 260 Platelet adhesion has been demonstrated to be strongly influenced by the
mechanosensitivity of VWF [55–58]. This protein uncoils and activates only under
certain flow conditions [59]. Previous studies have shown that VWF will unfold when
the shear rate or the rate of elongation exceeds a threshold [59,60]. Therefore, high
shear rate and elongation rate facilitate the binding of platelets and can lead to platelet
activation [61]. The appearing shear rate and elongational rate in the three cases are
investigated in Fig 10. The shear rate magnitude ˙γ is computed based on the 11/22 June 18, 2023 . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 10. Platelet aggregates geometries, shear rates and the rate of elongation. (a)-(c) Geometries of the platelet
aggregates under 800 s−1, 1600 s−1 and 4000 s−1 WSRs flow condition. (d)-(f) Cross-sectional shear rate profile under 800
s−1, 1600 s−1 and 4000 s−1 WSRs flow condition. (g)-(i) Cross-sectional elongation rate profile under 800 s−1, 1600 s−1 and
4000 s−1 WSRs flow condition. Fig 10. Platelet aggregates geometries, shear rates and the rate of elongation. (a)-(c) Geometries of the platelet
aggregates under 800 s−1, 1600 s−1 and 4000 s−1 WSRs flow condition. Effects of increasing WSR (d)-(f) Cross-sectional shear rate profile under 800
s−1, 1600 s−1 and 4000 s−1 WSRs flow condition. (g)-(i) Cross-sectional elongation rate profile under 800 s−1, 1600 s−1 and
4000 s−1 WSRs flow condition. corresponding flow rates: corresponding flow rates: ˙γ =
h
2
∂u
∂x
2
+ 2
∂v
∂y
2
+ 2
∂w
∂z
2
+
∂u
∂z + ∂w
∂x
2
+
∂u
∂y + ∂v
∂x
2
+
∂w
∂y + ∂v
∂z
2 i 1
2 ,
(7) (7) where u, v and w are the flow velocity in the x-, y- and z-directions, respectively. The
261
rate of the uni-axial elongation is defined as the magnitude of the diagonal elements of
262
the rate of the flow strain tensor [62,63]:
263 ˙ε =
s∂u
∂x
2
+
∂v
∂y
2
+
∂w
∂z
2
. (8) (8) According to [59,60,64], the threshold of elongation rate for VWF to unfold is much
264
smaller than that of the shear rate: when the shear rates reach 5000s−1 or elongation
265
flow strain rates are higher than 1000 s−1, the compact conformation of VWF will
266
change [60,65]. Hence, to observe the region where VWF unfolds in our cases, the color
267
bar maxima for results of the shear rate and the elongational flow are set to coincide
268
with the thresholds reported by these studies, namely 5000 s−1 and 1000 s−1,
269
respectively. The figure indicates that under a high shear rate condition (4000 s−1), the
270
region where the shear rate or the elongation rate exceeds the threshold, is considerably
271
larger than the region in the case of low shear rate flow conditions. This leads to a
272
higher potential for platelets to aggregate and adhere under high shear rates. 273 12/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Comparison of different ranges of porosity (b) Advection-dominated volume of ADP inside the
platelet aggregates under various shear flow conditions. (c) Advection-dominated volume of Ca2+, ADP and Factor X inside
the platelet aggregates under WSR of 1600 s−1. Comparison of different ranges of porosity 274 In this work, the porosity ranges are inferred from the local density of the platelets. The
275
measurement requires one to count the number of platelets manually in multiple regions
276
of the aggregate, which causes uncertainty (for further details see S1 File). Furthermore,
277
aggregates formed under different conditions might display different porosities. 278 In this work, the porosity ranges are inferred from the local density of the platelets. The
275
measurement requires one to count the number of platelets manually in multiple regions
276
of the aggregate, which causes uncertainty (for further details see S1 File). Furthermore,
277
aggregates formed under different conditions might display different porosities. 278
Therefore, a series of simulations based on different porosity ranges were performed to
279
quantify the possible influence of potential porosity ranges on the quantities of interest. 280
Different porosity values and different ranges of porosity were chosen and considered to
281
have a linear relation with fluorescence intensity. Fig 11 visualises the changes in
282
average blood flow velocity and advection-dominated volume inside the aggregates in
283
terms of a fixed range of porosity under three WSRs. With the same porosity settings,
284
both the average velocity and the advection-dominated volume are higher under a
285
higher WSR, which is expected. Simultaneously, the increase of the porosity, which
286
means more permeable aggregates, leads to an increase of not only the average velocity
287
but also more advection-dominated areas inside the aggregates. Moreover, the blood
288
flow behaviour under various ranges of porosity was also investigated and the results are
289
shown in Fig 12. With a wider porosity setting, both the average velocity within the
290
aggregates and the effect of advection on chemical transport increase. Overall, these
291
changes in the porosity lead to quantitative changes in the flow field, however, the
292
observed trends remain unaffected, implying the wider generality of the results. 293 Fig 11. Comparison of intrathrombus condition for different porosity values. (a) Average blood flow velocity
inside the platelet aggregates under various shear flow conditions. (b) Advection-dominated volume of ADP inside the
platelet aggregates under various shear flow conditions. (c) Advection-dominated volume of Ca2+, ADP and Factor X inside
the platelet aggregates under WSR of 1600 s−1. Fig 11. Comparison of intrathrombus condition for different porosity values. (a) Average blood flow velocity
inside the platelet aggregates under various shear flow conditions. Limitations Such a
314
phenomenon contributes to heterogeneity of viscosity in the lumen and therefore
315
influences the flow resistance and biological transport [68]. Also, the platelets and red
316
blood cells cannot penetrate platelet aggregates, which means the fluid inside the
317
aggregates is pure plasma. Its viscosity is smaller than whole blood [69,70]. A
318
simulation with plasma viscosity has been carried out for comparison. The result (S3
319
Fig) shows that there is no significant influence to the flow dynamics inside the
320
aggregates. Finally, the platelet aggregate pore wall presents barriers to the agonists,
321
resulting in a smaller agonist diffusion coefficient, which contributes to a larger P´eclet
322
number. Since the knowledge of how large the effect of the porous on the diffusion of
323
agonists is unknown, we used the diffusion coefficients that were measured in free flow. 324 Limitations 294 The proposed image-based computational model is based on several assumptions and
295
has some limitations. First, the porosity of the aggregates was inferred from the
296
fluorescence intensity of the images. However, the exposure time and exposure level in
297
the experiments can have an impact on the fluorescence intensity. How the fluorescence
298
intensity changes with the exposure time and level should also be investigated in more
299
detail to provide a more precise estimation of the platelet density. Second, the porosity
300
of the platelet aggregates is approximated by the ratio of the area occupied by platelets
301
on the images. This process requires manually counting the number of platelets, which
302
involves uncertainty and contributes to the uncertainty in the model outputs. However,
303 13/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Fig 12. Comparison of intrathrombus condition for different porosity ranges. (a) Average blood flow velocity
inside the platelet aggregates under various shear flow conditions. (b) Advection-dominated volume of ADP inside the
platelet aggregates under various shear flow conditions. (c) Advection-dominated volume of Ca2+, ADP and Factor X inside
the platelet aggregates under WSR of 1600 s−1. Fig 12. Comparison of intrathrombus condition for different porosity ranges. (a) Average blood flow velocity
inside the platelet aggregates under various shear flow conditions. (b) Advection-dominated volume of ADP inside the
platelet aggregates under various shear flow conditions. (c) Advection-dominated volume of Ca2+, ADP and Factor X inside
the platelet aggregates under WSR of 1600 s−1. the evaluation of interobserver variability was not performed. Therefore, a comparison
304
study based on different porosity settings was performed, and demonstrated how the
305
potential uncertainty in porosity ranges would affect quantities of interest, such as
306
average velocities and advection-dominated volume of agonists under three WSRs. Limitations No
307
qualitative changes have been observed due to the variety of porosity ranges. 308
Furthermore, the volume fraction result shown in Fig 6 follows our expectation on
309
platelet aggregates formed under different shear rates, i.e. the aggregates tend to be
310
denser when formed under higher shear rates. Moreover, in the computational model of
311
blood flow, the viscosity in the lumen was assumed to be constant. However, in actual
312
flow, red blood cells in the microchannel migrate toward the center of the lumen
313
resulting in a red blood cell-free layer close to the vessel wall [66,67]. Such a
314
phenomenon contributes to heterogeneity of viscosity in the lumen and therefore
315
influences the flow resistance and biological transport [68]. Also, the platelets and red
316
blood cells cannot penetrate platelet aggregates, which means the fluid inside the
317
aggregates is pure plasma. Its viscosity is smaller than whole blood [69,70]. A
318
simulation with plasma viscosity has been carried out for comparison. The result (S3
319
Fig) shows that there is no significant influence to the flow dynamics inside the
320
aggregates. Finally, the platelet aggregate pore wall presents barriers to the agonists,
321
resulting in a smaller agonist diffusion coefficient, which contributes to a larger P´eclet
322
number. Since the knowledge of how large the effect of the porous on the diffusion of
323
agonists is unknown, we used the diffusion coefficients that were measured in free flow. 324 the evaluation of interobserver variability was not performed. Therefore, a comparison
304
study based on different porosity settings was performed, and demonstrated how the
305
potential uncertainty in porosity ranges would affect quantities of interest, such as
306
average velocities and advection-dominated volume of agonists under three WSRs. No
307
qualitative changes have been observed due to the variety of porosity ranges. 308
Furthermore, the volume fraction result shown in Fig 6 follows our expectation on
309
platelet aggregates formed under different shear rates, i.e. the aggregates tend to be
310
denser when formed under higher shear rates. Moreover, in the computational model of
311
blood flow, the viscosity in the lumen was assumed to be constant. However, in actual
312
flow, red blood cells in the microchannel migrate toward the center of the lumen
313
resulting in a red blood cell-free layer close to the vessel wall [66,67]. Discussion In this work, two different microscopy image modalities were used to capture the
326
morphology and microstructure of platelet aggregates formed under in vitro blood
327
perfusion experiments. The information extracted from the images yields the geometry
328
and local porosity of the aggregates, that are subsequently applied in the computational
329
model. The image-based computational model enables an accurate prediction of
330
intrathrombus flow under various flow conditions. A separation of the core and shell of
331
the aggregates in terms of the platelet density can be observed in Fig 6. The core is
332
composed of densely packed platelets, where the transport of agonists is
333
diffusion-dominated, while the outer layer is a loose association of platelets. 334 14/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Furthermore, the blood flow velocity inside platelet aggregates observed has a
335
qualitative agreement with the result reported in [32]. However, a strip area with
336
relatively high flow velocity was found at the boundary between the shell and the core. 337
To understand what happens here, the magnitude of the blood flow velocity under three
338
WSRs was investigated. As Fig 13 shows, this situation occurs under all three WSRs. 339
This might be because the platelets in the core were excessively dense, causing the
340
circumferential flow around the core and contributing to the increase of the blood flow
341
velocity at the transition zone from shell to core. Correspondingly, the large kinetic
342
forces in the area were generated by the resistance when blood flowed from the shell to
343
the core, a much denser medium. This phenomenon may help to identify the boundary
344
between the shell and the core. 345 Fig 13. Velocity magnitude of the blood flow inside the platelet aggregates under three WSRs. (a) 800 s−1, (b)
1600 s−1 and (c) 4000 s−1. The arrows point out the high flow velocity area between the shell and the core. Fig 13. Velocity magnitude of the blood flow inside the platelet aggregates under three WSRs. (a) 800 s−1, (b)
1600 s−1 and (c) 4000 s−1. Discussion 381
The fluorescence intensity of the platelet labelling is used to infer the porosity of the
382
aggregates and the corresponding permeability is calculated via the Kozeny-Carman
383
equation. The imaged-base model allows us to study the details of hemodynamic
384
transport, such as the velocity, shear stress, and advection-diffusion of agonist transport
385
inside and around the platelet aggregates. Relatively high blood flow velocity and forces
386
were found at the transition from the shell to the core due to the compact distribution
387
of platelets in the interior core. The large forces in the area could contribute to the
388
activation of the platelets in the shell. Furthermore, the results demonstrate that both
389
the flow shear rate and aggregate microstructure have a substantial impact on the
390
transport of agonists. Finally, the shear rate and the rate of elongation flow have also
391
been investigated and the findings imply that there is a strong correlation between these
392
values and the shapes of growing platelet aggregates. Overall, the proposed
393
computational model incorporates the internal microstructure of the aggregates and
394
enables a more precise prediction of the hemodynamics in the platelet aggregates. Our
395
work lays the foundation for predicting shear-dependent aggregation and deformation
396
under different flow conditions, which could subsequently contribute to developing
397
shear-selective anti-thrombotic or anti-platelet drugs. 398 Conclusion In this work, we present an image-based computational blood flow model to simulate
378
the blood flowing through the platelet aggregates which are considered as porous media. 379
The shapes and the microstructures of the platelet aggregates are extracted from
380
microscopy image data of platelet aggregates formed in the blood perfusion experiments. 381
The fluorescence intensity of the platelet labelling is used to infer the porosity of the
382
aggregates and the corresponding permeability is calculated via the Kozeny-Carman
383
equation. The imaged-base model allows us to study the details of hemodynamic
384
transport, such as the velocity, shear stress, and advection-diffusion of agonist transport
385
inside and around the platelet aggregates. Relatively high blood flow velocity and forces
386
were found at the transition from the shell to the core due to the compact distribution
387
of platelets in the interior core. The large forces in the area could contribute to the
388
activation of the platelets in the shell. Furthermore, the results demonstrate that both
389
the flow shear rate and aggregate microstructure have a substantial impact on the
390
transport of agonists. Finally, the shear rate and the rate of elongation flow have also
391
been investigated and the findings imply that there is a strong correlation between these
392
values and the shapes of growing platelet aggregates. Overall, the proposed
393
computational model incorporates the internal microstructure of the aggregates and
394
enables a more precise prediction of the hemodynamics in the platelet aggregates. Our
395
work lays the foundation for predicting shear-dependent aggregation and deformation
396
under different flow conditions, which could subsequently contribute to developing
397
shear-selective anti-thrombotic or anti-platelet drugs. 398 Discussion The arrows point out the high flow velocity area between the shell and the core. he blood flow inside the platelet aggregates under three WSRs. (a) 800 s−1, (b)
ows point out the high flow velocity area between the shell and the core. In Table 3, no clear relation is found between advection-dominated volume and
346
WSRs. This is mainly due to the density difference of the aggregates formed under
347
three WSRs. As mentioned before, the platelet aggregate is denser under 4000 s−1
348
WSR, implying that there are more regions in the aggregate that large proteins, e.g. 349
Factor X, cannot advect by the fluid. Therefore, this leads to a smaller
350
advection-dominated volume for Factor X under 4000 s−1 WSR than that under 800
351
s−1 WSR. In contrast, for the lighter agonists such as Ca2+, the transport is not
352
significantly hampered even if the aggregate becomes relatively denser in the core. As a
353
result, advection dominates more area for the transport of lighter agonists inside the
354
platelet aggregates with the increase of the blood shear rate. 355 As mentioned before, if the shear rate or the rate of elongation exceeds a threshold,
356
the VWF will unfold and expose the A1 domain, hence supporting platelet attachment. 357
As shown in Fig 10, under high shear rate flow conditions, the shear rate and the rate of
358
elongation of most of the area around the aggregate exceed the threshold, which means
359
the aggregates are exposed to a large amount of unfolded VWF. This might contribute
360
to the easy attachment of VWF to the surface of aggregates, especially on the top of the
361
aggregates. It further explains the phenomenon that the aggregates are prone to grow
362
in parallel to the flow under a low shear rate while the ones formed under a high shear
363
rate show an aggregation tendency perpendicular to the flow direction. The results of
364
the shear rate and the rate of elongation might be further used to predict the
365
aggregation process over time. However, this observation on clot geometries is based on
366
a limited number of experiments. More experiments are required to show if it has
367
statistical significance. 368 In this work, we considered the aggregates as porous media and mainly focused on
3 15/22 June 18, 2023 . Discussion CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint the blood flow behaviour and its influence on the aggregates. However the aggregate
370
might deform, contract, or even break away during this dynamic process. A model
371
could be further developed to study the mechanical behaviour of the platelet aggregates. 372
The kinetic force exerted on the platelet aggregates by the blood flow calculated from
373
our model can be applied to this mechanical model. Combined with the experiments,
374
the break-away dynamics of the aggregates and the mechanical stress forces under
375
different environments can be further simulated and analyzed. 376 the blood flow behaviour and its influence on the aggregates. However the aggregate
370
might deform, contract, or even break away during this dynamic process. A model
371
could be further developed to study the mechanical behaviour of the platelet aggregates. 372
The kinetic force exerted on the platelet aggregates by the blood flow calculated from
373
our model can be applied to this mechanical model. Combined with the experiments,
374
the break-away dynamics of the aggregates and the mechanical stress forces under
375
different environments can be further simulated and analyzed. 376
Conclusion
377
In this work, we present an image-based computational blood flow model to simulate
378
the blood flowing through the platelet aggregates which are considered as porous media. 379
The shapes and the microstructures of the platelet aggregates are extracted from
380
microscopy image data of platelet aggregates formed in the blood perfusion experiments. References Mechanisms of platelet aggregation. Current
415
opinion in hematology. 2001;8(5):270—276. 416
doi:10.1097/00062752-200109000-00002. 417 7. Fogelson AL, Tania N. Coagulation under flow: the influence of flow-mediated
418
transport on the initiation and inhibition of coagulation. Pathophysiology of
419
Haemostasis and Thrombosis. 2005;34(2-3):91–108. doi:10.1159/000089930. 420 8. Yazdani A, Li H, Humphrey JD, Karniadakis GE. A general shear-dependent
421
model for thrombus formation. PLOS Computational Biology. 2017;13(1):1–24. 422
doi:10.1371/journal.pcbi.1005291. 423 9. Tokarev A, Sirakov I, Panasenko G, Volpert V, Shnol E, Butylin A, et al. 424
Continuous mathematical model of platelet thrombus formation in blood flow. 425
Russian Journal of Numerical Analysis and Mathematical Modelling. 426
2012;27(2):191–212. doi:doi:10.1515/rnam-2012-0011. 427 10. Mody NA, King MR. Platelet adhesive dynamics. Part I: characterization of
428
platelet hydrodynamic collisions and wall effects. Biophysical Journal. 429
2008;95(5):2539–2555. doi:https://doi.org/10.1529/biophysj.107.127670. 430 platelet hydrodynamic collisions and wall effects. Biophysical Journal. 429
2008;95(5):2539–2555. doi:https://doi.org/10.1529/biophysj.107.127670. 430
11. Mody NA, King MR. Platelet adhesive dynamics. Part II: high shear-induced
431
transient aggregation via GPIbα-vWF-GPIbα bridging. Biophysical Journal. 432
2008;95(5):2556–2574. doi:https://doi.org/10.1529/biophysj.107.128520. 433
12. Storti F, van Kempen THS, van de Vosse FN. A continuum model for platelet
434
l
f
ti
d
th I t
ti
l J
l f
N
i
l M th d i 11. Mody NA, King MR. Platelet adhesive dynamics. Part II: high shear-induced
431
transient aggregation via GPIbα-vWF-GPIbα bridging. Biophysical Journal. 432
2008;95(5):2556–2574. doi:https://doi.org/10.1529/biophysj.107.128520. 433 2008;95(5):2556 2574. doi:https://doi.org/10.1529/biophysj.107.128520. 433
12. Storti F, van Kempen THS, van de Vosse FN. A continuum model for platelet
434
plug formation and growth. International Journal for Numerical Methods in
435
Biomedical Engineering. 2014;30(6):634–658. 436
doi:https://doi.org/10.1002/cnm.2623. 437 12. Storti F, van Kempen THS, van de Vosse FN. A continuum model for platelet
434
plug formation and growth. International Journal for Numerical Methods in
435
Biomedical Engineering. 2014;30(6):634–658. 436
doi:https://doi.org/10.1002/cnm.2623. 437 13. Leiderman K, Fogelson AL. Grow with the flow: a spatial–temporal model of
438
platelet deposition and blood coagulation under flow. Mathematical Medicine and
439
Biology: A Journal of the IMA. 2010;28(1):47–84. doi:10.1093/imammb/dqq005. 440 14. Leiderman K, Fogelson AL. The influence of hindered transport on the
441
development of platelet thrombi under flow. Bulletin of Mathematical Biology. 442
2013;75(8):1255–1283. doi:10.1007/s11538-012-9784-3. 443 15. Welsh JD, Stalker TJ, Voronov R, Muthard RW, Tomaiuolo M, Diamond SL,
444
et al. A systems approach to hemostasis: 1. The interdependence of thrombus
445
architecture and agonist movements in the gaps between platelets. Blood. 446
2014;124(11):1808–1815. doi:10.1182/blood-2014-01-550335. 447 16. References Mechanisms of platelet aggregation. Current
415
opinion in hematology. 2001;8(5):270—276. 416
doi:10.1097/00062752-200109000-00002. 417
7. Fogelson AL, Tania N. Coagulation under flow: the influence of flow-mediated
418
transport on the initiation and inhibition of coagulation. Pathophysiology of
419
Haemostasis and Thrombosis. 2005;34(2-3):91–108. doi:10.1159/000089930. 420
8. Yazdani A, Li H, Humphrey JD, Karniadakis GE. A general shear-dependent
421
model for thrombus formation. PLOS Computational Biology. 2017;13(1):1–24. 422
doi:10.1371/journal.pcbi.1005291. 423
9. Tokarev A, Sirakov I, Panasenko G, Volpert V, Shnol E, Butylin A, et al. 424
Continuous mathematical model of platelet thrombus formation in blood flow. 425
Russian Journal of Numerical Analysis and Mathematical Modelling. 426
2012;27(2):191–212. doi:doi:10.1515/rnam-2012-0011. 427
10. Mody NA, King MR. Platelet adhesive dynamics. Part I: characterization of
428
platelet hydrodynamic collisions and wall effects. Biophysical Journal. 429
2008;95(5):2539–2555. doi:https://doi.org/10.1529/biophysj.107.127670. 430
11. Mody NA, King MR. Platelet adhesive dynamics. Part II: high shear-induced
431
transient aggregation via GPIbα-vWF-GPIbα bridging. Biophysical Journal. 432
2008;95(5):2556–2574. doi:https://doi.org/10.1529/biophysj.107.128520. 433
12. Storti F, van Kempen THS, van de Vosse FN. A continuum model for platelet
434
plug formation and growth. International Journal for Numerical Methods in
435
Biomedical Engineering. 2014;30(6):634–658. 436
doi:https://doi.org/10.1002/cnm.2623. 437
13. Leiderman K, Fogelson AL. Grow with the flow: a spatial–temporal model of
438
platelet deposition and blood coagulation under flow. Mathematical Medicine and
439
Biology: A Journal of the IMA. 2010;28(1):47–84. doi:10.1093/imammb/dqq005. 440
14. Leiderman K, Fogelson AL. The influence of hindered transport on the
441
development of platelet thrombi under flow. Bulletin of Mathematical Biology. 442
2013;75(8):1255–1283. doi:10.1007/s11538-012-9784-3. 443
15. Welsh JD, Stalker TJ, Voronov R, Muthard RW, Tomaiuolo M, Diamond SL,
444
et al. A systems approach to hemostasis: 1. The interdependence of thrombus
445
architecture and agonist movements in the gaps between platelets. Blood. 446
2014;124(11):1808–1815. doi:10.1182/blood-2014-01-550335. 447
16. Mirramezani M, Herbig BA, Stalker TJ, Nettey L, Cooper M, Weisel JW, et al. 448
Platelet packing density is an independent regulator of the hemostatic response
449
to injury. Journal of Thrombosis and Haemostasis. 2018;16(5):973–983. 450
doi:https://doi.org/10.1111/jth.13986. 451
17. Tomaiuolo M, Stalker TJ, Welsh JD, Diamond SL, Sinno T, Brass LF. A systems
452
approach to hemostasis: 2. Computational analysis of molecular transport in the
453
thrombus microenvironment. Blood. 2014;124(11):1816–1823. 454
doi:10.1182/blood-2014-01-550343. 455
18. Xu S, Xu Z, Kim OV, Litvinov RI, Weisel JW, Alber M. Model predictions of
456
deformation, embolization and permeability of partially obstructive blood clots
457 6. Savage B, Cattaneo M, Ruggeri Z. References 399 References
399
1. Ni H, Freedman J. Platelets in hemostasis and thrombosis: role of integrins and
400
their ligands. Transfusion and Apheresis Science. 2003;28(3):257–264. 401
doi:https://doi.org/10.1016/S1473-0502(03)00044-2. 402
2. Nesbitt WS, Westein E, Tovar-Lopez FJ, Tolouei E, Mitchell A, Fu J, et al. A
403
shear gradient–dependent platelet aggregation mechanism drives thrombus
404
formation. Nature Medicine. 2009;15(6):665–673. doi:10.1038/nm.1955. 405
3. Hou Y, Carrim N, Wang Y, Gallant RC, Marshall A, Ni H. Platelets in
406
hemostasis and thrombosis: Novel mechanisms of fibrinogen-independent platelet
407
aggregation and fibronectinmediated protein wave of hemostasis. The Journal of
408
Biomedical Research. 2015;29(jbr150602):437. doi:10.7555/JBR.29.20150121. 409
4. Jackson SP. The growing complexity of platelet aggregation. Blood. 410
2007;109(12):5087–5095. doi:https://doi.org/10.1182/blood-2006-12-027698. 411
5. Goto S, Ikeda Y, Sald´ıvar E, Ruggeri ZM. Distinct mechanisms of platelet
412
aggregation as a consequence of different shearing flow conditions. The Journal of
413
Clinical Investigation. 1998;101(2):479–486. doi:10.1172/JCI973. 414 1. Ni H, Freedman J. Platelets in hemostasis and thrombosis: role of integrins and
400
their ligands. Transfusion and Apheresis Science. 2003;28(3):257–264. 401
doi:https://doi.org/10.1016/S1473-0502(03)00044-2. 402 2. Nesbitt WS, Westein E, Tovar-Lopez FJ, Tolouei E, Mitchell A, Fu J, et al. A
403
shear gradient–dependent platelet aggregation mechanism drives thrombus
404
formation. Nature Medicine. 2009;15(6):665–673. doi:10.1038/nm.1955. 405 3. Hou Y, Carrim N, Wang Y, Gallant RC, Marshall A, Ni H. Platelets in
406
hemostasis and thrombosis: Novel mechanisms of fibrinogen-independent platelet
407
aggregation and fibronectinmediated protein wave of hemostasis. The Journal of
408
Biomedical Research. 2015;29(jbr150602):437. doi:10.7555/JBR.29.20150121. 409 4. Jackson SP. The growing complexity of platelet aggregation. Blood. 410
2007;109(12):5087–5095. doi:https://doi.org/10.1182/blood-2006-12-027698. 411 5. Goto S, Ikeda Y, Sald´ıvar E, Ruggeri ZM. Distinct mechanisms of platelet
412
aggregation as a consequence of different shearing flow conditions. The Journal of
413
Clinical Investigation. 1998;101(2):479–486. doi:10.1172/JCI973. 414 16/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint 6. Savage B, Cattaneo M, Ruggeri Z. References Mirramezani M, Herbig BA, Stalker TJ, Nettey L, Cooper M, Weisel JW, et al. 448
Platelet packing density is an independent regulator of the hemostatic response
449
to injury. Journal of Thrombosis and Haemostasis. 2018;16(5):973–983. 450
doi:https://doi.org/10.1111/jth.13986. 451 17. Tomaiuolo M, Stalker TJ, Welsh JD, Diamond SL, Sinno T, Brass LF. A systems
452
approach to hemostasis: 2. Computational analysis of molecular transport in the
453
thrombus microenvironment. Blood. 2014;124(11):1816–1823. 454
doi:10.1182/blood-2014-01-550343. 455 18. Xu S, Xu Z, Kim OV, Litvinov RI, Weisel JW, Alber M. Model predictions of
456
deformation, embolization and permeability of partially obstructive blood clots
457
under variable shear flow. Journal of The Royal Society Interface. 458
2017;14(136):20170441. doi:10.1098/rsif.2017.0441. 459 459 17/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint 19. Arrarte Terreros N, Tolhuisen ML, Bennink E, de Jong HWAM, Beenen LFM,
460
Majoie CBLM, et al. From perviousness to permeability, modelling and
461
measuring intra-thrombus flow in acute ischemic stroke. Journal of Biomechanics. 462
2020;111:110001. doi:https://doi.org/10.1016/j.jbiomech.2020.110001. 463 20. Du J, Kim D, Alhawael G, Ku DN, Fogelson AL. Clot permeability, agonist
464
transport, and platelet binding kinetics in arterial thrombosis. Biophysical
465
Journal. 2020;119(10):2102–2115. doi:https://doi.org/10.1016/j.bpj.2020.08.041. 466 21. Wufsus AR, Macera NE, Neeves KB. The hydraulic permeability of blood clots
467
as a function of fibrin and platelet density. Biophysical Journal. 468
2013;104(8):1812–1823. doi:https://doi.org/10.1016/j.bpj.2013.02.055. 469 22. Muthard RW, Diamond SL. Blood lots are rapidly assembled hemodynamic
470
sensors. Arteriosclerosis, Thrombosis, and Vascular Biology. 471
2012;32(12):2938–2945. doi:10.1161/ATVBAHA.112.300312. 472 23. Kim OV, Xu Z, Rosen ED, Alber MS. References ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint 33. Fedorov A, Beichel R, Kalpathy-Cramer J, Finet J, Fillion-Robin JC, Pujol S,
505
et al. 3D Slicer as an image computing platform for the Quantitative Imaging
506
Network. Magnetic Resonance Imaging. 2012;30(9):1323–1341. 507
doi:https://doi.org/10.1016/j.mri.2012.05.001. 508 34. Cignoni P, Callieri M, Corsini M, Dellepiane M, Ganovelli F, Ranzuglia G. 509
MeshLab: an open-source mesh processing tool. In: Scarano V, Chiara RD, Erra
510
U, editors. Eurographics Italian Chapter Conference. The Eurographics
511
Association; 2008. 512 35. Geuzaine C, Remacle JF. Gmsh: A 3-D finite element mesh generator with
513
built-in pre- and post-processing facilities. International Journal for Numerical
514
Methods in Engineering. 2009;79(11):1309–1331. 515
doi:https://doi.org/10.1002/nme.2579. 516 36. Kaviany M. Principles of heat transfer in porous media Springer. New York etc. 517
1991;. 518 37. Bear J, Bachmat Y. Introduction to modeling of transport phenomena in porous
519
media. vol. 4. Springer Science & Business Media; 2012. 520 38. Demirin H, Ozhan H, Ucgun T, Celer A, Bulur S, Cil H, et al. Normal range of
521
mean platelet volume in healthy subjects: Insight from a large epidemiologic
522
study. Thrombosis Research. 2011;128(4):358–360. 523
doi:https://doi.org/10.1016/j.thromres.2011.05.007. 524 39. Trudnowski RJ, Rico RC. Specific gravity of blood and plasma at 4 and 37 °C. 525
Clinical Chemistry. 1974;20(5):615–616. doi:10.1093/clinchem/20.5.615. 526 39. Trudnowski RJ, Rico RC. Specific gravity of blood and plasma at 4 and 37 °C. 525
Clinical Chemistry. 1974;20(5):615–616. doi:10.1093/clinchem/20.5.615. 526 40. Merrill EW, Pelletier GA. Viscosity of human blood: transition from Newtonian
527
to non-Newtonian. Journal of Applied Physiology. 1967;23(2):178–182. 528
doi:10.1152/jappl.1967.23.2.178. 529 41. Lee S, Jang S, Park Y. Measuring three-dimensional dynamics of platelet
530
activation using 3-D quantitative phase imaging. bioRxiv. 531
2019;doi:10.1101/827436. 532 42. Bath P, Butterworth R. Platelet size: measurement, physiology and vascular
533
disease. Blood coagulation & fibrinolysis : an international journal in haemostasis
534
and thrombosis. 1996;7(2):157—161. 535 43. Donea J, Huerta A. 4. In: Viscous incompressible flows. John Wiley & Sons, Ltd;
536
2003. p. 147–208. Available from:
537
https://onlinelibrary.wiley.com/doi/abs/10.1002/0470013826.ch4. 538 44. Hecht F. New development in FreeFem++. J Numer Math. 2012;20(3-4):251–265. 539 45. van der Vorst HA. References Fibrin networks regulate protein transport
473
during thrombus development. PLOS Computational Biology. 2013;9(6):1–10. 474
doi:10.1371/journal.pcbi.1003095. 475 24. Sinegre T, Milenkovic D, Teissandier D, Fully P, Bourdin J, Morand C, et al. 476
Impact of epicatechin on fibrin clot structure. European Journal of Pharmacology. 477
2021;893:173830. doi:https://doi.org/10.1016/j.ejphar.2020.173830. 478 25. Kozeny J. Uber kapillare Leitung der Wasser im Boden. Royal Academy of
479
Science, Vienna, Proc Class I. 1927;136:271–306. 480 26. Carman PC. Permeability of saturated sands, soils and clays. The Journal of
481
Agricultural Science. 1939;29(2):262–273. doi:10.1017/S0021859600051789. 482 27. Slaboch CL, Alber MS, Rosen ED, Ovaert TC. Mechano-rheological properties of
483
the murine thrombus determined via nanoindentation and finite element
484
modeling. Journal of the Mechanical Behavior of Biomedical Materials. 485
2012;10:75–86. doi:https://doi.org/10.1016/j.jmbbm.2012.02.012. 486 28. Kadri OE, Chandran VD, Surblyte M, Voronov RS. In vivo measurement of
487
blood clot mechanics from computational fluid dynamics based on intravital
488
microscopy images. Computers in Biology and Medicine. 2019;106:1–11. 489
doi:https://doi.org/10.1016/j.compbiomed.2019.01.001. 490 29. Wang W, Lindsey JP, Chen J, Diacovo TG, King MR. Analysis of early
491
thrombus dynamics in a humanized mouse laser injury model. Biorheology. 492
2014;51:3–14. doi:10.3233/BIR-130648. 493 30. Taylor JO, Witmer KP, Neuberger T, Craven BA, Meyer RS, Deutsch S, et al. In
494
vitro quantification of time dependent thrombus size using magnetic resonance
495
imaging and computational simulations of thrombus surface shear stresses. 496
Journal of Biomechanical Engineering. 2014;136(7). doi:10.1115/1.4027613. 497 31. Pinar IP, Arthur JF, Andrews RK, Gardiner EE, Ryan K, Carberry J. Methods
498
to determine the Lagrangian shear experienced by platelets during thrombus
499
growth. PLOS ONE. 2015;10(12):1–14. doi:10.1371/journal.pone.0144860. 500 32. Voronov RS, Stalker TJ, Brass LF, Diamond SL. Simulation of intrathrombus
501
fluid and solute transport ssing in vivo clot structures with single platelet
502
resolution. Annals of Biomedical Engineering. 2013;41(6):1297–1307. 503
doi:10.1007/s10439-013-0764-z. 504 18/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. References Iterative Krylov methods for large linear systems. Cambridge
540
Monographs on Applied and Computational Mathematics. Cambridge University
541
Press; 2003. 542 46. Balay S, Abhyankar S, Adams MF, Benson S, Brown J, Brune P, et al.. PETSc
543
Web page; 2022. https://petsc.org/. Available from: https://petsc.org/. 544 47. Sakariassen KS, Orning L, Turitto VT. The impact of blood shear rate on arterial
545
thrombus formation. Future Science OA. 2015;1(4). doi:10.4155/fso.15.28. 546 19/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint 48. Miyazaki Y, Nomura S, Miyake T, Kagawa H, Kitada C, Taniguchi H, et al. High
547
shear stress can initiate both platelet aggregation and shedding of procoagulant
548
containing microparticles. Blood. 1996;88(9):3456–3464. 549
doi:https://doi.org/10.1182/blood.V88.9.3456.bloodjournal8893456. 550 49. Reininger AJ, Heijnen HFG, Schumann H, Specht HM, Schramm W, Ruggeri ZM. 551
Mechanism of platelet adhesion to von Willebrand factor and microparticle
552
formation under high shear stress. Blood. 2006;107(9):3537–3545. 553
doi:https://doi.org/10.1182/blood-2005-02-0618. 554 50. Varga-Szabo D, Braun A, Nieswandt B. Calcium signaling in platelets. Journal of
555
thrombosis and haemostasis : JTH. 2009;7(7):1057—1066. 556
doi:10.1111/j.1538-7836.2009.03455.x. 557 51. Rickles FR, Falanga A. Molecular basis for the relationship between thrombosis
558
and cancer. Thrombosis Research. 2001;102(6):V215–V224. 559
doi:https://doi.org/10.1016/S0049-3848(01)00285-7. 560 52. Folie BJ, McIntire LV. Mathematical analysis of mural thrombogenesis. 561
Concentration profiles of platelet-activating agents and effects of viscous shear
562
flow. Biophysical Journal. 1989;56(6):1121–1141. 563
doi:https://doi.org/10.1016/S0006-3495(89)82760-2. 564 53. Levine HA, McGee MP, Serna S. Diffusion and reaction in the cell glycocalyx
565
and the extracellular matrix. Journal of Mathematical Biology. 2010;60(1):1–26. 566
doi:10.1007/s00285-009-0254-y. 567 54. Teeraratkul C, Mukherjee D. References Microstructure aware modeling of biochemical
568
transport in arterial blood clots. Journal of Biomechanics. 2021;127:110692. 569
doi:https://doi.org/10.1016/j.jbiomech.2021.110692. 570 55. Sadler JE. Biochemistry and genetics of von Willebrand factor. Annual Review
571
of Biochemistry. 1998;67(1):395–424. doi:10.1146/annurev.biochem.67.1.395. 572 56. Savage B, Almus-Jacobs F, Ruggeri ZM. Specific synergy of multiple
573
substrate–receptor interactions in platelet thrombus formation under flow. Cell. 574
1998;94(5):657–666. doi:https://doi.org/10.1016/S0092-8674(00)81607-4. 575 57. Ruggeri ZM, Orje JN, Habermann R, Federici AB, Reininger AJ. 576
Activation-independent platelet adhesion and aggregation under elevated shear
577
stress. Blood. 2006;108(6):1903–1910. doi:10.1182/blood-2006-04-011551. 578 58. Zhang X, Halvorsen K, Zhang CZ, Wong WP, Springer TA. Mechanoenzymatic
579
cleavage of the ultralarge vascular protein von Willebrand factor. Science. 580
2009;324(5932):1330–1334. doi:10.1126/science.1170905. 581 59. Sing CE, Alexander-Katz A. Elongational flow induces the unfolding of von
582
Willebrand factor at physiological flow rates. Biophysical Journal. 583
2010;98(9):L35–L37. doi:https://doi.org/10.1016/j.bpj.2010.01.032. 584 60. Schneider SW, Nuschele S, Wixforth A, Gorzelanny C, Alexander-Katz A, Netz
585
RR, et al. Shear-induced unfolding triggers adhesion of von Willebrand factor
586
fibers. Proceedings of the National Academy of Sciences. 2007;104(19):7899–7903. 587
doi:10.1073/pnas.0608422104. 588 61. van Rooij BJM, Z´avodszky G, Hoekstra AG, Ku DN. Biorheology of occlusive
589
thrombi formation under high shear: in vitro growth and shrinkage. Scientific
590
Reports. 2020;10(1):18604. doi:10.1038/s41598-020-74518-7. 591 20/22 June 18, 2023 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint 62. Petrie CJS. Extensional viscosity: A critical discussion. Journal of
592
Non-Newtonian Fluid Mechanics. 2006;137(1):15–23. 593
doi:https://doi.org/10.1016/j.jnnfm.2006.01.011. 594 62. Petrie CJS. Extensional viscosity: A critical discussion. Journal of
592
Non-Newtonian Fluid Mechanics. 2006;137(1):15–23. 593
doi:https://doi.org/10.1016/j.jnnfm.2006.01.011. 594
63. Spieker CJ, Z´avodszky G, Mouriaux C, van der Kolk M, Gachet C, Mangin PH,
595
et al. The effects of micro-vessel curvature induced elongational flows on platelet
596
adhesion. Annals of Biomedical Engineering. 2021;49(12):3609–3620. 597
doi:10.1007/s10439-021-02870-4. 598
64. Wang Y, Morabito M, Zhang XF, Webb E, Oztekin A, Cheng X. References Shear-induced
599
extensional response behaviors of tethered von Willebrand factor. Biophysical
600
Journal. 2019;116(11):2092–2102. doi:https://doi.org/10.1016/j.bpj.2019.04.025. 601
65. Kania S, Oztekin A, Cheng X, Zhang XF, Webb E. Predicting pathological von
602
Willebrand factor unraveling in elongational flow. Biophysical Journal. 603
2021;120(10):1903–1915. doi:https://doi.org/10.1016/j.bpj.2021.03.008. 604
66. Kim S, Ong PK, Yalcin O, Intaglietta M, Johnson PC. The cell-free layer in
605
microvascular blood flow. Biorheology. 2009;46:181–189. 606
doi:10.3233/BIR-2009-0530. 607
67. Fedosov DA, Caswell B, Popel AS, Karniadakis GE. Blood flow and cell-free
608
layer in microvessels. Microcirculation. 2010;17(8):615–628. 609
doi:https://doi.org/10.1111/j.1549-8719.2010.00056.x. 610
68. Zhang J, Johnson PC, Popel AS. Effects of erythrocyte deformability and
611
aggregation on the cell free layer and apparent viscosity of microscopic blood
612
flows. Microvascular Research. 2009;77(3):265–272. 613
doi:https://doi.org/10.1016/j.mvr.2009.01.010. 614
69. Nader E, Skinner S, Romana M, Fort R, Lemonne N, Guillot N, et al. Blood
615
rheology: key parameters, impact on blood flow, role in sickle cell disease and
616
effects of exercise. Frontiers in Physiology. 2019;10. doi:10.3389/fphys.2019.01329. 617
70. Windberger U, Bartholovitsch A, Plasenzotti R, Korak KJ, Heinze G. Whole
618
lood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian
619
species: reference values and comparison of data. Experimental Physiology. 620
2003;88(3):431–440. doi:https://doi.org/10.1113/eph8802496. 621 63. Spieker CJ, Z´avodszky G, Mouriaux C, van der Kolk M, Gachet C, Mangin PH,
595
et al. The effects of micro-vessel curvature induced elongational flows on platelet
596
adhesion. Annals of Biomedical Engineering. 2021;49(12):3609–3620. 597
doi:10.1007/s10439-021-02870-4. 598 64. Wang Y, Morabito M, Zhang XF, Webb E, Oztekin A, Cheng X. Shear-induced
599
extensional response behaviors of tethered von Willebrand factor. Biophysical
600
Journal. 2019;116(11):2092–2102. doi:https://doi.org/10.1016/j.bpj.2019.04.025. 601 65. Kania S, Oztekin A, Cheng X, Zhang XF, Webb E. Predicting pathological von
602
Willebrand factor unraveling in elongational flow. Biophysical Journal. 603
2021;120(10):1903–1915. doi:https://doi.org/10.1016/j.bpj.2021.03.008. 604 66. Kim S, Ong PK, Yalcin O, Intaglietta M, Johnson PC. The cell-free layer in
605
microvascular blood flow. Biorheology. 2009;46:181–189. 606
doi:10.3233/BIR-2009-0530. 607 /
67. Fedosov DA, Caswell B, Popel AS, Karniadakis GE. Blood flow and cell-free
608
layer in microvessels. Microcirculation. 2010;17(8):615–628. 609
doi:https://doi.org/10.1111/j.1549-8719.2010.00056.x. 610
68. Zhang J, Johnson PC, Popel AS. Effects of erythrocyte deformability and
611
aggregation on the cell free layer and apparent viscosity of microscopic blood
612
flows. Microvascular Research. 2009;77(3):265–272. 613
doi:https://doi.org/10.1016/j.mvr.2009.01.010. 614
69. Nader E, Skinner S, Romana M, Fort R, Lemonne N, Guillot N, et al. References Blood
615
rheology: key parameters, impact on blood flow, role in sickle cell disease and
616
ff
t
f
i
F
ti
i
Ph
i l
2019 10 d i 10 3389/f h
2019 01329 67. Fedosov DA, Caswell B, Popel AS, Karniadakis GE. Blood flow and cell-free
608
layer in microvessels. Microcirculation. 2010;17(8):615–628. 609
doi:https://doi.org/10.1111/j.1549-8719.2010.00056.x. 610 68. Zhang J, Johnson PC, Popel AS. Effects of erythrocyte deformability and
611
aggregation on the cell free layer and apparent viscosity of microscopic blood
612
flows. Microvascular Research. 2009;77(3):265–272. 613
doi:https://doi.org/10.1016/j.mvr.2009.01.010. 614 69. Nader E, Skinner S, Romana M, Fort R, Lemonne N, Guillot N, et al. Blood
615
rheology: key parameters, impact on blood flow, role in sickle cell disease and
616
effects of exercise. Frontiers in Physiology. 2019;10. doi:10.3389/fphys.2019.01329. 617 70. Windberger U, Bartholovitsch A, Plasenzotti R, Korak KJ, Heinze G. Whole
618
lood viscosity, plasma viscosity and erythrocyte aggregation in nine mammalian
619
species: reference values and comparison of data. Experimental Physiology. 620
2003;88(3):431–440. doi:https://doi.org/10.1113/eph8802496. 621 June 18, 2023 21/22 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted June 18, 2023. ;
https://doi.org/10.1101/2023.02.22.529480
doi:
bioRxiv preprint Supporting information 622 S1 Fig. Intensity of the fluorescence, corresponding porosity and the
623
permeability of the platelet aggregates under 800 s−1 and 4000 s−1 WSRs. 624
(a)-(c) Intensity of the fluorescence, corresponding porosity and permeability of the
625
platelet aggregate on the cross-sections of the aggregate under 800 s−1 WSR. (d)-(f)
626
Intensity of the fluorescence, corresponding porosity and permeability of the platelet
627
aggregate on the cross-sections of the aggregate under 4000 s−1 WSR. 628 S1 Fig. Intensity of the fluorescence, corresponding porosity and the
623
permeability of the platelet aggregates under 800 s−1 and 4000 s−1 WSRs. 624
(a)-(c) Intensity of the fluorescence, corresponding porosity and permeability of the
625
platelet aggregate on the cross-sections of the aggregate under 800 s−1 WSR. (d)-(f)
626
Intensity of the fluorescence, corresponding porosity and permeability of the platelet
627
aggregate on the cross-sections of the aggregate under 4000 s−1 WSR. 628 S2 Fig. Stress analysis of the blood flow and the platelet aggregate under
629
800 s−1 and 4000 s−1 WSRs. (a)-(b) The kinetic force exerted on the platelet
630
aggregate and the fluid shear stress on the surface of the platelet aggregate under 800
631
s−1 WSR. (c)-(d) The kinetic force exerted on the platelet aggregate and the fluid shear
632
stress on the surface of the platelet aggregate under 4000 s−1 WSR. 633
S3 Fig. Flow field at WSR of 1600 s−1 with plasma viscosity. (a) The velocity
634
field of the blood flow on a cross-section of the flow domain. (b) The velocity field of
635
the blood flow inside the platelet aggregate on the cross-sections. 636
S1 File. Platelet density measurement
637 S2 Fig. Stress analysis of the blood flow and the platelet aggregate under
629
800 s−1 and 4000 s−1 WSRs. (a)-(b) The kinetic force exerted on the platelet
630
aggregate and the fluid shear stress on the surface of the platelet aggregate under 800
631
s−1 WSR. (c)-(d) The kinetic force exerted on the platelet aggregate and the fluid shear
632
stress on the surface of the platelet aggregate under 4000 s−1 WSR. 633 S2 Fig. Stress analysis of the blood flow and the platelet aggregate under
629
800 s−1 and 4000 s−1 WSRs. (a)-(b) The kinetic force exerted on the platelet
630
aggregate and the fluid shear stress on the surface of the platelet aggregate under 800
631
s−1 WSR. Supporting information (c)-(d) The kinetic force exerted on the platelet aggregate and the fluid shear
632
stress on the surface of the platelet aggregate under 4000 s−1 WSR. 633 S3 Fig. Flow field at WSR of 1600 s−1 with plasma viscosity. (a) The velocity
634
field of the blood flow on a cross-section of the flow domain. (b) The velocity field of
635
the blood flow inside the platelet aggregate on the cross-sections. 636 S1 File. Platelet density measurement S1 File. Platelet density measurement 637 22/22 June 18, 2023
|
https://openalex.org/W2810368332
|
http://researchspace.bathspa.ac.uk/11371/1/11371.pdf
|
English
| null |
Monitoring Biological and Chemical Trends in Temperate Still Waters Using Citizen Science
|
Water
| 2,018
|
cc-by
| 9,597
|
Received: 11 April 2018; Accepted: 14 June 2018; Published: 25 June 2018 Abstract: The involvement of volunteers in the monitoring of the environment holds great potential
to gather information on a wider temporal and spatial scale than is currently possible. However,
the mass involvement of citizens in monitoring freshwater health is a relatively new field and
subject to uncertainty. Here, we examine 1192 samples collected across 46 temperate ponds (<2 ha)
and 29 temperate lakes (>2 ha) by 120 volunteers trained through the FreshWater Watch citizen
science programme to consider if the approach is able to (a) identify well established patterns in
water quality and biological indicators (i.e., fish), and (b) provide a potentially useful basis for the
identification of pollution sources in urban or peri-urban landscapes. Seasonal patterns observed
agreed well with established principles of nutrient dynamics, algal bloom seasonality, and broad
biological trends between ponds and lakes. Further, observational data collected by the volunteers
suggested plausible links between the presence of residential discharge and water level fluctuation
and significant increases in algal bloom observations between peri-urban and urban sites. We suggest
that citizen science can have a role to play in complementing regulatory monitoring efforts and that
local citizens should be empowered to become stewards of their local freshwater resources. Keywords: still waters; citizen observatories; water quality; urbanization; nutrient dynamics;
algal blooms water water water Water 2018, 10, 839; doi:10.3390/w10070839 Monitoring Biological and Chemical Trends in
Temperate Still Waters Using Citizen Science Ian Thornhill 1,2,* ID , Alice Chautard 3 and Steven Loiselle 1,4 ID 1
Earthwatch Institute (Europe), Mayfield House, 256 Banbury Road, Summertown, Oxford OX2 7DE, UK;
sloiselle@earthwatch.org.uk 2
College of Liberal Arts (CoLA), Bath Spa University, Newton St. Loe, Bath BA2 9BN, UK
3
School of Geography and the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK;
alice.chautard@gmail.com 2
College of Liberal Arts (CoLA), Bath Spa University, Newton St. Loe, Bath BA2 9BN, UK
3
School of Geography and the Environment, University of Oxford, South Parks Road, Oxford OX1 3QY, UK;
alice.chautard@gmail.com g
4
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2,
53100 Siena, Italy 4
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2,
53100 Siena, Italy 4
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via Aldo Moro 2,
53100 Siena, Italy *
Correspondence: ian.thornhill@live.co.uk; Tel.: +44-(0)122-587-6329 Received: 11 April 2018; Accepted: 14 June 2018; Published: 25 June 2018 water water 1. Introduction The number and importance of ponds and small lakes far outweighs the attention given to
their study and management [1]. Small waterbodies (<2 ha) contain a disproportionate amount
of freshwater biodiversity [2,3] and provide ecosystem services with important local and global
benefits [4]. Yet, the conventional conservation and monitoring priorities have been focused on
large lakes and reservoirs. For example, the EU Water Framework Directive 2000/16/EC excludes the
majority of small water bodies [5]. This dilemma presents an opportunity for the collection of ecological
information regarding these local water bodies using non-conventional monitoring approaches [6]. One such rapidly emerging approach is citizen science, the participation of ordinary members of the
public in scientific research [7,8]. However its effectiveness remains largely untested [9]. Seasonal fluctuations in nutrient status and algal growth patterns in temperate lakes are relatively
well understood thanks to long term lake monitoring (e.g., North Temperate Lakes) and shallow
lakes research [10]. In contrast to deep, stratified lakes, the intense sediment–water interface in Water 2018, 10, 839; doi:10.3390/w10070839 www.mdpi.com/journal/water www.mdpi.com/journal/water 2 of 15 Water 2018, 10, 839 shallow ponds and lakes ensures a rapid return of most sediment material into the water column [11]. In addition, the relatively high sediment temperatures in summer lead to an increase in mineralization
rates, and consequently to an increased release of phosphorous from the sediment into its soluble
form [12]. Thus, a summer peak in phosphate (P-PO4) concentrations is observed. By contrast, soluble
nitrate peak concentrations (N-NO3) typically occur during the winter months after a period of plant
senescence, with minima in early summer following aquatic vegetation growth. Algal growth patterns
reflect nutrient availability in the water column. In the first instance, internally loaded phosphate can
result in algal blooms during early summer [13]. Secondarily, an autumn bloom may be observed
under suitable temperature and light conditions in response to nutrients released from decaying plant
material [11]. In addition, fish presence is increasingly likely in larger water bodies [14,15]. These same
patterns should be easily identifiable through a citizen science approach. Shallow lakes are often considered more susceptible to local and global environmental change
than deeper water bodies, because of their limited volume and depth [10] such that the effect of
urbanization upon them could be profound and greatly interfere with natural processes [16], as
in urban streams [17]. 1. Introduction The presence of fish will be recorded with greater frequency in lakes (>2 ha) than in ponds (<2 ha). 3. The effect of urbanization would be more pronounced in lakes than in ponds. p
g
q
y
(
)
p
(
)
3. The effect of urbanization would be more pronounced in lakes than in ponds. 3. The effect of urbanization would be more pronounced in lakes than in ponds. Finally, we applied the field data to highlight any potential drivers of those water chemistry
factors that exhibited a clear difference between urban and peri-urban sites. Finally, we applied the field data to highlight any potential drivers of those water chemistry
factors that exhibited a clear difference between urban and peri-urban sites. 1. Introduction By contrast, the typically reduced catchment area of ponds allows for the
possibility that these ecosystems can avoid some of the impacts that plague water bodies with large
catchments [18,19]. Previously cited effects of urbanization upon the water quality of still waters are
nutrient concentration increases [19,20], greater frequency of algal blooms [21] as well as increased
trace metal concentrations and the presence of invasive species [16]. However, it is not clear whether
these effects hold true for smaller still waters such as ponds (<2 ha [22]) as they do lakes. In addition to supporting scientific research and environmental monitoring, citizen science also
provides potential benefits associated with environmental awareness and education and advocacy on
environmental issues [23,24]. FreshWater Watch (FWW) is the first global citizen science research project
studying freshwater ecosystem dynamics. Within FWW, 30 local projects in more than 20 countries
have been established; generating information on more than 1500 waterbodies, many of which are
small ponds and lakes, in both temperate and tropical regions that hold local cultural importance (https:
//freshwaterwatch.thewaterhub.org/). Through the participation of citizen scientists, researchers
have been able to gather data over a large spatial and temporal scale [25]. Using data collection at
such resolution, it should be possible to explore if expected seasonal dynamics, reported for large
waterbodies, are also the dominant trends in smaller water bodies and if drivers such as urbanization
or an elevated surface to volume ratio lead to modified seasonal dynamics. This study represents a first attempt to compare seasonal trends between lake and ponds using
citizen science gathered data. We explore the effect of increasing urbanization on the seasonal chemical
and biological conditions of ponds and lakes. We use a dataset of 1192 measurements from 75 temperate
still waters in three continents. Field data were gathered by trained citizen scientists and combined
with freely available, global datasets. Using this data, the following hypotheses were tested: 1. Seasonal fluctuations would be detected in four parameters indicative of water quality; nitrate
(N-NO3), phosphate (P-PO4), turbidity (NTU), and frequency of algal blooms. 1. Seasonal fluctuations would be detected in four parameters indicative of water quality; nitrate
(N-NO3), phosphate (P-PO4), turbidity (NTU), and frequency of algal blooms. 2. The presence of fish will be recorded with greater frequency in lakes (>2 ha) than in ponds (<2 ha). 3. The effect of urbanization would be more pronounced in lakes than in ponds. 2. 2.1. Study Area 1192 samples were used in the present study taken from 75 ponds (see Supplementary Material
Table S1) and lakes (mean 47,956 m2, min. 10 m2, max. 444,745 m2) within temperate broadleaf,
coniferous, and mixed forests, as defined within terrestrial ecosystems of the world [26], across 3 of 15
Material
adleaf Water 2018, 10, 839
1192 sampl
T bl
S1)
d l the continents of Europe, North America, and Oceania (Figure 1). Sites were located in urban and
peri-urban areas as part of the FWW programme and are mostly manmade having been built for a
range of purposes including recreation (e.g., fishing) and infrastructure (e.g., stormwater lagoons). All data are uploaded and open access (freshwaterwatch.thewaterhub.org/content/data-map). coniferous, and mixed forests, as defined within terrestrial ecosystems of the world [26], across the
continents of Europe, North America, and Oceania (Figure 1). Sites were located in urban and
peri-urban areas as part of the FWW programme and are mostly manmade having been built for a
range of purposes including recreation (e.g., fishing) and infrastructure (e.g., stormwater lagoons). All data are uploaded and open access (freshwaterwatch.thewaterhub.org/content/data-map). Figure 1. Distribution of 75 ponds and lakes used in the present study located in temperate biomes
across three continents (six countries). Figure 1. Distribution of 75 ponds and lakes used in the present study located in temperate biomes
across three continents (six countries). Figure 1. Distribution of 75 ponds and lakes used in the present study located in temperate biomes
across three continents (six countries). Figure 1. Distribution of 75 ponds and lakes used in the present study located in temperate biomes
across three continents (six countries). Data were collected by 120 trained citizen scientists (from >8000 total trained) using
standardized methods. Quality control against standard methods was conducted with
approximately 10% of the samples [27,28]. Measurements and observations of algal blooms were
also checked automatically and by local partner scientists. Individual sites were only included in the
analysis if they were sampled across the four seasons between 1 June 2014 and 31 May 2016 (Table 1). To identify seasonal shifts between different continents, average monthly temperatures (between the
year 2000 and 2012 [29]) were obtained for each locality (e.g., nearest town or city) and apportioned
into quarterly periods. This allowed for direct comparison of seasonal data between global sites. Data were collected by 120 trained citizen scientists (from >8000 total trained) using standardized
methods. 2.1. Study Area Quality control against standard methods was conducted with approximately 10% of the
samples [27,28]. Measurements and observations of algal blooms were also checked automatically and
by local partner scientists. Individual sites were only included in the analysis if they were sampled
across the four seasons between 1 June 2014 and 31 May 2016 (Table 1). To identify seasonal shifts
between different continents, average monthly temperatures (between the year 2000 and 2012 [29])
were obtained for each locality (e.g., nearest town or city) and apportioned into quarterly periods. This
allowed for direct comparison of seasonal data between global sites. Table 1. Data summary for FreshWater Watch temperate still water sites and samples (in
parentheses) between 1 June 2014 and 31 May 2016. North America
Europe
Oceania
Totals
<0.2 ha
0 (0)
15 (135)
0 (0)
15 (135)
>0.2 ha <2 ha
7 (113)
22 (543)
2 (29)
31 (685)
>2 ha <20 ha
2 (33)
20 (215)
1 (43)
23 (291)
>20 ha
1 (10)
5 (71)
0 (0)
6 (81)
Totals
10 (156)
63 (964)
3 (72)
75 (1192)
2.2. Field Measurements
E
h d
i
d
b
i
d
f
di i
h d
l
Table 1. Data summary for FreshWater Watch temperate still water sites and samples (in parentheses)
between 1 June 2014 and 31 May 2016. North America
Europe
Oceania
Totals
<0.2 ha
0 (0)
15 (135)
0 (0)
15 (135)
>0.2 ha <2 ha
7 (113)
22 (543)
2 (29)
31 (685)
>2 ha <20 ha
2 (33)
20 (215)
1 (43)
23 (291)
>20 ha
1 (10)
5 (71)
0 (0)
6 (81)
Totals
10 (156)
63 (964)
3 (72)
75 (1192)
2.2. Field Measurements Table 1. Data summary for FreshWater Watch temperate still water sites and samples (in
parentheses) between 1 June 2014 and 31 May 2016. Table 1. Data summary for FreshWater Watch temperate still water sites and samples (in parentheses)
between 1 June 2014 and 31 May 2016. 2.2. Field Measurements
E
h d t
t
t
2.2. Field Measurements 2.2. Field Measurements
E
h d t
t
t
2.2. Field Measurements Each dataset contained observations and measurements of ecosystem conditions, hydrology,
and water quality, collected using consistent methods. General ecosystem conditions included
Each dataset contained observations and measurements of ecosystem conditions, hydrology, and
water quality, collected using consistent methods. General ecosystem conditions included observations
of the land use/cover in the immediate surroundings of the sampling site, visible evidence of pollution
sources (e.g., discharge pipes) with estimates of their potential sources (urban or road runoff/drainage,
residential, industrial, other) and the presence of bank side vegetation. These observations were limited
to the immediate area of the sampling site, in general less than 25 m in both directions. Menu-based 4 of 15 Water 2018, 10, 839 observations of water color, the presence of and algal blooms were also recorded for each site and
supported by photographic documentation. Measurements of dissolved phosphate (P-PO4) and nitrate (N-NO3) concentrations were
performed from unfiltered samples using colorimetric methods. The method allowed for in situ
estimates of dissolved nutrients with exposure to reagents occurring within closed sample tubes,
a method appropriate for a mass citizen science programme. Nitrate was measured using a
naphthylethylenediamine method (Griess reagent) [30,31] in seven specific ranges from 0.2 to 0.5, 1, 2, 5,
and 10.0 mg/L. Phosphate used a low-range enzymatic method (enzyme and 4-aminoantipyrine) [32]
also in seven ranges (from 0.02 to 0.05, 0.1, 0.2, 0.5, and 1 mg/L) (Kyoritsu RIKEN, Tokyo, Japan). Turbidity (NTU, LOD 12–240) was measured using a calibrated Secchi tube [33,34], while water color
and algal blooms were compared to photographic references present in the program app and data
sheets. All data were collected within a quality assurance and control framework (Table 2). The presence of fish was recorded either directly or through evidence of fishing. The frequency
of sampling occasions with fish presence was used as a proxy for fish abundance. Participants are
also trained to differentiate between different macrophyte stands and vegetation complexity was
calculated as the mean of the percentage of samples at each site that recorded the presence or absence
of emergent, submerged, or floating vegetation. The presence of bare bank side, bank side grass, and
trees or shrubs and potential point pollution sources (urban/road, residential, industrial, other) were
expressed as binary variables as was the immediate dominant land use (predominately urban park,
urban residential). The sum of different potential point pollution sources was also calculated (0 to 4). 2.2. Field Measurements
E
h d t
t
t
2.2. Field Measurements Fluctuation in water level was assessed as the proportion of observations recorded as low or high on
a three-point scale (i.e., not average). The presence or absence of algal blooms was averaged across
samples within each season and each site as a proxy for algal bloom frequency [35]. Table 2. Quality assurance and control framework within the FreshWater Watch programme. Table 2. Quality assurance and control framework within the FreshWater Watch programme. Table 2. Quality assurance and control framework within the FreshWater Watch programme. Prevention
Detection
Training
Feedback
Train-the-trainer events
Automatic (qualitative and quantitative)
One-day experiential learning event
Principal Investigator (local partner)
Mandatory research quiz
Global research team (Earthwatch)
Recording form instruction
Quality control
Voluntary online modular learning
10% validity check by PI [28]
Method testing
Accredited laboratory [36,37]
Independent testing [33,38]
Manual database checking protocol [37]
Photographs 2.3. Statistical Methods To consider the presence of seasonal trends in water quality, at each site seasonal median values
were calculated for nitrate and phosphate measurements (mg/L, ordinal scale) and mean values for
turbidity (NTU, numerical continuous), thus removing any effect of uneven sampling. Study sites were then classified into two surface area categories to test for differences between ponds
and lakes. The classes related to a widely accepted pond definition whereby ponds are those bodies of
water that hold water for at least four months of the year and are between 1 m2 and 2 ha in surface area
that are typically shallow enough to allow for rooted macrophyte growth throughout [22,39]. Urbanization was calculated within a Geographical Information System (ESRI ArcMap 10.4) for a
1 km buffer from each water body using two metrics related to land-use. These were the percentage
cover of artificial surfaces, which was derived using the GLC-SHARE dataset [40] (range 0–100) and
population density from a gridded population of the world [41] (range 34.9 to 17,682 people/km2). Both global datasets have a 30 arc-second (~1 km2) resolution. Each metric was standardized (0 to
1 scale) and the mean of both used as a broad indicator of surrounding urbanization at each site. Those with urbanization values greater than the median (0.53) were thus considered urban (sites = 37), 5 of 15 Water 2018, 10, 839 with those less than this figure peri-urban (38). Data groupings (e.g., seasonal, pond vs. lake, urban vs. peri-urban) were tested for significant differences using non-parametric methods e.g., Kruskal–Wallis
test for multiple groups with the Dunn post hoc test where significant differences were present and
Mann–Whitney for paired comparisons. We also tested for differences in group variances using the
Brown–Forsythe test. All statistical analyses were carried out in R 3.3.2 [42]. y
y
To examine the underlying factors contributing to water quality differences between sites
within urban or peri-urban areas we used multiple linear regression incorporating the observational
components of the FWW recording form. All possible combinations were tested using an
AIC–information theoretic (AIC–IT) approach [43] limiting any single model to no more than three
predictor variables in order to retain statistical power. 3. Results We used a test set of 75 study sites distributed across three continents to analyze patterns relating to
seasonality, water body size, and the influence of urbanization. Median nitrate (N-NO3) concentration
of across all study sites was 0.10 mg/L (SD 0.94), however 10 European sites (nine UK and one
France) equaled or exceeded median concentrations of 1.0 mg/L. Median concentration of phosphate
(P-PO4) across all study sites was 0.016 mg/L (SD 0.036) with four sites (two UK and two Netherlands)
exceeding median concentrations of 0.1 mg/L. Mean turbidity was 24.1 NTU (SD 19.9) and algal
blooms were typically observed in one in three visits (31.5%). 2.3. Statistical Methods The AIC–IT approach is recognized as a solution
to overcoming problems of null hypothesis testing (e.g., a priori false null hypotheses and arbitrary
significance levels), and as a means for making inferences based on statistics weighted by support from
several models [44,45]. Multiple models are simultaneously compared using a likelihood measure (we
used AICc to account for small sample sizes) and an Akaike weight (wi), a measure of the probability
that the model i in question would be the best-fitting model of the candidate models, were the data
collected again under the same circumstances. The models are then ranked and the best set of models
identified using wi. Multi-model inferences can then be made through the use of wi by summing this
statistic across all models. The relative importance of predictor variables in this study was measured
using the cumulative probability (w + (j)), namely the sum of w values for all the models in which the
predictor of interest occurred. To assess model power and appropriateness the AIC-best predictors
were ran in a separate model and residual plots examined. Variance inflation factors (VIF) were
calculated to assess for covariance amongst predictors (where VIF > 2). 3.1. Temporal Trends in Water Quality Temporal patterns in water quality were comparable across all study regions (Figure 2) with
statistically significant peaks in nitrate concentration during winter with summer and autumn minima
(Figure 2a). Phosphate showed less variation across the seasons, although a spring–summer maxima
and autumn–winter minima were observed (Figure 2b). Observations in algal blooms peaked during the summer months where 42.5% of the datasets
contained observations of algal blooms, and were significantly less frequently observed in the winter
(Figure 2d). North American sites had notably more algal bloom observations during the summer
and autumn than European sites where blooms were more frequently observed in the winter (see
Supplementary Material Table S2). Turbidity followed a similar trend (Figure 2c). A weak but highly
significant correlation was observed at a site level between algal bloom frequency and turbidity (ρ 0.12,
P < 0.01). 6 of 15
idity (ρ Water 2018, 10, 839
significant corre
0 12 P < 0 01) Figure 2. Comparison of nutrient concentrations (a,b), turbidity (c) and algal bloom observations (d)
across seasons and continents within still waters (ponds and lakes). Lettering (e.g., A, B) denotes
significant differences between seasons (Kruskal–Wallis post hoc Dunn test, where upper case = P <
0.05, lower case = P < 0.1). Symbols are median seasonal values from individual sites: □ = Oceania, ○ =
North America, ∆ = Europe. Figure 2. Comparison of nutrient concentrations (a,b), turbidity (c) and algal bloom observations (d)
across seasons and continents within still waters (ponds and lakes). Lettering (e.g., A, B) denotes
significant differences between seasons (Kruskal–Wallis post hoc Dunn test, where upper case = P < 0.05,
lower case = P < 0.1). Symbols are median seasonal values from individual sites: □= Oceania, # = North
America, ∆= Europe. Figure 2. Comparison of nutrient concentrations (a,b), turbidity (c) and algal bloom observations (d)
across seasons and continents within still waters (ponds and lakes). Lettering (e.g., A, B) denotes
significant differences between seasons (Kruskal–Wallis post hoc Dunn test, where upper case = P <
0.05, lower case = P < 0.1). Symbols are median seasonal values from individual sites: □ = Oceania, ○ =
North America, ∆ = Europe. Figure 2. Comparison of nutrient concentrations (a,b), turbidity (c) and algal bloom observations (d)
across seasons and continents within still waters (ponds and lakes). 3.1. Temporal Trends in Water Quality Lettering (e.g., A, B) denotes
significant differences between seasons (Kruskal–Wallis post hoc Dunn test, where upper case = P < 0.05,
lower case = P < 0.1). Symbols are median seasonal values from individual sites: □= Oceania, # = North
America, ∆= Europe. 3.2. Ponds Versus Lakes
3.2. Ponds Versus Lakes Nutrient concentrations did not show differences between ponds and lakes (Table 3; Figure 3). Turbidity measurements were consistently and significantly (P < 0.05, Mann–Whitney) greater and
more wide ranging (P < 0.001, Brown–Forsythe) in ponds (mean 25.3 NTU) than in lakes (mean 17.5
Nutrient concentrations did not show differences between ponds and lakes (Table 3; Figure 3). Turbidity measurements were consistently and significantly (P < 0.05, Mann–Whitney) greater and more
wide ranging (P < 0.001, Brown–Forsythe) in ponds (mean 25.3 NTU) than in lakes (mean 17.5 NTU). U). Table 3. Comparison of average of and variance in nutrient concentrations, turbidity, and algal
Table 3. Comparison of average of and variance in nutrient concentrations, turbidity, and algal bloom
occurrence between ponds (<2 ha) vs. lakes (>2 ha). Numbers in parentheses are ±1 SD. NTU). Table 3. Comparison of average of and variance in nutrient concentrations, turbidity, and algal
bloom occurrence between ponds (<2 ha) vs. lakes (>2 ha). Numbers in parentheses are ±1 SD. Ponds
Lakes
Mann–Whitney p
Brown–Forsyth p
Table 3. Comparison of average of and variance in nutrient concentrations, turbidity, and algal bloom
occurrence between ponds (<2 ha) vs. lakes (>2 ha). Numbers in parentheses are ±1 SD. Ponds
Lakes
Mann–Whitney p
Brown–Forsyth p bloom occurrence between ponds (<2 ha) vs. lakes (>2 ha). Numbers in parentheses are ±1 SD. Ponds
Lakes
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.75)
0.1 (0.44)
0.55
0.54
Phosphate (mg/L)
0.01 (0.02)
0.01 (0.03)
0.86
0.85
Turbidity (NTU)
17.0 (22.9)
13.4 (9.9)
0.01 **
<0.001 **
Bloom frequency (%)
32.0 (27.6)
30.5 (26.9)
0.80
0.56
Note: Asterisks indicate significant difference where * = P < 0.5 and ** = P < 0.01. Ponds
Lakes
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.75)
0.1 (0.44)
0.55
0.54
Phosphate (mg/L)
0.01 (0.02)
0.01 (0.03)
0.86
0.85
Turbidity (NTU)
17.0 (22.9)
13.4 (9.9)
0.01 **
<0.001 **
Bloom frequency (%)
32.0 (27.6)
30.5 (26.9)
0.80
0.56
Note: Asterisks indicate significant difference where ** = P < 0.01. q
y (
)
(
)
(
)
erisks indicate significant difference where * = P < 0.5 and **
Note: Asterisks indicate significant difference where ** = P < 0.01. 7 of 15
7 of 15
7 of 15 Water 2018, 10, 839
Water 2018, 10, x F
Water 2018, 10, x F Figure 3. 3.2. Ponds Versus Lakes
3.2. Ponds Versus Lakes Comparison of nutrient concentrations, turbidity, and algal bloom observations across
seasons within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences
between ponds and lakes (Kruskal–Wallis post hoc Dunn test ** = P < 0 01 * = P < 0 05)
Figure 3. Comparison of nutrient concentrations, turbidity, and algal bloom observations across seasons
within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences between
ponds and lakes (Kruskal–Wallis post hoc Dunn test, ** = P < 0.01, * = P < 0.05). Figure 3. Comparison of nutrient concentrations, turbidity, and algal bloom observations across
seasons within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences
between ponds and lakes (Kruskal–Wallis post hoc Dunn test, ** = P < 0.01, * = P < 0.05). Figure 3. Comparison of nutrient concentrations, turbidity, and algal bloom observations across
seasons within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences
between ponds and lakes (Kruskal Wallis post hoc Dunn test ** = P < 0 01 * = P < 0 05)
Figure 3. Comparison of nutrient concentrations, turbidity, and algal bloom observations across seasons
within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences between
ponds and lakes (Kruskal–Wallis post hoc Dunn test, ** = P < 0.01, * = P < 0.05). Figure 3. Comparison of nutrient concentrations, turbidity, and algal bloom observations across
seasons within ponds (hollow boxes) or lakes (grey boxes). Asterisks denote significant differences
between ponds and lakes (Kruskal–Wallis post hoc Dunn test, ** = P < 0.01, * = P < 0.05). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. 3.2. Ponds Versus Lakes
3.2. Ponds Versus Lakes Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Significant differences were apparent in biological observations, with evidence of fish presence
being significantly more likely in larger water bodies. Conversely, complex macrophyte stands were
more frequently observed in the smallest water bodies (Figure 4). Figure 4. Comparison of physical characteristics between ponds (<2 ha) and lakes (>2 ha). Asterisks
indicate significant differences (Mann–Whitney test, ** P < 0.05, *** P < 0.01). Figure 4. Comparison of physical characteristics between ponds (<2 ha) and lakes (>2 ha). Asterisks
indicate significant differences (Mann–Whitney test, ** P < 0.05, *** P < 0.01). Water 2018, 10, 839 8 of 15 3.3. Influence of Urbanisation
Table 5 Comparison of a Peri-urban ponds had higher levels of turbidity compared to urban equivalents (Figure 5c);
however, this was not found to be significant (Table 4). There were no differences observed
between urban and peri-urban lakes. Both ponds and lakes that were located in more urban areas
showed a significant increase in algal bloom observations (P < 0.05, Mann–Whitney) (Figure 5d, and
Table 5). Other water quality factors did not appear to differ consistently or significantly in response
to urbanization. occurrence between urban vs. peri-urban lakes and ponds. Numbers in parentheses are ±1 SD. Peri-Urban
Urban
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.63)
0.1 (0.66)
0.44
0.71
Phosphate (mg/L)
0.01 (0.03)
0.01 (0.02)
0.38
0.15
Turbidity (NTU)
25.4 (22.6)
22.8 (16.8)
0.74
0.25
Bloom frequency (%)
24.7 (24.5)
38.6 (28.3)
0.03 **
0.22
Note: Asterisks indicate significant difference where ** = P < 0.01. Figure 5. Comparison of water quality trends across seasons between peri-urban (grey) and urban
(black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients,
mean (and standard error) for turbidity and algal bloom frequency. Figure 5. Comparison of trends in (a) nitrate, (b) phosphate, (c) turbidity, (d) algal bloom frequency,
across seasons between peri-urban (grey) and urban (black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients, mean (and standard error) for turbidity and
algal bloom frequency. Figure 5. Comparison of water quality trends across seasons between peri-urban (grey) and urban
(black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients,
mean (and standard error) for turbidity and algal bloom frequency. Figure 5. Comparison of trends in (a) nitrate, (b) phosphate, (c) turbidity, (d) algal bloom frequency,
across seasons between peri-urban (grey) and urban (black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients, mean (and standard error) for turbidity and
algal bloom frequency. Figure 5. Comparison of water quality trends across seasons between peri-urban (grey) and urban
(black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients,
mean (and standard error) for turbidity and algal bloom frequency. Figure 5. Comparison of trends in (a) nitrate, (b) phosphate, (c) turbidity, (d) algal bloom frequency,
across seasons between peri-urban (grey) and urban (black), ponds (circles), and lakes (squares). Median values (and inter-quartile range) for nutrients, mean (and standard error) for turbidity and
algal bloom frequency. Table 4. 3.3. Influence of Urbanisation
Table 5 Comparison of a Comparison of nutrient concentrations, turbidity, and algal bloom occurrence between urban
vs. peri-urban ponds. Numbers in parentheses are ±1 SD. Peri-Urban
Urban
Mann-Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.74)
0.1 (0.77)
0.19
0.59
Phosphate (mg/L)
0.01 (0.03)
0.01 (0.04)
0.07 *
0.11
Turbidity (NTU)
30.0 (27.1)
24.7 (19.0)
0.91
0.18
Bloom frequency (%)
25.1 (26.2)
38.6 (27.8)
0.09 *
0.54
Note: Asterisks indicate significant difference where * = P < 0.5. Note: Asterisks indicate significant difference where * = P < 0.5. 9 of 15 Water 2018, 10, 839 Table 5. Comparison of average of and variance in nutrient concentrations, turbidity and algal bloom
occurrence between urban vs. peri-urban lakes and ponds. Numbers in parentheses are ±1 SD. Peri-Urban
Urban
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.63)
0.1 (0.66)
0.44
0.71
Phosphate (mg/L)
0.01 (0.03)
0.01 (0.02)
0.38
0.15
Turbidity (NTU)
25.4 (22.6)
22.8 (16.8)
0.74
0.25
Bloom frequency (%)
24.7 (24.5)
38.6 (28.3)
0.03 **
0.22
Note: Asterisks indicate significant difference where ** = P < 0.01. Table 5. Comparison of average of and variance in nutrient concentrations, turbidity and algal bloom
occurrence between urban vs. peri-urban lakes and ponds. Numbers in parentheses are ±1 SD. Peri-Urban
Urban
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.63)
0.1 (0.66)
0.44
0.71
Phosphate (mg/L)
0.01 (0.03)
0.01 (0.02)
0.38
0.15
Turbidity (NTU)
25.4 (22.6)
22.8 (16.8)
0.74
0.25
Bloom frequency (%)
24.7 (24.5)
38.6 (28.3)
0.03 **
0.22
Note: Asterisks indicate significant difference where ** = P < 0.01. 3.4. Algal Bloom Frequency Across Urban and Peri-Urban Ponds and Lakes To identify contributing factors to the differences in algal bloom frequency between urban
and peri-urban ponds and lakes, multiple linear regression was used, incorporating observational
records taken by FWW participants following a multi-model approach. This resulted in 286 model
combinations (max. three predictors per model). Relative variable importance was calculated across all model combinations in relation to algal
bloom frequency in urban and peri-urban ponds and lakes (Table 6). The presence of potential point
sources of pollution, in particular those identified by citizen scientists as residential or road origin
occurred frequently in the top 10 models and were therefore important predictors of algal bloom
observations (Table 7). Another reoccurring driver was water level fluctuations. Interestingly, the
strength of the association of these drivers with algal blooms was much greater in urban areas (adj. R2 0.24, P < 0.05), than in peri-urban areas, where both variable importance and standardized model
coefficient values were low (adj. R2 0.008, P > 0.05). In urban areas, residential discharges showed
a positive association with algal bloom frequency while water level fluctuation and road discharges
were negative. Table 6. Comparison of nutrient concentrations, turbidity and algal bloom occurrence between urban
vs. peri-urban lakes. Numbers in parentheses are ±1 SD. Peri-Urban
Urban
Mann–Whitney p
Brown–Forsyth p
Nitrate (mg/L)
0.1 (0.47)
0.1 (0.41)
0.73
0.70
Phosphate (mg/L)
0.01 (0.04)
0.01 (0.2)
0.47
0.86
Turbidity (NTU)
18.7 (11.7)
19.2 (11.4)
0.72
0.85
Bloom frequency (%)
24.1 (22.6)
38.5 (30.4)
0.19
0.20 Table 6. Comparison of nutrient concentrations, turbidity and algal bloom occurrence between urban
vs. peri-urban lakes. Numbers in parentheses are ±1 SD. 10 of 15 Water 2018, 10, 839 Table 7. Multi-model inference results using an Akaike information criterion (AICc)—information
theoretic (AIC–IT) approach for algal observations in urban and peri-urban ponds and lakes. Table
limited to the predictors occurring in the top 10 models. Asterix indicates significance level within the
AIC-best model where P < 0.05. 4.1. Physical and Temporal Trends 4.1. Physical and Temporal Trends Data collected by citizen scientists engaged in a global study of freshwater quality identified
clear seasonal trends in nutrient status, with peak concentrations of nitrate (N-NO3) during winter
periods and phosphate (P-PO4) during spring and summer. Many studies have principally focused on
phosphorous owing to its assumed role as a key limiter to algal growth, however, bioavailable nitrogen
is also an important driver of aquatic vegetation dynamics [46]. In the present study, winter peaks in
nitrate conform to commonly observed patterns (e.g., New York lakes [47]) related to assimilation by
algae and other plants throughout the spring and summer growing period and subsequent release into
the water column during plant senescence when uptake by phytoplankton and denitrifying bacteria is
minimal [48,49]. Summer increases in phosphate accord well with studies of shallow artificial ponds in England [50]
and Danish lakes [12] where summer peaks in total phosphorus (TP) were observed. This is frequently
suggested to reflect internal loading mechanisms, to which shallow water bodies are more susceptible
than deeper water bodies owing to the intense sediment to water column interface [11]. The release
of P from the sediment is often in a biologically available form, more readily available for uptake by
phytoplankton as indicated by trends in algal bloom frequency [51]. Thus the combination of nutrient
sampling kits and observational parameter appeared appropriate for monitoring these trends (accept
hypothesis 1). Turbidity (NTU) and algal bloom frequency trends were comparable, likely because
of the underlying covariance in the parameters. However, whilst significant, the relationship was
not strong. 3.4. Algal Bloom Frequency Across Urban and Peri-Urban Ponds and Lakes Peri-Urban Ponds and Lakes
Urban Ponds and Lakes
Predictor
βj
w + (j)
#
Predictor
βj
w + (j)
#
Dis_Road
0.078
0.33
8
Dis_Res *
0.300
0.67
9
Lev_Flu
0.054
0.28
3
Lev_Flu *
−0.138
0.44
3
Dis_Tot
0.063
0.28
2
Dis_Road *
−0.185
0.43
5
Veg_Flt
−0.050
0.27
8
Dist_Tot
−0.021
0.34
5
Veg_Emer
−0.046
0.26
2
Bnk_Bar
−0.070
0.25
3
LU_For
−0.031
0.21
1
Veg_Flt
0.029
0.15
2
Fish
0.028
0.21
1
Bnk_Tree
−0.031
0.14
1
LU_UrbRes
−0.026
0.21
3
LU_UrbRes
0.028
0.14
2
Bnk_Grass
−0.025
0.20
-
LU_UP
−0.006
0.10
-
Dis_Res
0.020
0.19
-
Veg_Sub
0.001
0.09
-
Dis_Road = road discharge, Dis_Res = residential discharge, Lev_Flu = water level fluctuation, Dis_Tot = total
number of discharges, Veg_Flt = floating vegetation Veg_Emer = emergent vegetation, Bnk_Bar = bare bank, LU_For
= forested land-use, Bnk_Tree = bankside trees, LU_UrbRes = urban/residential land-use, Bnk_Grass = bank side
grass, LU_UP = urban park land-use, Veg_Sub = submerged vegetation. Asterisks indicate statistically significant
predictors where * = P < 0.5. Dis_Road = road discharge, Dis_Res = residential discharge, Lev_Flu = water level fluctuation, Dis_Tot = total
number of discharges, Veg_Flt = floating vegetation Veg_Emer = emergent vegetation, Bnk_Bar = bare bank, LU_For
= forested land-use, Bnk_Tree = bankside trees, LU_UrbRes = urban/residential land-use, Bnk_Grass = bank side
grass, LU_UP = urban park land-use, Veg_Sub = submerged vegetation. Asterisks indicate statistically significant
predictors where * = P < 0.5. 4.3. Influence of Urbanisation The influence of urbanization was most pronounced in the frequency of algal bloom observations
and there seemed to be a potentially greater influence in ponds (reject hypothesis 3). Eutrophic
conditions, a precursor to algal blooms are distinguishing component of the urban stream (urban
stream syndrome) [58,59]. This study indicates that it is a characteristic of urban ponds, contributing
to the global issue, often associated to lakes [60,61]. The association of algal blooms with a suite of observational parameters indicated that the
presence of residential discharges were a driver of algal bloom frequency, with hydrological variability
(water level fluctuations) likely to reduce the frequency of algal blooms in the most urban sites. Interestingly, the presence of road discharges were also associated with a reduction. Within our study
sites, the presence of residential discharges are more likely to represent phosphate availability as a
consequence of domestic waste water and household misconnections [62,63]. Conversely, the negative
effect of both water level fluctuation and the presence of road discharge could reflect dilution of
nutrients or influence trophic interactions resulting in higher grazing pressure [64,65]. The influence of
road discharge is less clear, however the chemical composition of road discharge has been identified to
both stimulate (e.g., sodium [66]) or inhibit algal growth (e.g., de-icing salts or herbicide [67]). Citizen observatories have previously been identified as a potentially powerful tool for the
monitoring of algal blooms [35,68] with a suite of applications now having being developed to
facilitate their record such as CyanoTRACKER (University of Georgia), Bloomin’ Algae (Centre for
Ecology and Hydrology), and bloomWatch (US EPA). The present study suggests that potential local
scale drivers may also be monitored by citizen scientists. 4.2. Pond vs. Lake Our study identified consistently higher levels of turbidity within ponds (<2 ha) than lakes. Reasons for this are uncertain, however, could relate to the susceptibility of shallow water bodies
to resuspension of sediment following disturbance, e.g., from benthivorous fish [52,53] or wind
action [54,55]. Fish presence was typically observed less frequently across repeat visits to smaller
water bodies (accept hypothesis 2), in agreement with several other studies of fish abundance [14,15];
however, fish were observed at least once in two-thirds (63%) of ponds. Although species were 11 of 15 Water 2018, 10, 839 not identified, the relative density of fish in ponds rather than abundance may be an over-riding
influence [56]. Higher macrophyte complexity was also observed within small ponds compared to lakes. A number of studies have highlighted the potential conservation value of ponds [2,3], which is closely
linked to macrophyte diversity [19,57]. The present study was limited to the presence of emergent,
submerged and floating vegetation based on visual observations, allowing for an underestimation in
larger, deeper water bodies, in particular regarding the presence of submerged vegetation. References 1. Downing, J.A.; Duarte, C.M. Abundance and size distribution of lakes, ponds and impoundments. In Encyclopedia of Inland Waters; Likens, G.E., Ed.; Elsevier: Oxford, UK, 2009; pp. 469–478. 1. Downing, J.A.; Duarte, C.M. Abundance and size distribution of lakes, ponds and impoundments. In Encyclopedia of Inland Waters; Likens, G.E., Ed.; Elsevier: Oxford, UK, 2009; pp. 469–478. 2. Williams, P.; Whitfielda, M.; Biggs, J.; Bray, S.; Fox, G.; Nicolet, P.; Sear, D. Comparative biodiversity of
rivers, streams, ditches and ponds in an agricultural landscape in Southern England. Biol. Conserv. 2004, 115,
329–341. [CrossRef] 2. Williams, P.; Whitfielda, M.; Biggs, J.; Bray, S.; Fox, G.; Nicolet, P.; Sear, D. Comparative biodiversity of
rivers, streams, ditches and ponds in an agricultural landscape in Southern England. Biol. Conserv. 2004, 115,
329–341. [CrossRef] 3. Davies, B.; Biggs, J.; Williams, P.; Whitfield, M.; Nicolet, P.; Sear, D.; Bray, S.; Maund, S. Comparative
biodiversity of aquatic habitats in the European agricultural landscape. Agric. Ecosyst. Environ. 2008, 125,
1–8. [CrossRef] 3. Davies, B.; Biggs, J.; Williams, P.; Whitfield, M.; Nicolet, P.; Sear, D.; Bray, S.; Maund, S. Comparative
biodiversity of aquatic habitats in the European agricultural landscape. Agric. Ecosyst. Environ. 2008, 125,
1–8. [CrossRef] 4. Tranvik, L.J.; Downing, J.A.; Cotner, J.B.; Loiselle, S.A.; Striegl, R.G.; Ballatore, T.J.; Dillon, P.; Finlay, K.;
Fortino, K.; Knoll, L.B.; et al. Lakes and reservoirs as regulators of carbon cycling and climate. Limnol. Oceanogr. 2009, 54, 2298–2314. [CrossRef] 5. Indermuehle, N.; Oertli, B.; Biggs, J.; Céréghino, R.; Grillas, P.; Hull, A.; Nicolet, P.; Scher, O. Pond conservation
in Europe: The European Pond Conservation Network ( EPCN ). SIL Proc. 2008, 30, 446–448. [CrossRef] 6. North American Lake Management Society the Secchi Dip-In. Available online: http://www.secchidipin.org
(accessed on 18 November 2017). 7. Bonney, R.; Ballard, H.; Jordan, R.; Mccallie, E.; Phillips, T.; Shirk, J.; Wilderman, C. Public Participation
in Scientific Research: Defining the Field and Assessing Its Potential for Informal Science Education. In A
CAISE Inquiry Group Report; Center for Advancement of Informal Science Education (CAISE): Washington,
DC, USA, 2009. 8. Dickinson, J.L.; Shirk, J.; Bonter, D.; Bonney, R.; Crain, R.L.; Martin, J.; Phillips, T.; Purcell, K. The current
state of citizen science as a tool for ecological research and public engagement. Front. Ecol. Environ. 2012, 10,
291–297. [CrossRef] 9. Gray, S.; Jordan, R.; Crall, A.; Newman, G.; Hmelo-Silver, C.; Huang, J.; Novak, W.; Mellor, D.; Frensley, T.;
Prysby, M.; et al. 5. Conclusions Funding: This research received no external funding. Funding: This research received no external funding. Acknowledgments: We sincerely acknowledge the efforts of the Citizen Science Leaders (CSLs) within the HSBC
Water Programme and Royal Dutch Shell plc for their dedication and enthusiasm. We also acknowledge the efforts
of Jeremy Biggs and Pascale Nicolet (Freshwater Habitats Trust; UK and France), Kim Anema (UNESCO-IHE
Institute for Water Education; Netherlands), Eren Turak (Office of Environment and Heritage; Australia), Wade
McGillis (Columbia University, USA), and Paul Frost (Canada; Trent University) for their assistance in training
and engaging the citizen scientists who participated in the study. Conflicts of Interest: The authors declare no conflict of interest. 5. Conclusions The incorporation of trained volunteers in the monitoring of inland water bodies presents a
potential to broaden our knowledge of their conditions and dynamics [69,70]. This is particularly
important for shallow lakes and ponds, which are often overlooked by national and international
legislation. In the present study, we demonstrate that observations and measurements of trained
citizen scientists can support the identification of temporal and spatial patterns in temperate ponds
and lakes. Most ponds and lakes within urban areas are artificial and often built as part of the supporting
infrastructure (e.g., storm water storage, sustainable urban drainage systems). From their local
aquatic ecosystems, human populations derive many services which support the economy and
wellbeing [71,72]. Direct drivers to the loss of ecosystem integrity will affect their functioning [73,74] and
are often difficult to identify. We show that the combination of GIS data with local-scale observational
data recorded by the citizen scientists helps to identify local drivers to ecosystem degradation. The results also confirm the role of size in influencing both water quality and ecosystem conditions,
again indicating the potential benefits of citizens to cover these often overlooked ecosystems. Overall, the approach proved promising to both ends, however, citizen science is not without
its limitations and its application is not always appropriate. Further, the engagement of citizens
can be time-consuming and further research is needed to understand the motivations of local
communities with regards to their freshwater resources. We recommend that citizen science provides 12 of 15 12 of 15 Water 2018, 10, 839 a useful complementary tool to regulatory and professional monitoring and could act as an early
warning system. Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4441/10/7/839/s1,
Table S1: Site summaries for ponds and lakes included within the analysis, Table S2: Average seasonal water
quality statistics (±1 SD) for temperate still waters across Oceania (OCN), North America (NAM), and Europe
(EUR). Median values displayed for nitrate (N-NO3) and phosphate (P-PO4), mean values for turbidity (NTU)
and frequency of algal observations (presence/absence). Author Contributions: I.T. conceived of the investigation here presented and carried out all statistical analysis
and helped train FWW participants. A.C. carried out preliminary investigations of still water FWW data and
helped edit and refine the manuscript. S.L. contributed significantly to the FWW citizen science programme and
provided advice regarding the aims and objectives of the present study as well as editing capacity. References Combining participatory modelling and citizen science to support volunteer conservation
action. Biol. Conserv. 2017, 208, 76–86. [CrossRef] 10. Beklio˘glu, M.; Meerhoff, M.; Davidson, T.A.; Ger, K.A.; Havens, K.; Moss, B. Preface: Shallow lakes in a fast
changing world. Hydrobiologia 2016, 778, 9–11. [CrossRef] 11. Scheffer, M. Ecology of Shallow Lakes, 1st ed.; Chapman and Hall: London, UK, 1998. 11. Scheffer, M. Ecology of Shallow Lakes, 1st ed.; Chapman and H 12. Jeppesen, E.; Jensen, J.P.; Søndergaard, M.; Lauridsen, T.; Pedersen, L.J.; Jensen, L. Top-down control in
freshwater lakes: The role of nutrient state, submerged macrophytes and water depth. Hydrobiologia 1997,
342, 151–164. [CrossRef] 13 of 15 13 of 15 Water 2018, 10, 839 13. Pettersson, K. Mechanisms for internal loading of phosphorus in lakes. Hydrobiologia 1998, 373, 21–25. [CrossRef] 14. Søndergaard, M.; Jeppesen, E.; Jensen, J.P.; Sondergaard, M. Pond or lake: Does it make any difference? Arch. Hydrobiol. 2005, 162, 143–165. [CrossRef] 15. Scheffer, M.; Van Geest, G.J.; Zimmer, K.; Jeppesen, E.; Sondergaard, M.; Butler, M.G.; Hanson, M.A.;
Declerck, S.; De Meester, L. Small habitat size and isolation can promote species richness: Second-order
effects on biodiversity in shallow lakes and ponds. Oikos 2006, 112, 227–231. [CrossRef] 16. Hassall, C. The ecology and biodiversity of urban ponds. Wiley Interdiscip. Rev. Water 2014, 1, 187–206. [CrossRef] 17. Walsh, C.J.; Roy, A.H.; Feminella, J.W.; Cottingham, P.D.; Groffman, P.M.; Morgan, R.P. The urban stream
syndrome: Current knowledge and the search for a cure. J. N. Am. Benthol. Soc. 2005, 24, 706–723. [CrossRef] 17. Walsh, C.J.; Roy, A.H.; Feminella, J.W.; Cottingham, P.D.; Groffman, P.M.; Morgan, R.P. The urban stream
syndrome: Current knowledge and the search for a cure. J. N. Am. Benthol. Soc. 2005, 24, 706–723. [CrossRef]
18. Hill, M.J.; Biggs, J.; Thornhill, I.; Briers, R.A.; Gledhill, D.G.; White, J.C.; Wood, P.J.; Hassall, C. Urban ponds
as an aquatic biodiversity resource in modified landscapes. Glob. Chang. Biol. 2016, 23, 986–999. [CrossRef]
[PubMed] 18. Hill, M.J.; Biggs, J.; Thornhill, I.; Briers, R.A.; Gledhill, D.G.; White, J.C.; Wood, P.J.; Hassall, C. Urban ponds
as an aquatic biodiversity resource in modified landscapes. Glob. Chang. Biol. 2016, 23, 986–999. [CrossRef]
[PubMed] 19. Thornhill, I.; Batty, L.; Death, R.G.; Friberg, N.R.; Ledger, M.E. Local and landscape scale determinants of
macroinvertebrate assemblages and their conservation value in ponds across an urban land-use gradient. Biodivers. Conserv. 2017, 26, 1065–1086. [CrossRef] 20. Wood, P.J.; Barker, S. References Old industrial mill ponds: A neglected ecological resource. Appl. Geogr. 2000, 20, 65–81. [CrossRef] 21. Peretyatko, A.; Teissier, S.; Backer, S.; Triest, L. Restoration potential of biomanipulation for eutrophic
peri-urban ponds: The role of zooplankton size and submerged macrophyte cover. Hydrobiologia 2009, 634,
125–135. [CrossRef] 22. Biggs, J.; Fox, G.; Nicolet, P.; Walker, D.; Whitfield, M.; Williams, P. A Guide to the Methods of the National Pond
Survey; Pond Action: Oxford, UK, 1998. 23. Conrad, C.C.; Hilchey, K.G. A review of citizen science and community-based environmental monitoring:
Issues and opportunities. Environ. Monit. Assess. 2011, 176, 273–291. [CrossRef] [PubMed] 24. Thornhill, I.; Loiselle, S.; Lind, K.; Ophof, D. The citizen science opportunity for researchers and agencies. Bioscience 2016, 66, 720–721. [CrossRef] 25. Whitelaw, G.; Vaughan, H.; Craig, B.; Atkinson, D. Establishing the Canadian Community Monitoring
Network. Environ. Monit. Assess. 2003, 88, 409–418. [CrossRef] [PubMed] 26. Olson, D.M.; Dinerstein, E.; Wikramanayake, E.D.; Burgess, N.D.; Powell, G.V.N.; Underwood, E.C.;
D’Amico, J.A.; Itoua, I.; Strand, H.E.; Morrison, J.C.; et al. Terrestrial ecoregions of the world: A new
map of life on Earth. Bioscience 2001, 51, 933–938. [CrossRef] 27. Thornhill, I.; Ho, J.G.; Zhang, Y.; Li, H.; Ho, K.C.; Miguel-Chinchilla, L.; Loiselle, S.A. Prioritising local action
for water quality improvement using citizen science; a study across three major metropolitan areas of China. Sci. Total Environ. 2017, 584–585, 1268–1281. [CrossRef] [PubMed] 28. Rice, E.W.; Baird, R.B.; Eaton, A.D.; Clesceri, L.S. Standard Methods for the Examination of Water and Wastewater,
22nd ed.; American Public Health Association: Washington, DC, USA, 2012. 29. World Weather Online World Weather Online (Monthly Climate Averages). Available online: http://www. worldweatheronline.com/ (accessed on 7 April 2018). 30. Ellis, P.S.; Shabani, A.M.H.; Gentle, B.S.; McKelvie, I.D. Field measurement of nitrate in marine and estuarine
waters with a flow analysis system utilizing on-line zinc reduction. Talanta 2011, 84, 98–103. [CrossRef]
[PubMed] 31. Strickland, J.D.H.; Parsons, T. A Practical Handbook of Seawater Analysis; Fisheries Research Board of Canada:
Ottawa, ON, Canada, 1972. 32. Berti, G.; Fossati, P.; Tarenghi, G.; Musitelli, C.; Melzi d’Eril, G.V. Enzymatic colorimetric method for the
determination of inorganic phosphorus in serum and urine. J. Clin. Chem. Clin. Biochem. 1988, 26, 399–404. [CrossRef] [PubMed] 33. Dahlgren, R.; Nieuwenhuyse, E.; Van Litton, G. Transparency tube provides reliable water-quality
measurements. Calif. Agric. 2008, 58, 149–153. [CrossRef] 34. Tyler, J.E. The Secchi disk. Limnol. Oceanogr. 1968, 13. [CrossRef] 34. Tyler, J.E. The Secchi disk. Limnol. Oceanogr. References 1968, 13. [CrossRef] 14 of 15 Water 2018, 10, 839 35. Castilla, E.P.; Cunha, D.G.F.; Lee, F.W.F.; Loiselle, S.; Ho, K.C.; Hall, C. Quantification of phytoplankton bloom
dynamics by citizen scientists in urban and peri-urban environments. Environ. Monit. Assess. 2015, 187. [CrossRef] [PubMed] 36. Biggs, J.; McGoff, E.; Ewald, N.; Dunn, F.; Nicolet, P.; Williams, P. Clean Water for Wildlife. In Using PackTest
Nitrate and Phosphate Test Kits to Find Clean Water and Assess the Extent of Pollution; Freshwater Habitats Trust:
Oxford, UK, 2016. 37. Miguel-Chinchilla, L.; Thornhill, I.; Baruch, A.; Loiselle, S.A. FreshWater Watch Methods (draft); Freshwater
Habitats Trust: Oxford, UK, 2017. 38. Muneoka, T. Evaluation of Nitrate Pollution in River Water at Agricultural Watershed; Freshwater Habitats Trust:
Oxford, UK, 2014; pp. 51–56. 39. Biggs, J.; Williams, P.; Whitfield, M.; Nicolet, P.; Weatherby, A. 15 years of pond assessment in Britain: Results
and lessons learned from the work of Pond Conservation. Aquat. Conserv. Mar. Freshw. Ecosyst. 2005, 15,
693–714. [CrossRef] 40. Latham, J.; Cumani, R.; Rosati, I.; Bloise, M. FAO Global Land Cover (GLC-SHARE) Beta-Release 1.0 Database;
Land and Water Division: Rome, Italy, 2014. 41. CIESIN. Gridded Population of the World, Version 4 (GPWv4). Preliminary Release 2 (2010) 2014. Available
online: http://sedac.ciesin.columbia.edu/data/collection/gpw-v4 (accessed on 14 June 2018). 42. R Core Team R: A Language and Environment for Statistical Computing; The R Foundation for Statistical
Computing: Vienna, Austria, 2017; ISBN 3-900051-07-0. 43. Burnham, K.P.; Anderson, D.R. Model Selection and Multimodel Inference: A Practical Information-Theoretic
Approach, 2nd ed.; Springer: New York, NY, USA, 2002; ISBN 0387953647. 44. Whittingham, M.J.; Stephens, P.A.; Bradbury, R.B.; Freckleton, R.P. Why do we still use stepwise modelling
in ecology and behaviour? J. Anim. Ecol. 2006, 75, 1182–1189. [CrossRef] [PubMed] 45. Grueber, C.E.; Nakagawa, S.; Laws, R.J.; Jamieson, I.G. Multimodel inference in ecology and evolution:
Challenges and solutions. J. Evol. Biol. 2011, 24, 699–711. [CrossRef] [PubMed] 6. Brönmark, C.; Hansson, L.-A. The Biology of Lakes and Ponds; Oxford University Press: Oxford, UK, 2008 47. Stewart, K.M.; Markello, S.J. Seasonal variations in concentrations of nitrate and total phosphorus, and
calculated nutrient loading for six lakes in western New York. Hydrobiologia 1974, 44, 61–89. [CrossRef] 48. Wetzel, R.G. Limnology:
Lake and River Ecosystems; Academic Press:
Cambridge, MA, USA, 2001;
ISBN 9780127447605. 49. Ansari, A.A.; Singh Gill, S.; Lanza, G.R.; Rast, W. Eutrophication: Causes; Springer: New York, NY, USA, 2011;
ISBN 9789048196241. 50. Bennion, H.; Smith, M.A. References Variability in the water chemistry of shallow ponds in southeast England, with
special reference to the seasonality of nutrients and implications for modelling trophic status. Hydrobiologia
2000, 436, 145–158. [CrossRef] 51. Nürnberg, G.K. Assessing internal phosphorus load—Problems to be solved. Lake Reserv. Manag. 2009, 25,
419–432. [CrossRef] 52. Breukelaar, A.W.; Lammens, E.H.R.R.; Breteler, J.G.P.K.; Tatrai, I. Effects of benthivorous bream (Abramis
brama) and carp (Cyprinus carpio) on sediment resuspension and concentrations of nutrients and
chlorophyll a. Freshw. Biol. 1994, 32, 113–121. [CrossRef] Cline, J.M.; East, T.L.; Threlkeld, S.T. Fish interactions with the sediment-water interface. Hydrobiologia 1994
275–276, 301–311. [CrossRef] 54. Globevnik, L.; Kirn, T. Small Water Bodies—Assessment of Status and Threats of Standing Small Water Bodies. IERS, 2009. Available online: http://icm.eionet.europa.eu/ETC_Reports/Assessment%20of%20status%
20and%20threats%20of%20standing%20small%20water%20bodies.pdf (accessed on 14 June 2018). IERS, 2009. Available online: http://icm.eionet.europa.eu/ETC_Reports/Assessment%20of%20status%
20and%20threats%20of%20standing%20small%20water%20bodies.pdf (accessed on 14 June 2018). 55. De Meester, L.; Declerck, S.; Stoks, R.; Louette, G.; Van de Meutter, F.; De Bie, T.; Michels, E.; Brendonck, L. Ponds and pools as model systems in conservation biology, ecology and evolutionary biology. Aquat. Conserv. Mar. Freshw. Ecosyst. 2005, 725, 715–725. [CrossRef] 56. Olin, M.; Rask, M.; Ruuhljärvi, J.; Kurkilahti, M.; Ala-Opas, P.; Ylönen, O. Fish community structure in
mesotrophic and eutrophic lakes of southern Finland: The relative abundances of percids and cyprinids
along a trophic gradient. J. Fish Biol. 2002, 60, 593–612. [CrossRef] 57. Gledhill, D.G.; James, P.; Davies, D.H. Pond density as a determinant of aquatic species richness in an urban
landscape. Landsc. Ecol. 2008, 23, 1219–1230. [CrossRef] 15 of 15 Water 2018, 10, 839 58. Wenger, S.J.; Roy, A.H.; Jackson, C.R.; Bernhardt, E.S.; Carter, T.L.; Filoso, S.; Gibson, C.A.; Hession, W.C.;
Kaushal, S.S.; Martí, E.; et al. Twenty-six key research questions in urban stream ecology: An assessment of
the state of the science. J. N. Am. Benthol. Soc. 2009, 28, 1080–1098. [CrossRef] 59. Walsh, C.J. Urban impacts on the ecology of receiving waters: A framework for assessment, conse
and restoration. USGS 2000, 431, 107–114. 60. Golterman, H.L.; Oude, N.T. Eutrophication of Lakes, Rivers and Coastal Seas. In Water Pollution; Springer:
Berlin/Heidelberg, Germany, 1991; pp. 79–124. 61. Brooks, B.W.; Lazorchak, J.M.; Howard, M.D.A.; Johnson, M.-V.V.; Morton, S.L.; Perkins, D.A.K.; Reavie, E.D.;
Scott, G.I.; Smith, S.A.; Steevens, J.A. Are harmful algal blooms becoming the greatest inland water quality
threat to public health and aquatic ecosystems? Environ. Toxicol. Chem. 2016, 35, 6–13. [CrossRef] [PubMed] 62. Ellis, J.B.B.; Butler, D. References Surface water sewer misconnections in England and Wales: Pollution sources and
impacts. Sci. Total Environ. 2015, 526, 98–109. [CrossRef] [PubMed] 63. Revitt, D.M.; Ellis, J.B. Urban surface water pollution problems arising from misconnections. Sci. Total Environ. 2016, 551–552, 163–174. [CrossRef] [PubMed] 64. Naselli-Flores, L.; Barone, R. Importance of water-level fluctuation on population dynamics of clad
in a hypertrophic reservoir (Lake Arancio, south-west Sicily, Italy). Hydrobiologia 1997, 223–232. [Cr 64. Naselli-Flores, L.; Barone, R. Importance of water-level fluctuation on population dynamics of cladocerans
in a hypertrophic reservoir (Lake Arancio, south-west Sicily, Italy). Hydrobiologia 1997, 223–232. [CrossRef]
65. Scheffer, M.; Hosper, S.H.; Meijer, M.L.; Moss, B.; Jeppesen, E. Alternative equilibria in shallow lakes. Trends Ecol. Evol. 1993, 8, 275–279. [CrossRef] 65. Scheffer, M.; Hosper, S.H.; Meijer, M.L.; Moss, B.; Jeppesen, E. Alternative equilibria in shallow
Trends Ecol. Evol. 1993, 8, 275–279. [CrossRef] 66. Ramakrishna, D.M.; Viraraghavan, T. Environmental impact of chemical deicers—A review. Water Air
Soil Pollut. 2005, 166, 49–63. [CrossRef] 67. Huang, X.; Pedersen, T.; Fischer, M.; White, R.; Young, T.M. Herbicide Runoff along Highways. 1. Field
Observations. Environ. Sci. Technol. 2004, 38, 3263–3271. [CrossRef] [PubMed] 68. Cunha, D.G.F.; Casali, S.P.; de Falco, P.B.; Thornhill, I.; Loiselle, S.A. The contribution of volunteer-based
monitoring data to the assessment of harmful phytoplankton blooms in Brazilian urban streams. Sci. Total Environ. 2017, 584–585, 586–594. [CrossRef] [PubMed] 69. Bonney, R.; Cooper, C.B.; Dickinson, J.; Kelling, S.; Phillips, T.; Rosenberg, K.V.; Shirk, J. Citizen Science:
A Developing Tool for Expanding Science Knowledge and Scientific Literacy. Bioscience 2009, 59, 977–984. [CrossRef] 70. Buytaert, W.; Zulkafli, Z.; Grainger, S.; Acosta, L.; Alemie, T.C.; Bastiaensen, J.; De Bièvre, B.; Bhusal, J.;
Clark, J.; Dewulf, A.; et al. Citizen science in hydrology and water resources: Opportunities for knowledge
generation, ecosystem service management, and sustainable development. Front. Earth Sci. 2014, 2, 1–21. [CrossRef] 71. Erwin, K.L. Wetlands and global climate change: The role of wetland restoration in a changing world. Wetl. Ecol. Manag. 2009, 17, 71–84. [CrossRef] 72. De Bello, F.; Lavorel, S.; Díaz, S.; Harrington, R.; Cornelissen, J.H.C.; Bardgett, R.D.; Berg, M.P.; Cipriotti, P.;
Feld, C.K.; Hering, D.; et al. Towards an assessment of multiple ecosystem processes and services via
functional traits. Biodivers. Conserv. 2010, 19, 2873–2893. [CrossRef] 73. Diaz, S.; Cabido, M. Vive la difference: Plant functional diversity matters to ecosystem processes. Trends Ecol. Evol. 2001, 16, 646–655. [CrossRef] 74. References Gessner, M.O.; Inchausti, P.; Persson, L.; Raffaelli, D.G.; Giller, P.S. Biodiversity effects on ecosystem
functioning: Insights from aquatic systems. Oikos 2004, 3, 419–422. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
|
https://openalex.org/W2557786761
|
https://www.revistas.usp.br/posfau/article/download/105108/119564
|
Portuguese
| null |
O lote e a maloca: territorialização indigenista, mudanças no saber – fazer arquitetônico e a evolução da paisagem nas aldeias indígenas. Um estudo de caso a partir dos Kaingáng
|
Pós. Revista do Programa de Pós-Graduação em Arquitetura e Urbanismo da FAUUSP
| 2,016
|
cc-by
| 9,875
|
o
Sandoval dos Santos
Amparo o
Sandoval dos Santos
Amparo Resumo Este ensaio, com base em pesquisa bibliográfica, apresenta a
evolução da moradia indígena ao longo dos séculos, tomando
por referência o processo de territorialização indigenista e o
avanço dos processos de urbanização e das formas
ocidentalizadas de construção sobre os espaços tradicionais da
aldeia. A partir da análise paisagística, suas referências serão o
lote, forma espacial que irá caracterizar a urbanização no
Brasil; e na outra ponta, a maloca, principal referência da
habitação tradicional indígena. Palavras-chave
Lote. Maloca. Espaço. Territorialização. pós v.23 n.40 • são paulo • outubro 2016
doi: http://dx.doi.org/10.11606/issn.2317-2762.v23i40p26-46 LA TRAMA Y LA CASA COMUNAL:
TERRITORIALIZACIÓN
INDIGENISTA, CAMBIO NO
SABER-HACER
ARQUITECTÓNICO Y LA
EVOLUCIÓN DEL PAISAJE DE LOS
PUEBLOS INDÍGENAS. UN
ESTUDIO DE CASO KAINGÁNG Resumen
Este ensayo, de basis bibliográfica, presenta la
evolución de la habitación indígena al longo de
los siglos, tendo por referência el processo de
territorialización indigenista y el avanzo de los
processos de urbanización y de las formas
ocidentalizadas de construción sobre los
espacios tradicionales de la aldeia. Desde la
analisis paisagística, sus referéncias son el
“lote”, forma espacial característica de la
urbanización em Brasil; y, de outro lado, la
“maloca”, referéncia principal de la habitación
tradicional indígena. artigos • p. 026-046 Apresentação: dos índios e suas terras Os homens só se apropriam do que faz sentido para suas vidas e esse sentido é,
sempre, criação social, e não das coisas em si e por si mesmas. Carlos Walter Porto-Gonçalves, 2003 A reflexão sobre o “lote” e a “maloca” parece central quando nos propomos a
compreender as formas de “habitar” contemporâneo dos povos ancestrais do
Brasil e da América Latina. Pensar o lote e a maloca deve vislumbrar, como
ponto de partida, uma análise paisagística do espaço da aldeia e os processos
sociais envolvidos em sua transformação1 . Durante muito tempo, e até os dias
atuais, o processo de territorialização nacional tem logrado, por meio de doxas,
reduzir e negar as contradições deste país ainda em busca de uma identidade. Rogério Haesbaert desenvolve um raciocínio muito interessante que nos ajuda a
compreender a dinâmica dos processos sociais em Geografia Humana,
especialmente suas noções de identidade territorial (2003), desterritorialização
(2011), precarização (2004), trans-territorialidade e antropofagia (2011),
conceitos muito importantes para compreender o universo social no qual se
situa a questão das Terras Indígenas no Brasil. Por sinal, trata-se de uma
questão mais que necessária, já que as terras indígenas representam
aproximadamente 13% do território nacional e constituem áreas ricas em
recursos culturais, minerais e florestais2 . As terras indígenas são conhecidas
também pelo alto índice de conflitos (que envolvem indígenas, produtores
rurais e camponeses diversos), mas também pela diversidade cultural, inscrita
em diversidades linguística, de habitus, de racionalidades, de formas de ver,
pensar, conceber e agir o mundo. Darcy Ribeiro, em seu livro O Povo Brasileiro, demonstra que a questão da
identidade tem sido cara aos brasileiros desde, sobretudo, os séculos XVI e
XVII, quando surgiram as primeiras gerações de brasileiros. Os primeiros
brasileiros tinham o pai português e a mãe indígena, fruto da violência sexual
inerente à “conquista”, mas, em seu sentido metafórico, esta expressão reduz
estes brasileiros a “filhos da terra”, predando os indígenas, também
simbolicamente, “vistos como selvagens”, seres próprios da natureza, cujos pais
lhes viram as costas. Numa convergência cronológica, grande parte dos artigos
que compõem A inconstância da Alma Selvagem e outros ensaios de
antropologia, do antropólogo Viveiros de Castro, se dedica a estudar os textos
seiscentistas. O autor chama de brasis aos sujeitos sociais que reconhecia
naqueles textos, destacando, principalmente, a antropofagia ritual tupinambá3
(CASTRO, 2003). Foram estes brasileiros quem territorializaram o Brasil. Abstract This paper, based on literature, shows the
development of indigenous housing over the
centuries, with reference to the process of
indigenist territorialization and the advance
of urbanization processes and westernized
ways of building on traditional spaces of the
village. From the landscape analysis, your
references will be the batch, spatial form
that will characterize urbanization in Brazil;
and at the other side, the longhouse, the
main reference of traditional indigenous
housing. Keywords
Batch. Longhouse. Space. Territorialization. Keywords
Batch. Longhouse. Space. Territorialization. Palabras clave
Lote. Maloca. Espacio. Territorialización. Palabras clave
Lote. Maloca. Espacio. Territorialización. artigos • p. 026-046 Apresentação: dos índios e suas terras A paisagem da aldeia Kaingáng5 contemporânea pode ser
compreendida a partir da lógica espacial conhecida entre os geógrafos por
rugosidade, em M. Santos, ou de residualidade, em H. Lefebvre. Local
histórico de assentamentos Kaingáng nos últimos 25 séculos – com fartos
vestígios arqueológicos6 – as terras Kaingáng, no contexto da fricção
interétnica7 e das políticas de territorialização8 , não foram demarcadas a partir
de suas territorialidades tradicionais, pautadas na lógica da mobilidade e do
ciclo roça/coleta/ritual. A forma geográfica contemporânea é dada pelo
entrecruzamento de duas lógicas escalares distintas (HAESBAERT, 2011). Para
Oliveira, as Terras Indígenas são antes unidades jurídicas que sociológicas, em
função da série de contradições que marcam o processo demarcatório nos
contextos políticos e legais (OLIVEIRA, 1998, p. 26). As Terras Indígenas, ao
serem demarcadas, em meio a fortes processos e tensões políticas, mormente
lhes reservou alguma área a partir de seus assentamentos (toldos) e lhes
restringiu a territorialidade, já que a inscrição típica da geografia “branca”
(ocidental) é o limite – instituído na cartografia geralmente por uma linha reta
– que limita dois espaços contíguos (estabelecendo o dentro e o fora). pós- Outras controvérsias demarcam o processo: a mais importante delas: os índios
não são “proprietários” da terra, sendo, antes, exclusivos usufrutuários (Art. 231 da Constituição Federal), e, portanto, impedidos tanto de sua alienação
quanto do gozo dos bens de seu subsolo. A degradação da maloca – no caso dos Kaingáng, da casa subterrânea – é a
degradação do kre, vocábulo cujas variações estão sempre relacionadas ao
artefato na língua Jê (cesto, balaio, semear, plantar, etc.9 ). No mesmo
momento em que suas terras eram reduzidas a meros fragmentos do que
historicamente foram, sobretudo no que tange à escala territorial, das caçadas
e migrações, os Kaingáng passaram a ser banidos dos espaços colonizados
justamente por possuírem10 suas terras. A despeito das limitações de extensão,
os Kaingáng passaram a conviver, também, com uma outra racionalidade
produtiva, voltada para a economia de mercado. Assim, indiretamente, o
design das Terras Indígenas, até hoje, está, de certo modo, determinado pela
economia de mercado. Tal afirmação se deve ao fato de estas Terras, que não
constituem territórios, estarem sujeitas a sistemas de controle impostos pela
instituição indigenista (atualmente FUNAI). Apresentação: dos índios e suas terras Os que avançaram pelo
litoral abrindo caminhos para o progresso bruto, simbolizado na imagem do pai
arbitrário e repressor, sem amor à terra nem aos filhos. Por meio das formas
geográficas é que este caminho foi aberto, por meio da destruição e redução
de povos inteiros, por meio de doenças, de cooptação política ou da guerra
declarada. O lote, neste sentido, é a forma-símbolo deste modelo de
dominação do território, já que, implantado pelo europeu na cidade colonial,
se expandirá primeiro em direção às quintas4 e, depois, irá demarcar o espaço
construído nas fazendas coloniais e, nas cidades fundadas no interior do país, pós v.23 n.40 • são paulo • outubro 2016 até, por fim, alcançar as aldeias indígenas, cuja lógica de ordenamento –
implantada primeiro pelos padres jesuítas e em seguida pelo SPI – será
bastante semelhante à lógica da fazenda, tendo como princípio a lógica da
produção, ou do que podemos chamar de “uso produtivo do espaço”
(AMPARO, 2015). A paisagem da aldeia Kaingáng5 contemporânea pode ser
compreendida a partir da lógica espacial conhecida entre os geógrafos por
rugosidade, em M. Santos, ou de residualidade, em H. Lefebvre. Local
histórico de assentamentos Kaingáng nos últimos 25 séculos – com fartos
vestígios arqueológicos6 – as terras Kaingáng, no contexto da fricção
interétnica7 e das políticas de territorialização8 , não foram demarcadas a partir
de suas territorialidades tradicionais, pautadas na lógica da mobilidade e do
ciclo roça/coleta/ritual. A forma geográfica contemporânea é dada pelo
entrecruzamento de duas lógicas escalares distintas (HAESBAERT, 2011). Para
Oliveira, as Terras Indígenas são antes unidades jurídicas que sociológicas, em
função da série de contradições que marcam o processo demarcatório nos
contextos políticos e legais (OLIVEIRA, 1998, p. 26). As Terras Indígenas, ao
serem demarcadas, em meio a fortes processos e tensões políticas, mormente
lhes reservou alguma área a partir de seus assentamentos (toldos) e lhes
restringiu a territorialidade, já que a inscrição típica da geografia “branca”
(ocidental) é o limite – instituído na cartografia geralmente por uma linha reta
– que limita dois espaços contíguos (estabelecendo o dentro e o fora). até, por fim, alcançar as aldeias indígenas, cuja lógica de ordenamento –
implantada primeiro pelos padres jesuítas e em seguida pelo SPI – será
bastante semelhante à lógica da fazenda, tendo como princípio a lógica da
produção, ou do que podemos chamar de “uso produtivo do espaço”
(AMPARO, 2015). Apresentação: dos índios e suas terras Assim, através da FUNAI, do controle do Estado e também, agora, através das
parcerias com ONGs ambientalistas que possuem propósitos e objetivos que
nem sempre se deve confundir com os interesses dos indígenas, as Terras
Indígenas obedecem a uma lógica de produção espacial na qual o controle que
delas fazem é meramente simbólico, uma espécie de territorialismo que não
discute autonomia, onde se reproduz uma lógica imposta de fora da aldeia,
lógica esta que, não raro, enviesa em direção ao arbitrário e ao autoritário. Assim, os conflitos em torno desta lógica de produção do espaço nas terras
indígenas produzem rupturas políticas que, com frequência, levam à formação
de novas aldeias – emã e acampamentos – varé (JAENISCH, 2010, p. 21). artigos • p. 026-046 Fica claro que as Terras Indígenas, demarcadas como que fragmentos, são
uma das estratégias de territorialização estabelecidas junto aos indígenas, e
que, em seu bojo, provoca, necessariamente, conflitos internos, sobretudo os
relacionados aos projetos de uso da terra demarcada, já que, diferentemente
do planejamento estatal que as vê como homogêneas, as comunidades
indígenas são marcadas pela heterogeneidade de projetos e visões de mundo,
as quais convergem apenas em determinados momentos específicos,
geralmente momentos de luta contra algum inimigo comum e externo à
comunidade. Essas narrativas, as do conflito interno às aldeias, que geram mesmo a
desterritorialização de indígenas por outros (indígenas), são frequentes entre
os Kaingáng, de modo que assumem também um caráter essencial na
produção social dos espaços das aldeias. Pauta esta lógica uma visão
funcionalista-mecanicista do território, na qual este é tomado como recurso,
que remete ao valor de troca mais que ao valor de uso. Isto porque somente a
partir de 1988 passou a se reconhecer aos indígenas o direito de viverem
conforme suas racionalidades próprias. 030 As Terras Indígenas, até então, possuíam uma gestão voltada à produção de
bens conforme a “vocação” da terra. Isto explica o fato de as Terras Kaingáng
terem sido, ao longo do século XX, violentamente exploradas como área
provedora de madeiras, já que com o avanço das colônias de produção rural
(soja, trigo, milho, etc.), passaram a abrigar as únicas florestas que restavam
na região. Tão logo se consumou essa degradação inicial das florestas, ou
concomitantemente o modelo de produção agrícola de larga escala, e,
pautado no uso de máquinas e agrotóxicos, adentrou as Terras Kaingáng,
estabelecendo um ciclo de produção existente até hoje. Apresentação: dos índios e suas terras A este ciclo
chamamos de rugosidade da forma na aldeia (Amparo, 2015), tomando o
termo rugosidade no sentido de Milton Santos, ou seja, no sentido de uma
leitura da forma arquitetônica e de sua relação sincrônica ou diacrônica com
as práticas sociais e os modos de vida contemporâneos. Assim, estas formas herdadas de um tempo anterior (às quais Lefebvre, por
sua vez, chama de “espaços residuais”: os galpões, os campos de cultivos),
continuam operantes na paisagem Kaingáng, sendo percebidas por qualquer
pessoa que possa analisá-la do ponto de vista morfológico. pós v.23 n.40 • são paulo • outubro 2016 A produção do espaço social indígena Não se pode afirmar que os espaços mentais dos indígenas não se aproximem
da geometria. As pinturas corporais, os artesanatos, enfim, todos os grafismos
e formas arquitetônicas, todas elas expressões de saberes e fazeres indígenas,
remetem, de certa forma, a uma geometrização do mundo. O que se observa,
contudo, é que a apropriação dessas formas é abstratamente construída no
sentido de um binarismo ou dualismo identitário, remetendo a um ou outro
clã formador (caso das sociedades duais). Os Kaingáng são uma sociedade
dual, ou seja, uma sociedade fundada na divisão do mundo entre dois grupos
principais, os Kamé e os Kanhru. Kamé é o fundador do povo Kaingáng. Consciente de sua incompletude no mundo, criou seu irmão Kanhru, para
ajudar-lhe a constituir o mundo. Cada qual criou seu conjunto de seres, pós v.23 n.40 • são paulo • outubro 2016 identificando-os a si e assim se fez o mundo. Kamé fez cobras, Kanhru fez
onças. Kamé fez a Araucária, Kanhru o Cedro, etc. Os que se identificam a
Kamé, assim, adotam a pintura em forma de linhas/traços; ao passo que os
Kanhru adotam as formas de círculos e pontos na pintura corporal, no grafismo
e no artesanato. 031
pós- identificando-os a si e assim se fez o mundo. Kamé fez cobras, Kanhru fez
onças. Kamé fez a Araucária, Kanhru o Cedro, etc. Os que se identificam a
Kamé, assim, adotam a pintura em forma de linhas/traços; ao passo que os
Kanhru adotam as formas de círculos e pontos na pintura corporal, no grafismo
e no artesanato. A geometrização do espaço abstrato dos indígenas toma formas muito peculiares
de mediação, que em muito pouco se equiparam às formas tomadas do
racionalismo cartesiano. Antes, elas voltam na forma de expressões artísticas, se
expressam por meio de pinturas, da música, dos objetos, ou mesmo da família e
do ritual. Quando falamos de “propriedade”, ela é bastante relativa no caso
indígena, na verdade, “posse”, prevalecendo na comunidade os sistemas de
parentesco e afinidade na divisão dos bens, do trabalho e do prestígio social. A
forma da aldeia, salvo em casos onde esta foi profundamente alterada, como no
caso Kaingáng, obedecia a uma certa simetria com essas relações de parentesco
e afinidade, estabelecendo, por exemplo, aldeias circulares ou em forma de
ferraduras (como nos clássicos estudos do antropólogo Lévi-Strauss sobre os
Bororo, ou de Cristina Sá, na arquitetura). Figura 1: Aspecto da paisagem
da Aldeia Votouro.
Foto do autor, 16 de Abril de
2005 O lote e maloca Se a reflexão desde a escala territorial nos permite compreender os processos
de fricção interétnica e a paulatina assimilação de práticas e formas espaciais
pelos Kaingáng em suas terras demarcadas estabelecendo formas que se forjam
a partir do “conflito entre a predação simbólica da alteridade (no sentido de
Eduardo Viveiros de Castro), da antropofagia e da hibridização” (HAESBAERT;
MONDARDO, 2010) e a territorialização indigenista (no sentido weberiano, da
administração pública, vide João Pacheco de Oliveira), as escalas residencial e
aldeã nos possibilitam observar uma evolução material na forma de construção
das habitações Kaingáng. O lote, com base nas reflexões realizadas pelo Prof. Nestor Goulart Reis (USP) em seu livro Quadro da Arquitetura no Brasil, é a
forma geométrica que irá demarcar o espaço urbano e a arquitetura no Brasil. Não é o momento de aprofundar ou resenhar suas análises, somente captar
esta ideia para compreender sua implicação prática nas formas de fazer
arquitetura que, a partir da colonização, se estabeleceram no Brasil. O sentido do lote, tal qual demarcado e conhecido, se dá na cidade, para
responder às limitações espaciais a serem suplantadas pelo urbanismo colonial;
mas sua expansão em direção ao campo não obedece ao mesmo princípio,
resultando, antes, uma mímeses de um saber-fazer já testado, e que, até certo
ponto, resolvia um problema urbano. É necessário ter em conta que a
arquitetura brasileira buscou adequar-se ao lote retangular, resultando esta
forma em referência para os projetos de arquitetura no Brasil, sendo ela
encontrada nas Casas Grandes das fazendas, em palácios e nas sedes dos
órgãos do Governo imperial e republicano. É o modelo naturalizado que
somente com o movimento modernista conhece um esforço de ruptura. Essa
reprodução, que alcançará as sedes dos postos indígenas, é explicada, então,
não como criação, mas como mímesis, já que nas fazendas e quintas não havia
o espaço limitante do lote para determinar a área a ser construída. A este modelo irá aderir a forma indígena implantada nos mais diferentes
rincões do Brasil, resultando, portanto, da mímesis de um projeto urbano
transposto para locais onde outras formas de saber-fazer inscreviam
arquiteturas e manejos territoriais historicamente construídos desde a
necessidade de resolver problemas específicos. O posto indígena (expressão do
lote) e a maloca passam a confrontar-se na paisagem, de norte a sul do país. A produção do espaço social indígena A noção de limite, tal qual como conhecemos, estabelecida por um esquema
racional-cartesiano, não parece inerente ao Kaingáng. Ao contrário, parece que
a realidade demarcada pelo limite se mostra por demais cara a estes indígenas;
é o que se depreende da literatura etnográfica e do cenário atual. Entretanto,
sabemos que a propriedade era bem demarcada e exercida pelos chefes e seus
grupos de afinidade. Somos, assim, induzidos a outra racionalidade ante o
espaço geográfico, a uma percepção que só recentemente passa a encontrar
acolhimento no contexto da geografia disciplinar (simbolismo/fenomenologia),
ao passo que, desde pelo menos Pierre Clastres, Carl Sauer e Darril Poasey, já
se estuda na etnologia. O sistema de cacicados não implica uma percepção
retilínea do território. Antes, os caciques Kaingáng constituem seus territórios
pelo domínio de áreas de exploração do pinhão. Estas árvores eram marcadas
com os grafismos indígenas e respeitadas pelos demais caciques. Com a
colonização, contudo, essas áreas passaram a ser cada vez mais devastadas e
transformadas em fazendas, lotes, polígonos..., enfim, propriedades artigos • p. 026-046 delimitadas. Portanto, a paisagem da aldeia Kaingáng reproduz, atualmente, a
lógica dessas limitações e conflitos que se travam no território. pós v.23 n.40 • são paulo • outubro 2016 Figura 2: Habitar Pirahã.
Foto do autor O lote e maloca A
dissimetria que se estabelece entre uma forma e outra é exatamente o fato de
o primeiro ter limites e o segundo não, haja visto a diversidade de malocas
reconhecidas na etnografia: circulares, retangulares, sibs (malocas coletivas
yanomamis), ou mesmo meras coberturas de palha e completo desapego pela
“casa”, como entre os Pirahã. Fruto da racionalidade “branca”, homogênea,
em sua supremacia ante as múltiplas racionalidades ancestrais e suas
expressões sem Estado, a evolução da paisagem na aldeia se dá em sincronia
com o movimento de negação do caráter histórico dos povos indígenas. Se dá
concomitante à naturalização de sua relação com a terra e a negação de sua
alteridade. pós v.23 n.40 • são paulo • outubro 2016 A evolução da maloca, a assimilação da forma do branco, tomada como
superior a partir da mímesis, se dá concomitante à política de integração do
indígena à sociedade nacional, tendo por base o instituto da tutela. Se dá a
partir do momento em que ele (o índio) é tido como um ser em transformação,
asseverando seu status provisório, de trânsito, numa história de curso único
que, negando-lhe o direito de ser índio, lhe toma como um ser em transição
para o seu futuro, que é destituir-se por completo de sua identidade, passando
a ser camponês (OLIVEIRA, 1998) ou, mais recentemente, um cidadão
marginalizado da periferia urbana. Assim seus cultivos, seus saberes e fazeres,
seus modos de habitar e viver, sua visão cosmológica, tudo isso deve ser
suprimido em benefício do progresso intocável, que impede o sujeito de
assumir os rumos de sua transformação social. A lógica dos resíduos de Lefebvre ou a lógica das rugosidades (de Milton
Santos), aplicadas dialeticamente aos indígenas, nos possibilita tomar as
formas ancestrais como rugosidades que lhes conferem uma sub-identidade, já
que ali persistem relembrando aquilo que eram, e que não deveriam mais ser;
ao mesmo tempo em que se pode observar os objetos espaciais introduzidos
nas suas aldeias como rugosidades de projetos dos brancos para suas terras,
com os quais não conseguem romper, por necessidades materiais e financeiras
contemporâneas. Não são quaisquer dificuldades que os indígenas da
atualidade enfrentam, particularmente os kaingang. Habitar kaingáng Categoria
Morfologia
Situação Jurídica e política
Razão dos conflitos
Sujeitos do conflito
Varé
Varé
Varé
Varé
Varé
Acampamento
Habitação precária, cobertas de lona,
geralmente à beira de estradas (des-
territorialização). Demanda / Reivindicação. Luta por terra e dignidade (re-existência). Indígenas x Fazendeiros, grileiros e
comunidade política “regional” (sem nos
aprofundar devidamente nos múltiplos
significados do termo “regional”). Emã
Emã
Emã
Emã
Emã
Assentamento, “toldo”, aldeia
Tendência à “modernização”/adoção da “forma”
indigenista/“predação simbólica da alteridade”, maior
autonomia em função da área territorial demarcada,
que, no entanto, tende a ser restrita e conflituosa
(reclusão /exclusão/contenção territorial). Demarcada/Regularizada (re-territorialização). Ordenamento e gestão Territorial (re-territorialização). 1. Conflitos Internos
2. Indígenas x posseiros
3. Indígenas x rendeiros da terra (no caso do RS, que
por meio de “parcerias” ilegais arrendam áreas
indígenas para plantações de soja);
3. Indígenas x outras etnias vivendo na mesma aldeia
(re-territorialização). Quadro 1: Tipologias de Varé e Emã (Acampamento e Assentamentos) entre os Kaingáng. encontram-se, portanto, nesta frente de ação, situando-se no sul do Brasil, na
modernidade colonial com poucas terras – fragmentadas e conflituosas. Esse é
o contexto no qual se discute, portanto, o habitar e o fazer arquitetônico
indígena. A estratégia de “ocupação” (ou varé), utilizada pelos indígenas, não é de todo
diferente das estratégias do MST – Movimento dos Trabalhadores Rurais Sem-
Terra. Nesta condição se situam dois acampamentos – Kandóia e Forquilha
Grande, no Rio Grande do Sul. Nestes acampamentos, os indígenas vivem em
barracos de lona, à beira da estrada, em condições precárias. Nestas
habitações vivem durante todo o tempo à espera da demarcação de suas terras,
causa que pode atravessar várias gerações. O emã (assentamentos ou toldos), por sua vez, corresponde às áreas já
demarcadas, nas quais já se tem certas garantias legais, por meio do usufruto
exclusivo, este, como um direito parcial. Seu fazer arquitetônico, colonizado,
torna-se híbrido, apropriando-se da técnica e das casas deixadas pelos
colonos. O acampamento (-varé) e os toldos (-emã, ou assentamentos) são
paisagens rurais diferenciadas não tanto em sua paisagem, mas em sua
organização política e social. Há relações intrínsecas entre os acampamentos
e os assentamentos, as quais correspondem a espaços de “exclusão”,
“contenção” ou mesmo “reclusão” (HAESBAERT, 2004). Habitar kaingáng Ao contrário dessa lógica de negação (“tornar invisível”) das formas indígenas,
a qual pode-se chamar de indigenismo “moderno-colonial”, novas lógicas
emergem no século XXI, articulando-se de muitas maneiras, com novas
bandeiras, como dignidade, direito à sexualidade e empodeiramento das
mulheres, que vão muito além das tradicionais bandeiras de esquerda (CRUZ,
2013). Assim, da luta reprimida de todos estes sujeitos insurgentes, temos a
pluralidade de ideias e correntes que irão determinar a apropriação dos
espaços sociais, orientada a manterem-se vivos, em sua plenitude, r-existir
(PORTO GONÇALVES, 2003; CRUZ, 2013). Os Kaingáng contemporâneos artigos • p. 026-046 ós
são
a
o
o t
o
6
encontram-se, portanto, nesta frente de ação, situando-se no sul do Brasil, na
modernidade colonial com poucas terras – fragmentadas e conflituosas. Esse é
o contexto no qual se discute, portanto, o habitar e o fazer arquitetônico
indígena. A estratégia de “ocupação” (ou varé), utilizada pelos indígenas, não é de todo
diferente das estratégias do MST – Movimento dos Trabalhadores Rurais Sem-
Terra. Nesta condição se situam dois acampamentos – Kandóia e Forquilha
Grande, no Rio Grande do Sul. Nestes acampamentos, os indígenas vivem em
barracos de lona, à beira da estrada, em condições precárias. Nestas
habitações vivem durante todo o tempo à espera da demarcação de suas terras,
causa que pode atravessar várias gerações. O emã (assentamentos ou toldos), por sua vez, corresponde às áreas já
demarcadas, nas quais já se tem certas garantias legais, por meio do usufruto
exclusivo, este, como um direito parcial. Seu fazer arquitetônico, colonizado,
torna-se híbrido, apropriando-se da técnica e das casas deixadas pelos
colonos. O acampamento (-varé) e os toldos (-emã, ou assentamentos) são
paisagens rurais diferenciadas não tanto em sua paisagem, mas em sua
organização política e social. Há relações intrínsecas entre os acampamentos
e os assentamentos, as quais correspondem a espaços de “exclusão”,
“contenção” ou mesmo “reclusão” (HAESBAERT, 2004). Seus conflitos
decorrem geralmente:
a) do restrito espaço para grandes comunidades;
b) do uso predatório e espoliativo (garimpo, desmatamento, cultivo de soja) por
indígenas ou invasores;
c) dos impactos decorrentes de grandes projetos como hidrelétricas ou
mineração (os Kaingáng do norte do Rio Grande do Sul foram recentemente
impactados com a construção da Pequena Central Hidrelétrica de
Monjolinho, na bacia do Rio Uruguai). O quadro a seguir apresenta uma tipologia entre Varé e Emã. Colonização Nas primeiras menções da literatura etnográfica, os Kaingáng aparecem
primeiro como sendo os Guaianás, por volta do século XVIII ao XIX, período no
qual eram ainda pouco conhecidos11 . Apenas em fins desse século e início do
século XX é que, através da análise linguística, se lhes descreve a eles – os
Kaingáng – e seus parentes – Xokreng –, como os Jê do Sul; sendo diferentes
dos Guarany. Parte importante dos estudos sobre os Kaingáng é realizada por
linguistas, sendo a língua destes indígenas uma das mais conhecidas dentre os
povos ameríndios. Alguns pesquisadores têm se dedicado ao estudo de aspectos
da história, das relações de poder, da religiosidade e da apropriação da
natureza pelos indígenas. Os estudos sobre o habitar Kaingáng se apoiam em
subsídios dos estudos realizados por arqueólogos, os quais descobriram e
delimitaram os sítios da Tradição Taquara, ajudando a compreender melhor o
habitar Kaingáng ante o massacre promovido pela expropriação colonial (a
“hecatombe” de outrora e o etnocídio contemporâneo). Segundo os arqueólogos, a casa subterrânea foi concebida pelos Kaingáng como
uma resposta adaptativa ao ambiente meridional, para o qual haviam migrado
há cerca de 2 mil e quatrocentos anos, procedendo do cerrado. Por esta razão,
os Kaingáng são também conhecidos como os Jê do Sul, ou Jê meridionais,
linguística e etnograficamente relacionados aos povos Jê; portanto, parentes dos
Craô, dos Timbira, dos Bororo, dos Xavante e dos Kayapó. Esta observação nos
permitirá entender a forma arquitetônica que veio em seguida. Consta que no
século XIX não havia mais registro das casas subterrâneas Kaingáng na
paisagem da região Sul. Ao invés destas, na paisagem dos toldos (aldeias) passa a prevalecer malocas
feitas à semelhança das casas que atualmente encontram-se entre os Timbira do
Brasil central. Certamente a memória ancestral deve ter sido crucial para o
desenvolvimento desta forma arquitetônica. Entretanto, a casa indígena do século
XIX era muito menos eficaz que a casa subterrânea no que diz respeito a conforto
climático. Uma análise dos relatos do século XIX demonstra que, exatamente
neste período, tanto os Kaingáng foram vitimados em guerras tribais quanto
também se verificaram enormes epidemias de gripes e tuberculose em suas
aldeias. Crê-se também que esta relação entre conforto ambiental, arquitetura e
mortandade indígena ainda não tenha sido devidamente conhecida12 . Habitar kaingáng Seus conflitos
decorrem geralmente: a) do restrito espaço para grandes comunidades; b) do uso predatório e espoliativo (garimpo, desmatamento, cultivo de soja) por
indígenas ou invasores; c) dos impactos decorrentes de grandes projetos como hidrelétricas ou
mineração (os Kaingáng do norte do Rio Grande do Sul foram recentemente
impactados com a construção da Pequena Central Hidrelétrica de
Monjolinho, na bacia do Rio Uruguai). O quadro a seguir apresenta uma tipologia entre Varé e Emã. O quadro a seguir apresenta uma tipologia entre Varé e Emã. Categoria
Morfologia
Situação Jurídica e política
Razão dos conflitos
Sujeitos do conflito
Varé
Varé
Varé
Varé
Varé
Acampamento
Habitação precária, cobertas de lona,
geralmente à beira de estradas (des-
territorialização). Demanda / Reivindicação. Luta por terra e dignidade (re-existência). Indígenas x Fazendeiros, grileiros e
comunidade política “regional” (sem nos
aprofundar devidamente nos múltiplos
significados do termo “regional”). Emã
Emã
Emã
Emã
Emã
Assentamento, “toldo”, aldeia
Tendência à “modernização”/adoção da “forma”
indigenista/“predação simbólica da alteridade”, maior
autonomia em função da área territorial demarcada,
que, no entanto, tende a ser restrita e conflituosa
(reclusão /exclusão/contenção territorial). Demarcada/Regularizada (re-territorialização). Ordenamento e gestão Territorial (re-territorialização). 1. Conflitos Internos
2. Indígenas x posseiros
3. Indígenas x rendeiros da terra (no caso do RS, que
por meio de “parcerias” ilegais arrendam áreas
indígenas para plantações de soja);
3. Indígenas x outras etnias vivendo na mesma aldeia
(re-territorialização). Quadro 1: Tipologias de Varé e Emã (Acampamento e Assentamentos) entre os Kaingáng. pós v.23 n.40 • são paulo • outubro 2016 pós v.23 n.40 • são paulo • outubro 2016 artigos • p. 026-046 Figura 5: Casa do Posto Indígena.
Fonte: Zuch-Dias, p. 251. Colonização Embora
muitos estudos se dediquem à moradia precária nas cidades, pouco se sabe sobre
as formas de habitação indígena e, principalmente, de que maneira a ação
indigenista determina suas tipologias, com a demarcação ou não da terra
indígena, forma jurídico-administrativa que determina a possibilidade de uma
habitação mais digna, ou mesmo o retorno da moradia subterrânea. Esta,
contudo, não é o caso das muitas comunidades Kaingáng situadas em beiras de
estradas, à espera do reconhecimento de seu direito legítimo13. Veiga & D’Ângelis, em artigo publicado em 2003, antecipam parte das
observações que também pudemos realizar: Podem-se encontrar, em comunidades Kaingang atuais, casas de alvenaria
com cobertura de telhas de cimento amianto (como “brasilit” ou
“eternit”), casas de madeira com o mesmo tipo de cobertura, ou cobertas
de zinco, ou cobertas com telhas de barro ou, ainda, cobertas de
“tabuinhas”. Mas também encontram-se, em muitas áreas, casas ou artigos • p. 026-046 ranchos de pau-a-pique, em geral com cobertura de folhas vegetais. É
claro, dada a situação de penúria de muitas famílias, encontram-se
Figura 3: Concepção de palhoça Kaingáng do século
XIX. Fonte: Zuch-Dias, p. 154. Figura 4: Concepção artística de uma casa subterrânea
(Adaptado de Fernando La Salvia). Fonte: Veiga, p. 40. Figura 5: Casa do Posto Indígena. Fonte: Zuch-Dias, p. 251. Figura 4: Concepção artística de uma casa subterrânea
(Adaptado de Fernando La Salvia). Fonte: Veiga, p. 40. Figura 3: Concepção de palhoça Kaingáng do século
XIX. Fonte: Zuch-Dias, p. 154. Figura 3: Concepção de palhoça Kaingáng do século
XIX. Fonte: Zuch-Dias, p. 154. Figura 4: Concepção artística de uma casa subterrânea
(Adaptado de Fernando La Salvia). Fonte: Veiga, p. 40. 036 pós- ranchos de pau-a-pique, em geral com cobertura de folhas vegetais. É
claro, dada a situação de penúria de muitas famílias, encontram-se
também nas áreas indígenas (ou em acampamentos indígenas nas
periferias de cidades), abrigos feitos de lona, papelão, compensados e
outros materiais de aproveitamento. Mais raramente, hoje em dia,
encontram-se também em algumas áreas casas com parede de trançado
de taquaruçu. (VEIGA & D’ÂNGELIS, 2003, p. 216). ranchos de pau-a-pique, em geral com cobertura de folhas vegetais. É
claro, dada a situação de penúria de muitas famílias, encontram-se
também nas áreas indígenas (ou em acampamentos indígenas nas
periferias de cidades), abrigos feitos de lona, papelão, compensados e
outros materiais de aproveitamento. Colonização Mais raramente, hoje em dia,
encontram-se também em algumas áreas casas com parede de trançado
de taquaruçu. (VEIGA & D’ÂNGELIS, 2003, p. 216). No século XX, houve, com o indigenismo do SPI, algum progresso no
reconhecimento à terra, ainda que no contexto da integração indígena por
meio do campesinato, como aponta João Pacheco de Oliveira. Assim,
rapidamente instituiu-se moradias indígenas inspiradas nas casas do posto
indígena e nas casas dos colonos regionais, por mímesis. Certamente em
função dos problemas apontados no tópico anterior, os Kaingáng adotaram
estas formas como estratégia de sobrevivência, operando, portanto, uma
predação simbólica destas formas. Isto não significa que a adoção destas
resolva os problemas do habitar Kaingáng. Ao contrário. Resolve um primeiro
problema, o de resistir ao frio numa região de clima temperado, mas estabelece
uma nova lógica de divisão do espaço interno da moradia, em quartos. O fogo
perde seu lugar central e passa a um espaço periférico, ainda que se tente por
inúmeras formas dar-lhe um lugar privilegiado. Compromete ainda a autonomia
de sua edificação, demandando agora de recursos monetários para obtenção
de “materiais” (telhas, pregos, etc.). Todas as formas da aldeia seguem sendo edificadas tomando por paradigma o
retângulo que estabeleceu o lote. Essas formas, atualmente, já não são
somente a do posto indígena, mas do posto de saúde, das igrejas católicas e pós v.23 n.40 • são paulo • outubro 2016 Figura 6: Sucessão da moradia
e territorialização Kaingáng ao
longo dos séculos. Esquema do
autor (com desenhos de Beber
e Zuch-Dias). evangélicas, das escolas, etc. A evolução da paisagem nas aldeias, portanto,
tomadas nas escalas da casa e da aldeia, nos revelam a assimilação destas
formas de construir, em detrimento das tecnologias ancestrais, saberes-fazeres
específicos que, conhecendo a ruptura, retiram também parte da autonomia
indígena ante o mundo socialmente construído. As paisagens das aldeias
Kaingáng revelam a apropriação capitalista deste espaço por um modo de
saber-fazer voltado para a integração do indígena, para sua transformação em
camponês, para a inclusão produtiva de suas terras e para a ruptura com suas
cosmovisões de mundo, que são pautadas na mitologia e na ancestralidade. Figura 6: Sucessão da moradia
e territorialização Kaingáng ao
longo dos séculos. Esquema do
autor (com desenhos de Beber
e Zuch-Dias). artigos • p. 026-046 Reflexão sobre alteridade: o saber-fazer e
a possibilidade/necessidade de
epistemologização do outro A pesquisa sobre as realidades contemporâneas dos povos indígenas, além de
observações empíricas a partir de suas lutas, deve remeter também a uma
construção conceitual, teórica e epistemológica. Conhecer os povos indígenas,
desde a perspectiva cosmológica e holística de suas visões de mundo, demanda
estar aberto a paradigmas como a transdisciplinaridade e a transculturalidade,
vislumbrando horizontes metodológicos como a dialógica, a horizontalidade e a
heterogeneidade, tal qual propõe Paulo Freire (1969). O transbordo de multiplicidades se dá não somente a partir do princípio opaco
da reflexão conceitual. Antes, este transbordo paradigmático emerge desde a
realidade concreta dos indígenas, que nos remete a estudos da antropologia14 . É neste contexto que cremos que tais realidades devem ser criticamente
avaliadas, aderindo ao paradigma da complexidade, ou, mais precisamente, na
reflexão sobre razão, racionalidade e racionalização e suas múltiplas formas de
expressão (MORIN, 1994). artigos • p. 026-046 O paradigma da complexidade ecoa e sofistica as propostas já apresentadas
anteriormente (por exemplo, o “pensamento mágico”, de Paulo Freire ou a
“predação simbólica da alteridade”, em Castro). Alteridade e complexidade
como pontos de partida conduzem a uma reflexão sobre as limitações das
abordagens folclóricas (dos conhecidos “estudos de Folk”), nas quais com a
homogeneização global, provocada pelo capitalismo em sua fase pós-industrial,
os povos indígenas e comunidades tradicionais estariam fadadas a desaparecer,
se aproximando de concepções equívocas, tais quais o “fim da história”
(Barthez) ou “da geografia (Virilio)”15 . Essas são, portanto, concepções das
quais pretendemos nos afastar, por constituírem, a nosso ver, um anacronismo
fatalista que apresenta as dificuldades de compreensão dos sujeitos sociais do
campo no Brasil e as formas geográficas que elas produzem e nas quais sua
alienação é re-produzida. Uma possibilidade metodológica a ser pensada diz respeito à nossa abordagem,
focada nos saberes e nos fazeres dessas populações, entendidas como formas
de apreensão e representação do mundo, mas também como conhecimentos
que viabilizam a sua transformação, ou seja, que atuam no espaço social
concreto. Assim, quando nos referimos a “saber” e a “fazer”, atribuímos a
ambas as noções da perspectiva dialética, necessária para, em seu processo de
re-produção social, constituírem o conhecimento. Assim, nem o saber, nem o
fazer são conhecimentos, mas a complexidade dialética que desenvolvem. Figura 7: Localização das TIs Kaingáng no sul do Brasil. (Mapa do autor) Figura 7: Localização das TIs Kaingáng no sul do Brasil. Reflexão sobre alteridade: o saber-fazer e
a possibilidade/necessidade de
epistemologização do outro (Mapa do autor) Figura 7: Localização das TIs Kaingáng no sul do Brasil. (Mapa do autor) pós v.23 n.40 • são paulo • outubro 2016 O saber não se sabe a si mesmo, assim como o fazer também não faz sem
saber. Estão mais imersos na economia da natureza que na economia de
mercado, pelo que respondem ao princípio marxista: “A flor produz sem saber
que produz” (MARX). Entretanto, o homem, individual ou coletivamente,
produz saberes (e sabores) a serem experimentados. Esses saberes e sabores,
como bem demonstrou Lévi-Strauss, se legitimam somente no projeto de sua
eficácia social, lócus de sua replicação cotidiana. Sua con-formação em fazer
está, portanto, relaciona à sua eficácia no contexto social que o produziu. Há uma grande diferença entre “saber”, “fazer” e “conhecimento”. O saber
ocupa a esfera do plano, do projeto. O fazer ocupa a esfera de objetivação
social de determinado saber e se efetiva por meio do trabalho (entendido no
plano de transformação da natureza e não meramente do ponto das relações
capitalistas). Nem todo saber se transforma em fazer, mas todo o fazer é,
necessariamente, a objetivação de um saber, uma “coisa” produzida a partir de
uma ideia-conceito, aqui nos servindo como exemplo a cestaria Kaingáng, cujo
artesanato lhes é típico e ao mesmo tempo peculiar e exclusivo, portanto, um
saber e um fazer complexo (PRADO JÚNIOR, 1980; MOREIRA, 2010). pós- Por conhecimento, na perspectiva dos povos indígenas e das comunidades
tradicionais, compreende-se o conjunto dos saberes, sabores e fazeres
produzidos por sujeitos sociais concretos, situados no tempo histórico e tendo
na produção, na reprodução e na transformação do espaço geográfico a real
dimensão de sua existência. Por sua vez, ao se “fazerem” no espaço, ao
objetivarem-se em formas, grafias, eles se tornam objetos de crítica e evolução
do saber que o instituiu, no sentido de sua adequação à realidade social de
onde vem sua significação. Por esta razão, então, é que este saber-fazer se
torna conhecimento, por se conformar não numa relação entre teoria e prática,
mas numa dialética da práxis, na qual tanto saber quanto fazer estão
constantemente submetidos à análise crítica por parte de um ente legitimador,
no caso, a realidade concreta e os sujeitos sociais. Finalmente, cabe aqui definir, com mais exatidão, a quais sujeitos nos
referimos quando falamos em indígenas. Reflexão sobre alteridade: o saber-fazer e
a possibilidade/necessidade de
epistemologização do outro Para o antropólogo Eduardo Viveiros
de Castro: Devemos começar então por distinguir as palavras “índio” e “indígena”,
que muitos talvez pensem ser sinônimos, ou que “índio” seja só uma
forma abreviada de “indígena”. Mas não é. Todos os índios no Brasil são
indígenas, mas nem todos os indígenas que vivem no Brasil são índios. Índios são os membros de povos e comunidades que têm consciência —
seja porque nunca a perderam, seja porque a recobraram — de sua
relação histórica com os indígenas que viviam nesta terra antes da
chegada dos europeus. Foram chamados de “índios” por conta do famoso
equívoco dos invasores que, ao aportarem na América, pensavam ter
chegado na Índia. “Indígena”, por outro lado, é uma palavra muito antiga,
sem nada de “indiana” nela; significa “gerado dentro da terra que lhe é
própria, originário da terra em que vive” [1]. Há povos indígenas no Brasil,
na África, na Ásia, na Oceania, e até mesmo na Europa. (...) O antônimo
de “indígena” é “alienígena”, ao passo que o antônimo de índio, no Brasil,
é “branco”, ou melhor, as muitas palavras das mais de 250 línguas índias
faladas dentro do território brasileiro que se costumam traduzir em
português por “branco”, mas que se refere a todas aquelas pessoas e
instituições que não são índias. (CASTRO , 2016) artigos • p. 026-046 0
TTTTTer
er
er
er
ermo
mo
mo
mo
mo
Povos (ou comunidades)
indígenas
Nações indígenas
Tribos
Sociedades indígenas
Coroados, gentis
Bugres, xavantes, selvagens
Leitura
Leitura
Leitura
Leitura
Leitura
Uso comum no contexto de estados nacionais, leitura
da comunidade como “gemmeinschaft” (influencia
weberiana). Movimentos por dignidade e território, sobretudo da
América Latina, mas também no Brasil. Está em
questão não apenas o direito à terra, mas à
continuidade de seus modos de “r-existir”. Noção colonialista, apresenta precisão limitada e
geradora de confusões de entendimento. Influência sociológica, não aceita por alguns autores,
como Cunha (2009.)
Textos seiscentistas, marcados por perspectiva teológica
(principalmente dos Jesuítas). Bandeirantes antigos e colonos sulistas no Brasil
central e Amazônia, agindo em contextos de violências
costumeiras, políticas e simbólicas que associadas à
expropriação e desterritorialização (pensadas a partir de
Marx e Haesbaert). Referência
Referência
Referência
Referência
Referência
Agências Indigenistas (FUNAI e
ONGs). Pensamento decolonial
latinoamericano (CRUZ, 2013;
PORTO -GONÇALVES, 2003). Textos antigos de cronistas e
relatórios de campo, muito
difundido no senso comum
Ex.: Pacheco de Oliveira, Manuela
Carneiro da Cunha. Reflexão sobre alteridade: o saber-fazer e
a possibilidade/necessidade de
epistemologização do outro Viveiros de Castro, Manuela
Carneiro da Cunha. Cidades regionais, redes sociais. Quadro 2: Termos relacionados aos povos ancestrais e as diferentes leituras. Cremos que se pode aderir sem maiores restrições à proposta deste autor, já
que contempla a situação particular dos Kaingáng. Cremos que se pode aderir sem maiores restrições à proposta deste autor, já
que contempla a situação particular dos Kaingáng. pós v.23 n.40 • são paulo • outubro 2016 Algumas palavras, para não concluir Atento à transformação do espaço das aldeias, deve-se considerar não apenas a
transformação de sua realidade geográfica, por meio da transformação dos
“fazeres”, no sentido de sua descrição e evolução, mas, sobretudo, dedicar-se
à compreensão dos saberes que orientam este fazer, imergindo nas
epistemologias que subsidiam a compreensão deste conhecimento e
identificação das diferentes racionalidades que atuam na produção do espaço
geográfico. Mas como imergir em tais realidades, baixo a sofisticação do etnocentrismo,
que coloniza mesmo os redutos mais recônditos do pensamento crítico-
libertário? Evidentemente, não existe um manual para escapar às armadilhas
metodológicas da ciência branca, ocidental e colonizadora. Mas, assim mesmo,
a horizontalidade e a dialógica parecem nos indicar caminhos para esta
construção, ao mesmo tempo em que fornecem as “superfícies de emergência”
(FOUCAULT, 2008) que viabilizam o surgimento de uma “epistemologia da
participação”, ou, mais precisamente, formas de dialogar com as comunidades
indígenas sem induzi-las ou direcioná-las, mas buscando compreender as
contradições inerentes à sua existência social concreta, sempre que possível,
problematizando-as, possibilitando que os mesmos possam, eventualmente,
superá-las. Aqui é importante salientar que a história dos povos indígenas é pós v.23 n.40 • são paulo • outubro 2016 uma história de lutas, que em diferentes momentos adotaram estratégias e
alianças para viabilizarem sua existência ou assegurar direitos mínimos, como a
terra, a dignidade e seus modos de re-existir. Como tem demonstrado Cruz, as
lutas sociais contemporâneas estão para além das tradicionais bandeiras de
esquerda e apontam para temas com o bem viver, o direito à diferença, etc. (CRUZ, 2013). Portanto, não se trata de modificar o curso de suas lutas, mas
de ajudar-lhes a desvendar eventuais armadilhas conceituais operatórias, que
atuam no sentido da restrição de direitos. Esta análise deve estar embebida na fonte do pensamento crítico latino-
americano, embebida no espírito crítico e libertário de pensadores como Paulo
Freire e, sobretudo, Darcy Ribeiro. É possível pensar a dialógica como a
possibilidade de refletir o conhecimento como produção social a partir da
concretude da realidade social. Esta não poderá se manifestar senão por meio
da valorização da vivência e da experiência dos sujeitos que produzem esta
realidade, e essas vivências podem ser captadas por meio da valorização da
narrativa e da possibilidade de serem cartografadas. Ambos processos
dialógicos que retiram o técnico de seu lugar de conforto historicamente
construído no contexto da ciência cartesiana. Figura 8: Local do fogo nas
diferentes moradias indígenas.
Ilustração do autor, com base
em ilustrações e desenhos de
Beber, Zuch-Dias e material
fotográfico. Algumas palavras, para não concluir O técnico, o cartógrafo, o
pesquisador, o educador geógrafo é colocado ante a realidade a partir da
dialética da produção social do espaço, marcada por estes saberes e fazeres. Por meio da interpretação crítica deste conhecimento, ele o reinventa numa
atividade dialógica, ou seja, se insere no processo de produção/reprodução
destes saberes e fazeres e, por conseguinte, da produção/reprodução do
conhecimento e da transformação do espaço e da sociedade, através de um
processo cíclico e dinâmico. 041
- Desvela-se, portanto, uma “epistemologia da participação”, epistemologia
dialética, horizontal e dialógica, na qual temos que considerar os sujeitos de
sua construção. Seu princípio norteador é a emergência do conceito desde a
realidade e seus atores sociais concretos. Seu papel é evitar a manipulação e o
direcionamento, lugares-comuns a que tem sido levado as ciências sociais baixo Figura 8: Local do fogo nas
diferentes moradias indígenas. Ilustração do autor, com base
em ilustrações e desenhos de
Beber, Zuch-Dias e material
fotográfico. Figura 8: Local do fogo nas
diferentes moradias indígenas. Ilustração do autor, com base
em ilustrações e desenhos de
Beber, Zuch-Dias e material
fotográfico. artigos • p. 026-046 o paradigma da colonização e da tecnocracia. Nestes temos as formas de
habitar e a habitação indígena emergem como um relevante objeto de estudo,
seja no sentido de acentuar a peculiaridade de seu saber-fazer e atribuir-lhe
valor ímpar; seja no sentido de anunciar a predação simbólica de formas
criadas por outra lógica territorial, no caso, a lógica indigenista e sua forma
simbólica, o lote. Há ainda uma outra dimensão a ser considerada, a dimensão ética da
produção deste conhecimento, esta incorrendo, sobretudo, na necessidade de
que o conhecimento antes de expressar ou manifestar a “vontade” dos
sujeitos sociais concretos, atue no sentido de dar-lhes voz e visibilidade às
suas causas concretas, possibilitando-lhes um olhar crítico e libertário sobre
as condições de existência e sobre os próprios sujeitos em questão. Neste
sentido, nós, pesquisadores, temos, certamente, muito a contribuir com os
povos indígenas, por meio da compreensão dessas condições de existências e
dos múltiplos saberes, fazeres e conhecimentos que se manifestam por meio
destas. Mas devemos também realizar a crítica de suas contradições. Notas 1 Este texto foi produzido a partir de pesquisa exclusivamente bibliográfica, por provocação da
Profa. Dra. Dinah Papi Guimaraenz, da Escola de Arquitetura e Urbanismo da Universidade Federal
Fluminense (Niterói-RJ), a quem externo meu agradecimento pelo convite para participação no
Seminário Estéticas Transculturais na Universidade Latino-Americana, realizado em maio de 2015
na UniRio, sob sua coordenação e do professor e filósofo Jacques Poulain (Unesco/Université Paris
8). Agradeço também aos Professores Antonio Carlos Carpintero, pela orientação durante o curso
de mestrado, na FAU-UnB, tendo me apresentado à obra do Prof. Nestor Goulart, amizade valiosa e
duradoura construída desde então; ao Professor Márcio Piñom de Oliveira (UFF), por dar vazão às
minhas pretensões intelectuais, estimulando minha pesquisa; à Profa. Flávia Martins, pela
“introdução” ao Lefebvre; ao Carlos (Walter Porto-Gonçalves), pelo diálogo antigo, retomado mais
recentemente com a discussão de algumas dessas ideias em sua disciplina Movimentos Sociais e
Territorialidades; ao professor Valter Carmo Cruz e Denilson Araujo, pela leitura crítica deste
trabalho; ao Ruy pela amizade ao Rogério Haesbaert, pelo ensinamento. Além disso, aos
companheiros geógrafxs Patrícia Moreira Luis Mendes, Marcio D’Arrochella, Leandro Tartaglia,
Luciano Nascimento, Bruno Malheiros e Tatiane Costa, e, pelo diálogo recente em Geografia
Humana, e principalmente, ao Marcos Mondardo, cujo diálogo gira diretamente em torno o objeto
deste artigo. Evidentemente, apesar da construção coletiva de ideias, muitas delas podem não ter
sido inteira ou devidamente apropriadas aqui, exceto quando explicitamente codificada em
citações ou referências. Incoerências e contradições devem ser imputadas ao autor. 2 Sem mencionar o rico patrimônio arqueológico, que desperta pouco interesse entre a população
de um modo geral. 3 Haesbaert e Mondardo fazem apontamentos sobre a antropofagia nas regiões de fronteira,
destacando a precarização e o hibridismo. Entretanto, ao estudarem o Brasil contemporâneo,
remetem à antropofagia no sentido do modernismo brasileiro. 3 Haesbaert e Mondardo fazem apontamentos sobre a antropofagia nas regiões de fronteira,
destacando a precarização e o hibridismo. Entretanto, ao estudarem o Brasil contemporâneo,
remetem à antropofagia no sentido do modernismo brasileiro. 4 Referência à quinta parte de um lote urbano estabelecido pelos portugueses no Brasil colonial, na
qual se encontra, geralmente, o quintal. 5 Por tradição, os etnólogos se referem aos povos indígenas sempre no singular, jamais utilizando o
plural, mesmo quando se utiliza o artigo no plural, com opor exemplo: “os Kaingáng”, ou “os
Terena”, “os Guarany”. Algumas palavras, para não concluir Conhecer a evolução da forma territorial (a
Terra Indígena) e da forma arquitetônica (a casa subterrânea, a maloca ou a
casa do tipo indigenista) é, acreditamos, penetrar nas narrativas
contemporâneas que remetem aos horizontes de re-existência desses sujeitos
históricos, sociais e geográficos. Por fim, quando propomos refletir a evolução da paisagem nas aldeias a partir
dos Kaingáng, cremos que esta reflexão extrapola o caso Kaingáng, sendo ao
mesmo tempo inviável estabelecer, ao menos no espaço de uma comunicação
oral, um aprofundamento de outras realidades indígenas/indigenistas. O que
sabemos, como afirma Pacheco de Oliveira, é que: O destino dos povos e culturas indígenas, tal qual o de qualquer outro
grupo étnico ou nação, não está escrito previamente em nenhum lugar. Seu aspecto primitivo, sua vulnerabilidade e sua presumida tendência à
extinção jamais foram componentes naturais de sua existência senão que
resultados da atuação das elites coloniais que lhes impuseram formas de
dominação que transformaram diferenças culturais, religiosas e políticas
em ‘marcas’ de um subordinação, cristalizando novas hierarquias e
estabelecendo um discurso hegemônico. (OLIVEIRA, 1998, p. 8) Algumas palavras, para não concluir Antes do “planejamento”, no “ordenamento” ou na “Gestão territorial” –
instrumentos técnicos e positivistas cujas racionalidades naturalizam e
reduzem as lutas sociais –, passamos a atuar na esfera dos múltiplos
territórios (territórios materiais e territórios identitários-culturais, como em
Haesbsaert, mas também territórios da comunicação e do diálogo trans e
intercultural) e suas diferentes apropriações expressas por meio de razões,
racionalidades e racionalizações. Deve-se caminhar em direção a uma análise
ontológica e complexa, para refletir sobre tais temas. Se, como afirma a
antropóloga Manuela Carneiro da Cunha, os índios são considerados seres
sem história (CUNHA, 2009, p. 128), o conhecimento de suas antigas
formas de habitação e sociabilidade, aquelas que remontam a bem antes do
processo de colonização, prenunciam o combate contra esta cruzada
ideológica, que é a de tomá-los como seres sem história e, portanto,
igualmente sem futuro, mesmo porque sabemos que a história indígena tem
sido meramente fruto da negligencia intelectual de uma ciência histórica e
geograficamente situada. Pode-se observar ainda, que para os indígenas, o
processo de desterritorialização ao qual estão sujeitos implica na precarização
da existência, como demonstrou Haesbaert (2011, p. 66). Ainda que se
mantenha a historicidade relacionada à identidade territorial, a “predação
simbólica da alteridade”, no caso, efetivada por meio da assimilação da
habitação indigenista, implica a perda do controle do processo de construção
da moradia pelos indígenas, em termos de materiais e técnicas, com a
consequente redução do “laço territorial” das comunidades (se temos em
mente uma noção de território construída a partir da integração das
diferentes dimensões sociais, tal qual em Haesbaert (2011, p. 52). Tal
situação impõe-lhes uma condição de dependência e subordinação em
relação aos agentes indigenistas (OLIVEIRA, 1998). O que não se pode afirmar, todavia, é que a integração subordinada dos
indígenas ao modo capitalista de produção, fruto do processo de
territorialização nacional, com a adoção nas aldeias da antiga casa
subterrânea por malocas e, em seguida, por casas do tipo indigenista,
implique o desaparecimento da ancestralidade pelo consumo, mas pós v.23 n.40 • são paulo • outubro 2016 meramente uma predação simbólica da alteridade (nos termos de Castro): a
apropriação daquilo que o universo de fora da aldeia propicia para a re-
existência (PORTO-GONÇALVES, 2003) contemporânea dos Kaingáng e
qualquer outra nação indígena. artigos • p. 026-046 Notas 15 Haesbaert, refletindo tais concepções nas ciências sociais, realiza importante análise sobre as
tendências teóricas indicadas e seus problemas conceituais. (HAESBAERT, 2003). Notas Destaca-se assim o caráter específico de cada nação, diferentemente de
“os índios” ou “os indígenas”, termos genéricos. artigos • p. 026-046 6 Um grupo de arqueólogos e historiadores do sul do país vem se dedicando ao levantamento destes
dados. As conclusões conhecidas sobre estes trabalhos estão reunidas em Amparo (2015). 6 Um grupo de arqueólogos e historiadores do sul do país vem se dedicando ao levantamento destes
dados. As conclusões conhecidas sobre estes trabalhos estão reunidas em Amparo (2015). 7 Roberto Cardoso de Oliveira, 1963 (apud OLIVEIRA, J.P., 1998). 8 Haesbaert, 2004; Oliveira, 1998. 9 O vocábulo kré significa “casa”, dentre os Kaiapó-Mebengokré, parentes meridionais dos
Kaingáng. (WISEMEAN, 2011). 9 O vocábulo kré significa “casa”, dentre os Kaiapó-Mebengokré, parentes meridionais dos
Kaingáng. (WISEMEAN, 2011). 10 Vale lembrar que, aqui, o termo exato faz menção à posse e não à propriedade. Os indígenas têm
direito ao “usufruto exclusivo”, nos termos do Artigo 231 da Constituição Federal. Contudo a gestão
destas terras – que são públicas – cabe a um órgão específico do Governo Federal, a FUNAI. 10 Vale lembrar que, aqui, o termo exato faz menção à posse e não à propriedade. Os indígenas têm
direito ao “usufruto exclusivo”, nos termos do Artigo 231 da Constituição Federal. Contudo a gestão
destas terras – que são públicas – cabe a um órgão específico do Governo Federal, a FUNAI. 11 Emblemático, neste sentido, o fato de as áreas habitadas pelos Kaingáng terem sido descritas com
“sertões desconhecidos”, no Mappa Chorográphico da Província de São Paulo, de D. Muller. (BEIER, 2015) 12 Negligência que os pesquisadores dos Jê do Sul constituem exceção, já que pesquisadores como
Almeida; Tomasinno (2015) e Fernandes (2003) e sobretudo Veiga & D’Ângelis (2003) têm
contribuído grandemente para o conhecimento sobre as formas antigas e contemporâneas de
moradia Kaingáng. 13 Enquanto finalizava a revisão deste texto, uma MILÍCIA financiada por produtores rurais do Mato
Grosso do Sul atacou um acampamento, na cidade de Caaraô. Em nota, a FAMASUL – Federação
Agropecuária do Mato Grosso do Sul – diz “lamentar a morte do indígena”, mas erra o nome da
vítima fatal, Clodiodi Aquileu Rodrigues Souza (seu nome, Aquileu, trocado cuidadosamente por
Aguille, para sugerir uma origem paraguaia, termo de origem castelhana). 14 Sobretudo na antropologia, ver o índio “hiper-real” de Alcida Ramos, a fricção interétnica
(CARDOSO DE OLIVEIRA apud OLIVEIRA, J. P., 1998). Referências Bibliográficas ALMEIDA, L. K.; TOMASINO, K. Territórios e territorialidades Kaingáng: a reinvenção dos espaços
e das formas de sobrevivência após a conquista. Mediações. Revista de Ciências Sociais, UEL,
v.19, n. 2, p. 18-42, 2014. AMPARO, S. Sobre a organização espacial dos Kaingáng, uma sociedade indígena Jê meridional. Ed. Luminária Acadêmica, Rio de Janeiro, 2015. BEIER, J. R. Daniel Pedro Muller e a trajetória de seu Mappa Choirographico da provincia de
São Paulo (1835-1842). Revista Brasileira de Cartografia, v. 67, n.4, p. 817-836, Rio de
Janeiro, 2015. BEBER, M. V. O sistema de assentamentos dos grupos ceramistas do planalto sul-brasileiro: o
caso da tradição Taquara-Itararé. Tese (Doutorado em Estudos Históricos Latino-Americanos). Programa de Pós-Graduação em História da Universidade do vale do Rio Sinos, São Leopoldo,
2004. CASTRO, E. V. A inconstância da alma selvagem e outros ensaios de antropologia. São Paulo:
CosacNaify, 2003. CASTRO, E. V, E. Os involuntários da Pátria: aula Pública em comemoração ao dia do índio, Rio
de Janeiro, abril de 2016. CRUZ, V. C. Das lutas por redistribuição de terra às lutas pelo reconhecimento de territórios:
uma nova gramática das lutas sociais? In: ACSERALD, H. (Org.). Cartografia social, terra e
território. Rio de Janeiro: IPPUR/UFRJ, 2013, v. 1, p. 119-176. CUNHA, M. C. Cultura com aspas e outros ensaios. São Paulo: CosacNaify, 200 FERNANDES, R. C. Terra, tradição e identidade: os Kaingang da Aldeia Condá no contexto da
UHE Foz do Chapecó. In: SANTOS, S. C. & NACKE, A.. (Org.). Hidrelétricas e povos indígenas. Florianópolis: Letras Contemporâneas, 2003, p. 158-174. pós v.23 n.40 • são paulo • outubro 2016 FREIRE, P. Extensão ou comunicação. Rio de Janeiro: Paz & Terra, 1969. FOUCAULT, Michel. A arqueologia do saber. 7ª ed., Rio de Janeiro: Livraria Forense
Universitária, 2008. HAESBAERT, R. Precarização, reclusão e exclusão territorial. Terra Livre, Associação dos
Geógrafos Brasileiros, Ano 20, v. 2, n. 23, p.35-52, 2004. HAESBAERT, R. Concepções de território para entender a des-territorialização. In SANTOS, M. &
BECKER, B. et al. Território, territórios: ensaios de ordenamento territorial. Rio de Janeiro:
Lamparina, 2011, p. 43-71. HAESBAERT, R. & MONDARDO, M. Transterritorialidade e antropofagia: territorialidades de
trtãnsito numa perspectiva latino-americana e brasileira. In: GEOgraphia, PPGEO-UFF, Niterói, v. 12, n. 24, p. 19-50, 2010. JAENISCH, D. A Arte Kaingáng da produção de objetos, corpos e pessoas. Dissertação
(Mestrado em Antropologia Social). pós- SANTOS, M. Pensando o espaço do homem. 4ª ed. São Paulo: Edusp, 2004.
VEIGA, J. & D’ÂNGELIS, W. Habitação e acampamentos Kaingáng hoje e no passado.
UNOCHAPECÓ/ARGOS,), Chapecó-SC, 2003. VEIGA, J. Aspectos Fundamentais da Cultura
Kaingáng. Campinas: Nimuendaju, 2006. p.213-242 (Cadernos do CEOM, N. 18)
ZUCH-DIAS, J. A tradição Taquara e sua ligação com o índio Kaingáng. Dissertação (Mestrado
em Estudos Históricos Latino-Americanos). Programa de Pós-Graduação em História.
Universidade do Vale do Rio Sinos. São Leopoldo, 2004.
WISEMEAN, U. Dicionário Kaingáng-Português/Português-Kaingáng, 2ª. ed., Curitiba: Esperança,
2011. Sandoval dos Santos Amparo
Professor de Geografia Humana e Ensino de Geografia da Universidade do Estado
do Pará.
CV: http://lattes.cnpq.br/0675773110915998
sanamparo@gmail.com pós v.23 n.40 • são paulo • outubro 2016 Referências Bibliográficas Programa de Pesquisa e Pós-Graduação em Antropologia
Social da Universidade Federal do Rio Grande do Sul, Porto Alegre, 2010. LEFEBVRE, H. La producción del espacio. Madrid: Entrelinhas, 2013. LEVI-STRAUSS, C. Antropologia Estrutural II. 6.ed. Rio de Janeiro: Tempo Brasileiro, 2003. MARX, K. Para a crítica da economia política, 2ª. ed. São Paulo: Martins Fontes, 1983. MARX, K. Teses sobre Feuerbach. In Ludwig Feuerbach e o fim da filosofia Alemã clássica. Lisboa: Progresso, 1982, p. 69-72. OLIVEIRA, J. P. O nosso governo: os Ticuna e o regime tutelar. São Paulo: Marco Zero/CNPQ. 1988. OLIVEIRA, J. P. (Org.). Indigenismo e Territorialização; poderes, rotinas e saberes coloniais no
Brasil contemporâneo. Rio de Janeiro: Contra-Capa Livraria e Editora, 1998. Série Territórios
Sociais. OLIVEIRA, J. P. & FREIRE, C. A. R. A presença indígena na formação do Brasil. UNESCO /
Ministério da Educação, Brasília: 2006. Coleção Educação Para Todos. PRADO JR., C. Dialética do Conhecimento. 6ª. ed. São Paulo: Brasiliense, 1980. PORTO-GONÇALVES, C. W. A geograficidade do social: uma contribuição para o debate
metodológico sobre estudos de conflito e movimentos sociais na América Latina. In: SEOANE, J. Movimientos sociales y conflictos en América Latina..... Buenos Aires: CLACSO, 2003. 288 p. (Programa OSAL) RAMOS, A. R. O índio hiper-real. Revista Brasileira de Ciências Sociais, v. 10, n. 28, jun. 1995. REIS, N. Quadro da arquitetura no Brasil. 9ª. ed. São Paulo: Perspectiva,. 2000. RIBEIRO, D. O povo brasileiro: a formação e o sentido do Brasil. São Paulo: Companhia das
Letras, São Paulo, 2006. RODRIGUES, A. D. Línguas Indígenas: para o conhecimento das línguas brasileiras. 4ª ed., São
Paulo: Loyola, 2002. SÁ, C. Habitações indígenas. Encontros com a Civilização Brasileira, Rio de Janeiro, n. 12,
p.129-42, 1979. SÁ, C. A aldeia Karajá de Santa Isabel do Morro. Revista Projeto, São Paulo, n. 23, p.19-23,
1980. SÁ, C. Aldeia de São Marcos: transformações da habitação de uma comunidade Xavante. Dissertação (Mestrado em Arquitetura e Urbanismo) – Faculdade de Arquitetura e Urbanismo. Universidade de São Paulo, São Paulo, 1982. SANTOS, M. Por uma Geografia Nova: da crítica da geografia a uma geografia crítica. São
Paulo: Hucitec, 1978. SANTOS, M. Espaço e método. São Paulo: Hucitec, 1984. SANTOS, M. Metamorfoses do espaço habitado. São Paulo: Hucitec, 1989. artigos • p. WISEMEAN, U. Dicionário Kaingáng-Português/Português-Kaingáng, 2ª. ed., Curitiba: Esperança,
2011. Referências Bibliográficas 026-046 Nota do Editor
Data de submissão: 29/9/2015
Aprovação: 27/06/2016
Revisão: Janayara Araujo Lima Sandoval dos Santos Amparo
Professor de Geografia Humana e Ensino de Geografia da Universidade do Estado
do Pará. sanamparo@gmail.com
|
https://openalex.org/W4365816674
|
https://www.qeios.com/read/ZYNEHS/pdf
|
English
| null |
Review of: "Knowledge among Health care workers (HCWs) regarding biomedical waste management (BMW) during COVID-19 Pandemic"
| null | 2,023
|
cc-by
| 975
|
Jomell Santiago1 1 Nueva Ecija University of Science and Technology Potential competing interests: No potential competing interests to declare. General Comments: i. Most parts of this paper requires major revisions about the content and its format. Recheck if this is the final copy of
the paper to be submitted and intend to be published since many revisions/changes were observe. ii. The data was insufficient to satisfy the topic/title of the research paper. ii. The data was insufficient to satisfy the topic/title of the research paper. sufficient to satisfy the topic/title of the research paper. Overall, the manuscript requires serious major revisions , and the reviewer will not recommend the acceptance of this
paper unless all the necessary revisions were provided. Specific Comments: 1. In the abstract, a revision is required which includes a portion or explain why the study was conducted or what is the
purpose of this study, the other parts of the methodology that must be included must be inserted such as the sampling,
data analysis and how the data was gathered. Also, briefly explain or state the conclusion in the last part and combine
the stanzas into a single stanza. 1. In the abstract, a revision is required which includes a portion or explain why the study was conducted or what is the
purpose of this study, the other parts of the methodology that must be included must be inserted such as the sampling,
data analysis and how the data was gathered. Also, briefly explain or state the conclusion in the last part and combine
the stanzas into a single stanza. 2. In the introduction, include why the study is important or why it should be conducted by explaining its necessity and
also add more literature which state the present problem about the knowledge of health care workers (HCWs) and how
and what are the possible solution regarding this. Also, improve the format or make the paper more organize. 2. In the introduction, include why the study is important or why it should be conducted by explaining its necessity and
also add more literature which state the present problem about the knowledge of health care workers (HCWs) and how
and what are the possible solution regarding this. Also, improve the format or make the paper more organize. 3. In the methodology, explain how the data analysis or how the data gathered was analyze and the statistical tools
utilized. Qeios, CC-BY 4.0 · Review, April 15, 2023 Review of: "Knowledge among Health care workers (HCWs)
regarding biomedical waste management (BMW) during
COVID-19 Pandemic" Jomell Santiago1 Jomell Santiago1 Jomell Santiago1 Also, explain how the questionnaire was made. If it was researchers' made, explain how its validity and
reliability was assessed. If the other parts were adopted to literatures, cite where it is derived or adapted. In the result,
it is mentioned that a 15 general awareness questions were ask about biomedical waste, waste generated in COVID
19 wards, diseases spread by biomedical waste, COVID 19 modes of transmission, biomedical waste management
handling rules 2016 rules, general management of biomedical waste during COVID 19 pandemic, and biomedical
waste management in SKIMS. Do you think that a 15 items can mentioned about topics/subject? 3. In the methodology, explain how the data analysis or how the data gathered was analyze and the statistical tools
utilized. Also, explain how the questionnaire was made. If it was researchers' made, explain how its validity and
reliability was assessed. If the other parts were adopted to literatures, cite where it is derived or adapted. In the result,
it is mentioned that a 15 general awareness questions were ask about biomedical waste, waste generated in COVID
19 wards, diseases spread by biomedical waste, COVID 19 modes of transmission, biomedical waste management
handling rules 2016 rules, general management of biomedical waste during COVID 19 pandemic, and biomedical
waste management in SKIMS. Do you think that a 15 items can mentioned about topics/subject? 4. For the result, improve the format/appearance of the tables and include a legend which will tell what does it mean
when the p-value is less than 0.05. In the discussion, make a separate discussion for each tables/data presented and
include either a supporting or a contrasting statement about the result obtained. Also, there is a statement “Doctors
and nurses have better knowledge, positive attitude attitude and good practices compared to paramedics and sanitary 4. For the result, improve the format/appearance of the tables and include a legend which will tell what does it mean
when the p-value is less than 0.05. In the discussion, make a separate discussion for each tables/data presented and
include either a supporting or a contrasting statement about the result obtained. Qeios ID: ZYNEHS · https://doi.org/10.32388/ZYNEHS Jomell Santiago1 Also, there is a statement “Doctors
and nurses have better knowledge, positive attitude attitude and good practices compared to paramedics and sanitary Qeios ID: ZYNEHS · https://doi.org/10.32388/ZYNEHS 1/2 Qeios, CC-BY 4.0 · Review, April 15, 2023 staff regarding infection waste management and were found statistically significant.” Recheck the table in the result if
the table for this statement was included since the tables only include the frequency and their mean score, and the
difference in their qualification. The comparison between the doctors, nurses, technicians, sanitary workers were
missing. To sum it up, make the necessary major revision in this part of the paper. staff regarding infection waste management and were found statistically significant.” Recheck the table in the result if
the table for this statement was included since the tables only include the frequency and their mean score, and the
difference in their qualification. The comparison between the doctors, nurses, technicians, sanitary workers were
missing. To sum it up, make the necessary major revision in this part of the paper. 6. The references were insufficient. Add more recent references from reputable source/journals. Qeios ID: ZYNEHS · https://doi.org/10.32388/ZYNEHS 2/2
|
https://openalex.org/W2767177702
|
http://ijarcs.info/index.php/Ijarcs/article/download/4691/4160
|
English
| null |
EFFICIENTLY RECOGNIZING THE FACE USING HYBRID APPROACH OF GAUSSIAN FILTERING AND SIFT KEY ALGORITHM
|
International journal of advanced research in computer science
| 2,017
|
cc-by
| 6,142
|
INTRODUCTION distinctive size, position, lightning conditions, and outward
appearance. Thus it may be hard to consider different
pictures of a comparable face under different conditions. Facial acknowledgment frameworks tend to fuse a pre-
dealing with organize where the picture is institutionalized
concerning the beforehand specified combination i.e. the
picture is changed to some standard size and position etc. Size and position are by and large easy to oversee yet the
others are troublesome. Because of expanding security requests, its potential
business or law authorization applications confront
acknowledgment has turned into an extremely dynamic
territory of research lately. The acknowledgment procedure
of this framework incorporated the area of components like
eyebrows, eyes, noses, computation of separations and
proportions to a typical reference point and layout
coordinating.[2]Despite the fact that there are various face
acknowledgment calculations which function correctly in
obliged conditions, confront acknowledgment is as yet an
open and extremely difficult issue in genuine applications. Among oppose acknowledgment calculations, appearance-
based methodologies are the most prominent. These
methodologies use the pixel force or power inferred
highlights. A few such frameworks have been effectively
created and introduced. Be that as it may, appearance-based
techniques don't perform well in some true circumstances,
where the inquiry test confront appearance is fundamentally
unique in relation to the preparation confront information,
because of varieties in posture, lighting and articulation. p
g
p
Size and position are by and large easy to oversee yet the
others are troublesome. Face acknowledgment gives the forefront progresses in
business law authorization and military applications. It has
furthermore drawn huge interest and thought from various
experts all through the past two decades because of its
potential applications in the regions of observation and
customer approval. The face is a champion among the most
attractive biometrics, and it has in like manner been the most
essential method for recognition that human use in their
visual associations. The issue with confirmation systems in
light of unique mark, voice, iris and the present quality
structure (DNA unique mark) has been the issue of data
obtainment or procurement. It is an appealing component of the front line human-PC
interfaces. PCs that can see outward appearances and
respond to the sentiments of individuals properly enable
better human machine correspondence. Appearance of the
face relies upon the geometry, surface, shape and
development of facial components. DOI: http://dx.doi.org/10.26483/ijarcs.v8i8.4691 DOI: http://dx.doi.org/10.26483/ijarcs.v8i8.4691 Volume 8, No. 8, September-October 2017 International Journal of Advanced Research in Computer Science
RESEARCH PAPER
Available Online at www.ijarcs.info
EFFICIENTLY RECOGNIZING THE FACE USING HYBRID APPROACH OF
GAUSSIAN FILTERING AND SIFT KEY ALGORITHM Bhavna Saini
Assistant Professor
SSIET, Dinanagar,India Sapna Bharti
M.tech Scholar
SSIET, Dinanagar,India Abstract:Automatic Face Recognition System becomes the challenging problem due to its requirement in various applications like defense,
surveillance and access control.It helps to identify the face image correctly and store them in database. In real time applications appearance of
face changes due to different pose, expression, affine transformations and illumination etc. In our research paper to obtain accurate results and
good performance Modified SIFT (Scale Invariant Feature Transform) technique is used. By using modified SIFT approach difference between
original and query database is identified. The proposed approach is efficient in performance and provides experimental results on the basis of
developed algorithm for accurately recognizing the images from dataset. Keywords- Face Recognition, SIFT, noise, HSIFT © 2015-19, IJARCS All Rights Reserved FACE RECOGNITION There is an extraordinary presentable variety in the way
facial appearance is translated for recognition by a
programmed framework. At present various distinctive
frameworks are being worked on, and which is most fitting
may rely upon the application area. A noteworthy contrast in
approaches is whether to speak to the presence of the face,
or the geometry. I have looked at the two methodologies; in
any case most frameworks today utilize a mix of both
appearance and geometry. Geometry is hard to measure with
any exactness, especially from a solitary still picture,
however gives more power against masks and maturing. Appearance data is promptly acquired from a face picture
yet is more subject to super
ficial variety, especially from
posture and behavior changes. By and by for most purposes,
even appearance-based frameworks must measure some
geometrical parameters with a specific end goal to
determine a 'shape free' portrayal that is autonomous of
articulation and stance relics. [5] A feature extraction calculation removes features from the
information. It makes those new features in light of changes
or mixes of the first information. A feature determination
calculation chooses the best subset of the information
feature set. It disposes of non-applicable features. Feature
determination
is
frequently
performed
after
feature
extraction. In this way, features are separated from the face
pictures, at that point an ideal subset of these features is
chosen. The dimensionality decrease process can be
implanted in some of these means, or performed before
them. Face recognition is utilized to perform two principle
essential tasks i.e. check and ID. Verification is the way
toward confirming a man to perceive an obscure individual
alongside a claim of personality and to guarantee whether
the individual is same is called verification. Recognizable
proof is a procedure to distinguish a man by looking at
his/her face with the given database [1]. Face recognition
frameworks are for the most part used to recognize people,
for get to control, for security purposes and there is
numerous other utilization of face recognition framework, a
couple of which are given underneath: This is accomplished by
finding facial land stamps and
twisting the face to an authoritative unbiased posture and
appearance. Facial features are likewise critical for
geometric methodologies and for tying down neighborhood
portrayals. FEATURE EXTRACTION/SELECTION Feature extraction includes a few stages - dimensionality
lessening,
feature
extraction
and
feature
choice. Dimensionality lessening is a fundamental undertaking in
any example recognition framework. The execution of a
classifier relies upon the measure of test pictures, number of
features and classifier multifaceted nature. This prerequisite
ought to be satis
fied when constructing a classifier. The
more perplexing the classifier, the bigger ought to be the
said proportion. This "reprove" is one reason why it's critical
to keep the quantity of features as little as could be expected
under the circumstances. The other principle reason is the
speed. The classifier will be speedier and will utilize less
memory. Additionally, a substantial arrangement of features
can bring about a false positive when these features are
excess. Eventually, the quantity of features must be
precisely picked. Too less or repetitive features can prompt
lost exactness of the recognition framework. Figure 1: Face Detection Process In some satisfying frameworks, face detection is preventing
by obliging the client. Most frameworks utilize a mix of
skin-tone and face surface to decide the area of a face and
utilize a picture pyramid to enable faces of differing sizes to
be distinguished.[4] Progressively, frameworks are being
produced to identify faces that are not full-frontal. Prompts,
for example, development and individual detection can be
utilized to limit faces for recognition. Commonly
interpretation, scale and in-plane turn for the face are
assessed at the same time, alongside pivot inside and out
when this is considered. INTRODUCTION For unique finger impression the concerned individual
should keep his/her finger in fitting position and in case of
speaker recognition the beneficiary should be kept in
genuine position and partition from the speaker. Regardless,
the technique for obtaining face pictures is nonintrusive and
the face can be used as a biometric property. Face Recognition winds up plainly difficult task if there
should
arise
an
occurrence
of
changing
outward
appearances. In such case focal inclination are required to be
utilized as a part of request to have a main issue speaking to
regular face behavior out of huge number of appearances. This point help in recognizable proof of face when looked at
against preparing set of pictures. The face looks of a man
are proficient medium to pass on points, emotions and the
internal viewpoint. One of the issues that any facial
acknowledgment framework must be fit to manage various
pictures of a comparative face pictures which have [3](Mukhedkar 2015)Face is a comprehensive part of
individuals. Face recognition is basic not in view of the
capacity of its piece of potential applications in investigate
fields also in light of the limit of its answer which would
help in settling other portrayal issues like question
recognition. © 2015-19, IJARCS All Rights Reserved 239 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 for instance the Fourier Transform, a de connecting change
to an option premise where great portrayals of the
remarkable attributes of a picture can be made from just a
couple of low-arrange coefficients in spite of disposing of a
hefty portion of the higher-arrange terms[15]. Figure 1: Face Detection Process
Detec
tion
Featu
re
Extrac
Face
Reco
gnitio
Imag
e
Outp
ut FACE RECOGNITION •In brilliant card applications where the face picture can be
put away in a keen card, scanner tag or attractive stripe, to
verify the live picture and the put away picture. managing such issues. [7] (Liu et al. 2014) proposed that idealistic faces created
better personality recognition concerning shake faces,
paying little regard to whether they were tried in a
comparative picture or another photo showing a neutral
attitude. None of the other passionate expressions made
quantifiable preferred standpoint for recognition memory. Notwithstanding,
the
point
by
point
investigations
additionally demonstrate that the upside of glad expression
on
character
recognition
may
not
be
comparably
recognizable from all other enthusiastic expressions. [8] (Ma n.d.) Proposed technique increases the BU-4DFE
dataset by adding distinctive lighting conditions to 3D
pictures of subjects performing diverse facial appearances. They build up a photo handling pipeline to amend the
effects of brightening on the photos, wanting to secure high
arrangement rate even in brutal lighting conditions. By then
they test the pipeline on two estimations: grouping precision
in perspective of a LDA model and SIFT key point
repeatability. The photo handling pipeline enhanced order
precision when performing LDA to recognize pictures in
dim lighting conditions. They didn't discover noteworthy
change in key point discovery. [7] (Liu et al. 2014) proposed that idealistic faces created
better personality recognition concerning shake faces,
paying little regard to whether they were tried in a
comparative picture or another photo showing a neutral
attitude. None of the other passionate expressions made
quantifiable preferred standpoint for recognition memory. Notwithstanding,
the
point
by
point
investigations
additionally demonstrate that the upside of glad expression
on
character
recognition
may
not
be
comparably
recognizable from all other enthusiastic expressions. •Labeling the face pictures in video called video Indexing. •For the order of sex. •For the order of sex. •In numerous restorative field like psychiatry for stress and
discouragement discovery. Any undesirable thing in a picture is considered as a
commotion. Since the execution of any face recognition
system relies upon the nature of caught picture of the face to
be perceived. The de-noising calculation expels the noise
part while safeguarding the first picture structures. In the
vast majority of the applications, the nearness of commotion
in picture prompts awful execution of consequent picture
preparing undertakings which are entirely subject to the
achievement of de-noising operation. FACE RECOGNITION Face appearance portrayal plans can be isolated
into neighborhood and worldwide, contingent upon whether
the face is spoken to all in all, or as a progression of little
locales. Most worldwide methodologies depend on a
primary parts portrayal of the face picture powers. This
portrayal conspire was conceived
first for face picture
pressure purposes and along these lines utilized for
recognition purposes. The last authored the term Eigen faces
for this sort of portrayal. A face picture is spoken to as a
vector of forces and this vector is then approximated as an
entirety of premise vectors (Eigen faces) figured by key part
investigation from a database of face pictures. These
primary segments speak to the regular varieties seen
amongst faces and give a brief epitome of the presence of a
specimen face picture, and a reason for its examination with
other face pictures. This important segments portrayal is, as •Security of structures, air terminals, seaports, programmed
teller machines (ATM) machines and fringe checkpoints. •Used in different territories of teach for contrast a substance
and an arrangement of element. g
•For Surveillance through CCTVs to search for known
culprits p
y
p
p
pressure purposes and along these lines utilized for
recognition purposes. The last authored the term Eigen faces
for this sort of portrayal. A face picture is spoken to as a
vector of forces and this vector is then approximated as an
entirety of premise vectors (Eigen faces) figured by key part
investigation from a database of face pictures. These
primary segments speak to the regular varieties seen
amongst faces and give a brief epitome of the presence of a
specimen face picture, and a reason for its examination with
other face pictures. This important segments portrayal is, as •General character confirmation at banks, organizations and
different associations. •Examination for improper records. •Examination for improper records. •Significant in procedure of some VIP is coming in the inn. •Criminal
equity
frameworks
through
post-occasion
investigation and legal applications. •Investigations of picture database like looking of picture
databases like authorized drivers advantage beneficiaries,
missing kids, migrants and so on. © 2015-19, IJARCS All Rights Reserved 240 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 extends the extension for different scientists and effectively
managing such issues. FACE RECOGNITION The issue in face
recognition framework is the recognition of loud face
picture. The recognition of loud picture is exceptionally
troublesome task and this may come about into wrong
recognition. Then again, it is important to guarantee that de-
noising system must not twist the valuable data in the
picture and should protect picture subtle elements and
surface while expelling the noise. It is exceptionally
important to expel commotion in the pictures previously
applying any further picture preparing assignment, for
example, picture recognition, edge location, picture division
and so on. recognizable from all other enthusiastic expressions. [8] (Ma n.d.) Proposed technique increases the BU-4DFE
dataset by adding distinctive lighting conditions to 3D
pictures of subjects performing diverse facial appearances. They build up a photo handling pipeline to amend the
effects of brightening on the photos, wanting to secure high
arrangement rate even in brutal lighting conditions. By then
they test the pipeline on two estimations: grouping precision
in perspective of a LDA model and SIFT key point
repeatability. The photo handling pipeline enhanced order
precision when performing LDA to recognize pictures in
dim lighting conditions. They didn't discover noteworthy
change in key point discovery. They build up a photo handling pipeline to amend the
effects of brightening on the photos, wanting to secure high
arrangement rate even in brutal lighting conditions. By then
they test the pipeline on two estimations: grouping precision
in perspective of a LDA model and SIFT key point
repeatability. The photo handling pipeline enhanced order
precision when performing LDA to recognize pictures in
dim lighting conditions. They didn't discover noteworthy
change in key point discovery. [9](Anon n.d.) extended the prevalence of Face Recognition
(FR) systems have expanded in light of their utilization in
boundless
applications,
for
instance,
biometric
(distinguishing evidence and validation), security (Banks,
air terminals, and so on.) and surveillance (missing
youngsters or finding outlaw crooks) structures, and in
addition picture and video ordering systems. FR has been a
solid field of research since the 1990s, however still a long
route from is dependable and more procedures are being
developed each year. FR investigate region primary
challenges are, a couple of individuals faces recognition
won't not fill in and in addition for others (for instance, long
hair or facial hair, feelings, lighting, and foundation may
give additional inconvenience). FACE RECOGNITION Most by far of the
exploration specialists solidly assume that feelings of a man
expect the critical part in essential leadership. The commotion may enter in a picture as a result of camera
movement, poor camera sensors, environmental conditions
or boisterous transmission medium. Electronic commotion
related with a capacity recovery framework can likewise be
the reason of debasement of picture. It is an extremely
normal issue. A picture can likewise get defiled amid the
securing forms, or while its stockpiling and recovery. There are diverse sorts of noise. Commotion might be sorted
into substitutive noise e.g., salt and pepper noise, irregular
esteemed motivation noise, and so forth and added
substance noise e.g., added substance white gaussiannoise. The substitutive commotion of low and normal noise
densities can be effortlessly evacuated with the assistance of
de-noising plans accessible in the writing. The middle
channel performs exceptionally well to remove drive
commotion at low thickness. There are numerous other de-
noising plans are proposed which are well skilled in
expelling motivation noise at low to high commotion
densities. Then again added substance commotion is
extremely hard to expel on the grounds that it debases every
one of the pixels display in the picture. The Mean channel
can be connected to expel added substance noise however it
additionally
obscure
the
picture
while
evacuating
commotion. p
p
p
[10] (Neeru and Kaur 2016a) provided the recognition of a
face under different feelings is a testing subject. The work
done in this paper is twofold. In any case, Local binary
Pattern (LBP) and center symmetric Local binary Pattern
(CS-LBP) has been connected to separate the nearby parallel
components of the photo. Second, Euclidean distance,
histogram convergence and chi-square separation is utilized
for recognition of face. The execution is assessed on the
Japanese Female Facial Expression (JAFFE) database and
results are analyzed the extent that recognition rate and time
taken for handling. It has been watched that CS-LBP gives
best recognition rate rather finished LBP in the event of
different appearances of face. © 2015-19, IJARCS All Rights Reserved LITERATURE SURVEY [11] (Neeru and Kaur 2016b) build up a completely
programmed face recognition calculation. Scale Invariant
Feature Transform (SIFT) has effortlessly been utilized as a
piece of face recognition. In this paper, a Modified SIFT
(MSIFT) approach has been proposed to enhance the
recognition execution of SIFT. In this paper, the work is
done in three phases. To begin with, the smoothing of the
photo has been finished utilizing DWT. Second, the
computational multifaceted nature of SIFT in descriptor
figuring is diminished by subtracting normal from each [6] (Murtaza et al. 2013) presented an entire review of face
recognition led under shifting outward appearances. In order
to
examine
distinctive
systems,
movements
based,
demonstrate based and muscles-based methodologies have
been utilized as a piece of demand to deal with the outward
appearance and recognition calamity. The investigation has
been finished by assessing different existing calculations
while looking at their results when in doubt. It additionally © 2015-19, IJARCS All Rights Reserved 241 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 procedures. The human face is dynamic having particular
appearance at unmistakable time. The human face is 3D
protest
displaying
distinctive
outward
appearance. Projection from 3D to 2D is basic in the examination
procedure as a few subtle elements might be lost. So a few
credits must be converged in existing ways to deal with
improve face acknowledgment process. The proposed
framework
handles
the
outward
appearance
acknowledgment with ideal acknowledgment rate. The
destinations are recorded as takes after descriptor instead of standardization. Third, the calculation
is made programmed by utilizing Coefficient of Correlation
(CoC) as opposed to utilizing the separation proportion
(which requires client communication). [11] (Chennamma and Rangarajan 2010) proposed another
approach for face unmistakable evidence from controlled
facial pictures in light of SIFT parts. The proposed approach
is differentiated and Eigen faces and showed its prevalence
through trials. In this paper, they concentrated just on mug
shot recognizable proof. As an expansion, they inspected the
usage of SIFT components for recuperation of right face
with various sorts of face delegates. •
Extracting the components from the preparation set
utilizing adjusted SIFT keys with middle channel
for
effective
extraction
taking
out
low
differentiation pixels.. [12] (Seo and Park 2014) took care of 2D pictures be
removing segments of the photo. Features extraction
instrument uses SIFT (Scale invariant Feature change). FEATURE EXTRACTION This is a procedure by which features are separated from
starting arrangement of preparing pictures. Dimensionality
is incredibly decreased by the utilization of feature
extraction. Basically excess data is wiped out thus speed of
acknowledgment is significantly upgraded. For extricating
the features SIFT (Scale Invariant Feature change) is
utilized. Filter for the most part utilize Gaussian channel to
choose potential key point yet in proposed work middle
channel is utilized to choose key focuses. However every
one of the features removed may not respect grouping
comes about. Thus feature selection is required to be
performed. g
DATASET COLLECTION In order to prove worth of research corresponding to face
recognition two datasets are used from the internet. YALE DATASET: This dataset contains 15 image set of
distinct Male individuals with 11 images per individual. Images are in GIF format. Gray scale images of individual
are loaded within this training set. YALE DATASET: This dataset contains 15 image set of
distinct Male individuals with 11 images per individual. Images are in GIF format. Gray scale images of individual
are loaded within this training set. NLPR DATASET: This dataset contains 450 Male and
Female face images. The image format is JPEG. So
conversion from JPEG to GIF is required. Each image has
896x592 pixels. NLPR DATASET: This dataset contains 450 Male and
Female face images. The image format is JPEG. So
conversion from JPEG to GIF is required. Each image has
896x592 pixels. [16] (Fallis 13AD) depicted that Individuals are normally
recognized by their faces. Headways in the past couple of
decades have engaged human to thusly do the conspicuous
evidence process. Early face recognition calculation used
fundamental geometric models. Directly, face recognition
get ready uses the advanced factual science and organizing
procedures. Changes and improvements in face recognition
development in the midst of 10 to 15 past years have pushed
it to the present status. Face recognition is applicable for
both examination and ID. This audit gave another face
recognition technique in light of SIFTS components. Upgrading SIFT calculation, this survey focused on face
recognition. Comes to fruition demonstrated the prevalence
of the proposed calculation over the SIFT. The results gotten
from various tests exhibited that the proposed calculation,
with 98.75% accuracy and run time of 4.3 seconds, is more
capable and correct than various calculations yet the run
time was shy of what others. LITERATURE SURVEY Channel is used to address neighborhood incorporates as a
descriptor. Variety in facial pictures is perceived by the use
of probability thickness work found out from SIFT. Benchmark datasets are used for distinguishing proof of
facial varieties through proposed method. •
After extracting the components, performing
determination of ideal elements related with
significant appearance of face with scale invariant
element choice. •
Enhancing precision of result created and thought
about against other classifier. [13] (Gupta and Garg 2014) SIFT used for face recognition
on different invariants and accuracy was figured. Close
examination among various invariants was moreover a bit of
this paper by giving the two figures and estimations. Through the examination it is demonstrated that they deduce
that SIFT works amazingly well for articulation and camera
determination. It gives normal results when pictures are of
different lighting up levels. Channel is fitting to check
pictures having about same edification, with different
position, articulation, embellishments etc.. •
Enhancing execution of outward appearance
detection
and
contrasting
it
and
diverse
methodologies as far as acknowledgment rate. Proposed Methodology Proposed Methodology The proposed work is described in this section. The
proposed work takes the optimal properties of various
algorithms along with genetic algorithm to produce optimal
rate corresponding to face recognition. [14] (Mohanraj et al. 2016) proposed Hybrid SIFT plan to
see face in video under different edification and position
variety. The proposed system uses light modification
channel of Retina Modeling for pre-taking care of and
Histogram of Oriented Gradients for face area, as it has
lesser false positive rates. Remembering the true objective to
beat the lighting up issue, the main SIFT is changed in this
paper with settled point of reference localizer to stamp the
key concentrations from which the presentation task of SIFT
is done and a light invariant part vector is gotten. Feature selection with RWBGA selection is required to be performed. Features like mean,
criticized deviation, Entropy, RMS, difference, smoothness,
Kurtosis, IDM, Contrast, connection, Energy, Homogeneity
are shaped particular classes. The features produced through
extraction process checked against the class passage. On the
off chance that feature don't exist in any class at that point
feature
is
rejected. Consequently
dimensionality
is
additionally lessened. In third step extraction of face elements to arrange the
picture utilizing Genetic calculation with roulette Wheel
Selection. In this chromosome assurance handle occurs on
the start of roulette wheel. Roulette wheel assurance should
be possible by the crucial bit of the decision technique is to
stochastic accomplice select from one time to make
commence of the general population to come. The need is
that the fittest individuals have a more conspicuous shot of
survival than weaker ones. This reproduces nature in that
fitter individuals will tend to have an unrivaled probability
of survival and will proceed to outline the mating pool for
the general population to come. Weaker individuals are not
without plausibility. In nature such individuals may have
innate coding that may exhibit supportive to who and what
is to come. After that assurance of the fittest has been
finished. Proposed Methodology Proposed methodology used hybrid approach of buffer
mechanism along with HSIFT and RWBGA. The steps
which are performed can be described as: Apply Face Acquisition and selection procedure to select
particular test image from Training set. g
g
1. Apply modified median filter to reduce noise if any
from the image 2. Test_Image = median2(Test_Image) Flowchart Flowchart 3. After Applying Pre-processing, apply HSIFT for
feature extraction Results
YALE DATASET RESULTS 4. Apply genetic approach with roulette wheel
selection for feature selection 5. Repeat the above listed steps until termination
criteria is satisfied or optimal result is obtained Feature extraction with HSIFT Feature extraction with HSIFT Besides include Selection utilizing Modified SIFT key will
be assessed. . Face acknowledgment is done by restricting
the objective work which prompts assurance of perfect
course of action of trusty centers or SIFT Points. The
technique shields the close-by information from different
facial points of view for mapping neighboring commitment
to its looking at yield, achieving low dimensional depiction
for encoding the associations of the data. The proposed
procedures
Hexagonal
Descriptor
Particle
Swarm
Optimization with Knowledge-Crowding (HDPSO-KC)
overcomes from adjacent optima and upgrades overall chase
plan and communitarian work. The system in like manner
covers the issue of shedding the particles in denser zones in
Pareto front scattering. (Chennamma and Rangarajan
2010)The proposed system is affirmed with benchmark
datasets for examining the execution over various methods. Lowe's system for picture feature period changes a photo
into an extensive social occasion of feature vectors, each of
which is invariant to picture understanding, scaling, and
turn, mostly invariant to lighting up changes and generous to
adjacent geometric reshaping. These components share
practically identical properties with neurons in basic Visual
cortex that are encoding central structures, shading and
advancement for question recognizable proof in primate
vision. Key regions are portrayed as maxima and minima of
the outcome of qualification of Gaussians limit associated in
scale space to a movement of smoothed and resampled
pictures. Low multifaceted nature candidate centers and
edge response centers along an edge are discarded. Winning
acquaintances are doled out with restricted key focuses. These methods ensure that the key focuses are all the more
consistent to match and affirmation. Channel descriptors
generous to neighborhood relative twisting are then gotten
by considering pixels around a traverse of the key range,
darkening and resampling of close-by picture presentation
planes. OBJECTIVE Feature selection extraordinarily decreases dimensionality to
upgrade rate of acknowledgment. However every one of the
features separated may not add to arrangement comes about. Thus features that add to various significant classes feature Face detection is a system of recognizing particular outward
appearance by the utilization of picture preparing © 2015-19, IJARCS All Rights Reserved 242 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 © 2015-19, IJARCS All Rights Reserved Results The following table shows the recognition rate of various
training sets in YALE dataset using different techniques Table 1: Comparison of results of various techniques with
purposed methodology using YALE dataset purposed methodology using YALE dataset
Traini
ng Set
Propos
ed
PCA
DWT
DWT+P
CA
DWT+
CC
Traini
ng
Set1
97.608
7
89.78
01
93.05
41
93.0401
89.8831
Traini
ng
Set2
94.022
3
93.20
43
94.68
42
94.6662
93.1981
Traini
ng
Set3
73.065
2
55.92
79
64.24
06
64.2447
57.6053
Traini
ng
Set4
91.023
2
71.34
55
89.23
43
87.0873
88.6089
Traini
ng
Set5
95.114
3
80.98
50
88.89
0
85.8792
86.8769
Traini
ng
Set6
82.230
4
70.57
44
83.34
24
81.0122
82.9024
Traini
ng
Set7
83.530
4
72.47
59
78.23
44
75.8549
77.3624
Traini
ng
Set8
80.145
8
71.59
84
79.25
48
78.8451
79.5214
Traini
ng
Set9
85.245
6
72.54
8
84.25
46
82.1245
85.3654
Traini
ng
Set10
90.254
8
80.26
58
88.36
54
86.4578
87.3654
Traini
ng
Set11
75.595
4
63.98
74
70.58
74
68.4825
70.2525
Traini
ng
Set12
86.245
7
72.58
44
84.24
98
81.9548
82.4579
Traini
ng
Set13
98.245
8
80.25
48
87.63
25
85.6642
86.9548
Traini
ng
Set14
97.897
4
85.65
48
95.64
87
93.1452
94.2455
Traini
ng
Set15
95.548
7
87.24
58
94.55
78
92.3258
93.1424
Traini
ng
Set16
94.025
4
85.58
75
91.01
45
90.2547
92.5874 0
20
40
60
80
100
120
Training Set1
Training Set4
Training Set7
Training Set10
Training Set13
Training Set16
Training Set19
Training Set22
Training Set25
Proposed
PCA
DWT
DWT+PCA
DWT+CC Figure 2: Comparison of results of various techniques with
purposed methodology using YALE dataset Figure 2: Comparison of results of various techniques with
purposed methodology using YALE dataset CONCLUSION AND FUTURE SCOPE The proposed approach uses modified SIFT(involving
median filter) with enhanced genetic algorithm involving
roulette wheel selection model in order to detect the face
effectively. Set of 11 distinct feature classes are used. Feature selection is used to reduce the search space further. Genetic algorithm is used in order to optimally select the
feature. For this purpose roulette wheel selection is
performed. Recognition rate and accuracy is significantly
enhanced through proposed technique(SIFT+GA). In other
words optimal features are selected after extraction and then
evaluation is performed using GA. The proposed approach
analyses the object from different viewpoints and then
object matching highest key point is selected as a destination
image. All the critical features are selected and hence
discrimination ability is high. The feature selection is based
on objective function value. Better convergence in terms of
recognition rate is observed through proposed technique. p
Genetic algorithm is used in order to optimally select the
feature. For this purpose roulette wheel selection is
performed. Recognition rate and accuracy is significantly
enhanced through proposed technique(SIFT+GA). In other
words optimal features are selected after extraction and then
evaluation is performed using GA. The proposed approach
analyses the object from different viewpoints and then
object matching highest key point is selected as a destination
image. All the critical features are selected and hence
discrimination ability is high. The feature selection is based
on objective function value. Better convergence in terms of
recognition rate is observed through proposed technique. © 2015-19, IJARCS All Rights Reserved Results YALE DATASET RESULTS This dataset contains 15 image set of distinct Male
individuals with 11 images per individual[17] .(Yang et al. © 2015-19, IJARCS All Rights Reserved 243 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 arch in Computer Science, 8 (8), Sept–Oct 2017,239-245
Traini
ng
Set17
82.564
8
75.85
48
80.54
84
78.6954
80.3254
Traini
ng
Set18
81.369
5
73.14
58
80.65
98
78.9548
80.6985
Traini
ng
Set19
96.548
6
85.55
66
92.65
87
90.8654
91.2354
Traini
ng
Set20
97.214
5
86.58
96
92.47
85
91.8569
92.7894
Figure 2: Comparison of results of various techniques with
purposed methodology using YALE dataset
CONCLUSION AND FUTURE SCOPE
The proposed approach uses modified SIFT(involving
median filter) with enhanced genetic algorithm involving
roulette wheel selection model in order to detect the face
effectively. Set of 11 distinct feature classes are used. Feature selection is used to reduce the search space further. Genetic algorithm is used in order to optimally select the
0
20
40
60
80
100
120
Training Set1
Training Set4
Training Set7
Training Set10
Training Set13
Training Set16
Training Set19
Training Set22
Training Set25
Proposed
PCA
DWT
DWT+PCA
DWT+CC Traini
ng
Set17
82.564
8
75.85
48
80.54
84
78.6954
80.3254
Traini
ng
Set18
81.369
5
73.14
58
80.65
98
78.9548
80.6985
Traini
ng
Set19
96.548
6
85.55
66
92.65
87
90.8654
91.2354
Traini
ng
Set20
97.214
5
86.58
96
92.47
85
91.8569
92.7894 Traini
ng
Set17
82.564
8
75.85
48
80.54
84
78.6954
80.3254
Traini
ng
Set18
81.369
5
73.14
58
80.65
98
78.9548
80.6985
Traini
ng
Set19
96.548
6
85.55
66
92.65
87
90.8654
91.2354
Traini
ng
Set20
97.214
5
86.58
96
92.47
85
91.8569
92.7894 2004) Images are in GIF format. Gray scale images of
individual are loaded within this training set. The database
contains 5760 single light source pictures of 10 subjects
each observed under 576 review conditions (9 postures x 64
brightening conditions). [18] (Belhumeur et al. 1997) For
each subject in a specific represent, a picture with
encompassing (foundation) brightening was likewise caught. REFERENCES 1. Belhumeur, P.N., Hespanha, J.P. & Kriegman, D.J., 1997. Eigenfaces vs. fisherfaces: Recognition using class specific
linear projection. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 19(7), pp.711–720. 2. Chennamma,
H.R. &
Rangarajan,
L.,
2010. Face
Identification from Manipulated Facial Images Using SIFT. 2010 3rd International Conference on Emerging Trends in
Engineering and Technology, pp.192–195. Available at:
http://ieeexplore.ieee.org/lpdocs/epic03/wrapper.htm?arnum
ber=5698318. 244 Sapna Bharti et al, International Journal of Advanced Research in Computer Science, 8 (8), Sept–Oct 2017,239-245 3. Lu, X.L.X., Wang, Y.W.Y. & Jain, A.K., 2003. Combining
classifiers
for
face
recognition. 2003
International
Conference on Multimedia and Expo., 3, pp.13–16. information for face recognition? Pattern Recognition, 36(3),
pp.649–655. information for face recognition? Pattern Recognition, 36(3),
pp.649–655. 12. Yang, J. et al., 2004. Two-Dimensional PCA: A New
Approach to Appearance-Based Face Representation and
Recognition. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 26(1), pp.131–137. 4. Mukhedkar, M.M., 2015. Fast Face Recognition Based on
Wavelet Transform on PCA. , (Icesa), pp.761–764. 5. Tian, Y. et al., 2003. Do singular values contain adequate
information for face recognition? Pattern Recognition, 36(3),
pp.649–655. g
pp
13. Belhumeur, P.N., Hespanha, J.P. & Kriegman, D.J., 1997. Eigenfaces vs. fisherfaces: Recognition using class specific
linear projection. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 19(7), pp.711–720. pp
6. Yang, J. et al., 2004. Two-Dimensional PCA: A New
Approach to Appearance-Based Face Representation and
Recognition. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 26(1), pp.131–137. 14. Chennamma,
H.R. &
Rangarajan,
L.,
2010. Face
Identification from Manipulated Facial Images Using SIFT. 2010 3rd International Conference on Emerging Trends in
Engineering and Technology, pp.192–195. Available at:
http://ieeexplore.ieee.org/lpdocs/epic03/wrapper.htm?arnum
ber=5698318. 7. Belhumeur, P.N., Hespanha, J.P. & Kriegman, D.J., 1997. Eigenfaces vs. fisherfaces: Recognition using class specific
linear projection. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 19(7), pp.711–720. 8. Chennamma,
H.R. &
Rangarajan,
L.,
2010. Face
Identification from Manipulated Facial Images Using SIFT. 2010 3rd International Conference on Emerging Trends in
Engineering and Technology, pp.192–195. Available at:
http://ieeexplore.ieee.org/lpdocs/epic03/wrapper.htm?arnum
ber=5698318. 15. Lu, X.L.X., Wang, Y.W.Y. & Jain, A.K., 2003. Combining
classifiers
for
face
recognition. 2003
International
Conference on Multimedia and Expo., 3, pp.13–16. 16. Mukhedkar, M.M., 2015. Fast Face Recognition Based on
Wavelet Transform on PCA. , (Icesa), pp.761–764. pp
17. Tian, Y. et al., 2003. Do singular values contain adequate
information for face recognition? Pattern Recognition, 36(3),
pp.649–655. 9. Lu, X.L.X., Wang, Y.W.Y. & Jain, A.K., 2003. Combining
classifiers
for
face
recognition. © 2015-19, IJARCS All Rights Reserved REFERENCES 2003
International
Conference on Multimedia and Expo., 3, pp.13–16. 18. Yang, J. et al., 2004. Two-Dimensional PCA: A New
Approach to Appearance-Based Face Representation and
Recognition. IEEE Transactions on Pattern Analysis and
Machine Intelligence, 26(1), pp.131–137. p
pp
10. Mukhedkar, M.M., 2015. Fast Face Recognition Based on
Wavelet Transform on PCA. , (Icesa), pp.761–764. 11. Tian, Y. et al., 2003. Do singular values contain adequate © 2015-19, IJARCS All Rights Reserved 245
|
W3113644722.txt
|
https://brill.com/downloadpdf/journals/ges/64/3-4/article-p294_37.pdf
|
fr
|
Lynn, Michael R.: Popular science and public opinion in eighteenth-century France. Manchester and New York, Manchester University Press, 2006. IX, 177 p. (Studies in early modern European history). £ 50.–. ISBN 0-7190-7373-1.
|
Gesnerus
| 2,007
|
cc-by-sa
| 1,046
|
L’entreprise repose sur un constat: le peu de place laissé à la médecine dans les
ouvrages de référence traitant de l’Antiquité gréco-romaine. Une lacune que nous
avons souvent déplorée, mais qui s’explique historiquement par l’orientation qui a
été celle des études antiques. Un philologue italien ne qualifiait-il pas récemment
encore la production médicale antique de «pattumiera»! Les esprits ont heureusement évolué au cours de ces dernières décennies. Grâce aux efforts conjugués de
philologues, historiens, médecins, philosophes – car il s’agit par excellence d’études
interdisciplinaires – cette littérature commence à acquérir sa vraie place dans l’histoire de la culture gréco-romaine.
L’ouvrage se présente sous la forme d’un lexique comprenant environ mille
articles d’ampleur fort différente. Ils traitent essentiellement de la médecine antique
(personnes, doctrines, pratiques, instruments, etc.); ils abordent aussi des réalités
apparemment étrangères à la médecine mais qui peuvent avoir quelque relation avec
la santé et la maladie. C’est ainsi qu’à côté de rubriques consacrées à la pathologie
humorale, à la vivisection, à l’anatomie ou encore à l’éthique hippocratique, on découvre de façon au premier abord surprenante des articles consacrés aux insectes, au
christianisme, à l’infibulation, aux peintures murales dans les catacombes ou même
à la Chine! Mais cet apparent disparate recouvre toujours un lien avec la médecine
antique. Ainsi l’article «Chine» met en évidence les analogies dans la doctrine des
éléments entre médecine grecque et médecine chinoise, tout en précisant qu’on ne
peut déceler une influence directe de l’une sur l’autre. Autre exemple: l’article sur les
peintures murales dans les catacombes fait le point sur les diverses interprétations de
la fameuse fresque de via Latina traditionnellement connue sous le nom de «la leçon
d’anatomie».
Ce volume couvre un espace chronologique qui va de la Grèce archaïque à la fin
de l’Antiquité et un espace culturel qui englobe la Grèce et Rome. Mais il ne s’interdit pas des «excursions» dans d’autres périodes (p. ex. Byzance, Moyen Age latin)
ou d’autres mondes (Egypte, Mésopotamie, Islam), dans la mesure où existe un lien
avec la médecine gréco-romaine: mais il s’agit alors d’un aperçu offrant une perspective d’ensemble plutôt que d’une information particulière.
Comme dans toute entreprise collective, la qualité de l’information varie en fonction des auteurs. Mais elle est en règle générale d’un très bon niveau avec un seul
bémol à propos de la bibliographie sommaire accompagnant chaque article: on y
constate une propension certaine à citer les mêmes références dont les auteurs se
trouvent être les propres collaborateurs de l’ouvrage. Il arrive même que l’auteur
de l’article cite principalement si ce n’est exclusivement ses propres œuvres dans la
bibliographie. Celui qui n’a jamais péché jettera la première pierre!
Philippe Mudry, Lausanne
Lynn, Michael R.: Popular science and public opinion in eighteenth-century France.
Manchester and New York, Manchester University Press, 2006. IX, 177 p. (Studies in
early modern European history). £ 50.–. ISBN 0-7190-7373-1.
Questo libro è la versione rielaborata della tesi di laurea di Lynn alla University of
Wisconsin-Madison (dir. D. Sella). Esplora il processo di diffusione delle conoscenze
294
Downloaded from Brill.com 05/19/2024 11:06:14AM
via Open Access. This is an Open Access article distributed under the terms
of the prevailing CC-BY-NC license at the time of publication.
http://creativecommons.org/licenses/by-sa/4.0
scientifiche nella società parigina della seconda metà del XVIIIo secolo e si propone
di mettere in evidenza lo sviluppo di un’opinione pubblica legata a una cultura scientifica urbana. Sin dalle prime pagine, l’autore ci guida in un percorso che dalla
Rive Gauche, quartiere delle università e frequentato dagli intellettuali, termina in
Boulevard du Temple, zona dei teatri e capoluogo dei divertimenti dove, lontano
dalle accademie, intraprendenti volgarizzatori mettono in scena le recenti scoperte
scientifiche in maniera frivola e accattivante. Sfruttando ampiamente i numerosi
annunci presenti nella stampa periodica, Lynn dipinge una Parigi dove, a ogni ora del
giorno, si possono seguire corsi di fisica sperimentale, ascoltare delle conferenze sulle
potenzialità dell’elettricità, instaurare conversazioni nei numerosi luoghi dove gli
amanti della scienza si riuniscono, quali i musées, istituzioni che possono essere
descritte come un ibrido tra il salotto letterario e l’accademia scientifica, e ben analizzate dall’autore.
Oltre a esplorare gli aspetti legati alla nascita di un opinione pubblica, – nel quinto
capitolo ci mostra come, su un argomento complesso quale la rabdomanzia, intervengano non soltanto la chiesa e gli scienziati ma anche dei semplici cittadini – Lynn
si interroga sulla dimensione economica della popolarizzazione scientifica. Punto
forte del suo lavoro, punto che meriterebbe un ulteriore approfondimento, è la dimostrazione che all’interesse crescente nei confronti della scienza sperimentale è legato
un florido commercio di libri e di tutto il materiale necessario al fine di riprodurre a
casa propria le esperienze di fisica. La prova: Madame de Pompadour possedeva non
meno di cinquanta strumenti scientifici e alcuni periodici inserivano delle rubriche
per favorirne la vendita o lo scambio. I costi di iscrizione ai corsi, alle conferenze e
alle dimostrazioni (ben descritte nel sesto capitolo quelle per i voli in mongolfiera)
sono altrettanti elementi che Lynn prende in considerazione nella sua analisi.
Se le forme, la geografia e gli aspetti economici della socializzazione scientifica sono ben esposti, meno approfondita è l’analisi che l’autore fa del pubblico che
assiste alle manifestazioni. La nobiltà, la borghesia medio-alta e gli studenti rientrano
certamente tra i fruitori di queste numerose offerte. Per quanto riguarda i ceti mediobassi del popolo parigino, quasi nessuna indicazione è fornita, se non quella che precisa che alcune delle rappresentazioni di Boulevard du Temple erano accessibili per
pochi spiccioli e altre erano gratuite per le donne.Troppo raramente l’autore ci fa sentire la voce e le opinioni del pubblico.
Dobbiamo a Lynn un lavoro stimolante, non esaustivo, ma che solleva numerose
riflessioni soprattutto sulle relazioni tra scienza e consumismo.
Miriam Nicoli, Lausanne
Marinozzi, Silvia; Fornaciari, Gino: Le mummie e l’arte medica nell’evo moderno.
Per una storia dell’imbalsamazione artificiale dei corpi umani nell’evo moderno.
Roma, Università La Sapienza, 2005. 341 p. Ill. (Medicina nei secoli. Supplemento, 1).
I 40.–. ISBN 88-87242-72-0.
Questo libro, accessibile ai non specialisti, presentato al lettore da una prefazione di
Ezio Fulcheri, professore di anatomia patologica, restituisce alla storia della mummificazione un’originaria dimensione antropologica di pratica sociale. Si tratta di
295
Downloaded from Brill.com 05/19/2024 11:06:14AM
via Open Access. This is an Open Access article distributed under the terms
of the prevailing CC-BY-NC license at the time of publication.
http://creativecommons.org/licenses/by-sa/4.0
|
|
https://openalex.org/W4394573011
|
https://gastro.zaslavsky.com.ua/index.php/journal/article/download/579/803
|
Ukrainian
| null |
Experience of using different schemes of eradication therapy for Helicobacter pylori infection and their effectiveness in Ukraine
|
Gastroenterologìa/Gastroenterologìâ
| 2,024
|
cc-by
| 4,991
|
Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract DOI: https://doi.org/10.22141/2308-2097.58.1.2024.579 DOI: https://doi.org/10.22141/2308-2097.58.1.2024.579 УДК 616.33-002.44-022:579.835.12-085(477)
DOI: https://doi.org
Чернявський В.В. , Павловський Л.Л. , Решотько Д.О. Національний медичний університет імені О.О. Богомольця, м. Київ, Україна Чернявський В.В. , Павловський Л.Л. , Решотько Д.О. Національний медичний університет імені О.О. Богомольця, м. Київ, Україна © 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.
Для кореспонденції: Павловський Леонід Леонідович, доктор філософії, асистент кафедри внутрішньої медицини № 1, Національний медичний університет імені О.О. Богомольця,
бульв. Т. Шевченка, 13, м. Київ, 01601, Україна; e-mail: Leonya545@gmail.com; тел.: +380 (50) 216-02-71
For correspondence: Leonid Pavlovskyi, PhD, Assistant at the Department of Internal Medicine № 1, Bogomolets National Medical University, T. Shevchenko boulevard, 13, Kyiv, 01601, Ukraine; e-mail:
Leonya545@gmail.com; phone: +380 (50) 216-02-71
Full list of authors information is available at the end of the article. For citation: Gastroenterologìa. 2024;58(1):1-5. doi: 10.22141/2308-2097.58.1.2024.579 Резюме. Актуальність. Резистентність Helicobacter pylori (Hр) до антибактеріальних препаратів зросла
за останні роки. Передусім це пов’язано з необґрунтованим використанням антибіотиків, як продемон
струвала остання пандемія CОVID-19. Вибір оптимальної схеми та тривалості лікування є актуальним
питанням сьогодення. Мета: ретроспективне дослідження ефективності 14-денних схем ерадикації Нр, що
використовувались протягом 2022–2023 року, і порівняння їх ефективності та безпеки з 10-денними схема
ми, що застосовувалися протягом 2020–2021 року в Україні. Матеріали та методи. Ретроспективно були
проаналізовані дані 242 пацієнтів (123 чоловіки та 119 жінок) віком від 18 до 65 років з хронічним гастритом
і пептичною виразкою ДПК та шлунка, асоційованими з Нр. Всі пацієнти були проліковані стандартними
схемами згідно з рекомендаціями Маастрихтських консенсусів 5 і 6 перегляду. Нр-інфекція підтверджувалась
швидким уреазним тестом, визначенням фекального антигену та гістологічно. Результати. У результаті до
слідження виявлено, що 10-денні схеми потрійної терапії мали ефективність на рівні 80–81 %. Ефективність
14-денної потрійної терапії з езомепразолом та лансопразолом була вірогідно вищою порівняно з 10-денною
схемою — 85 та 86 % відповідно (p < 0,05). Проте частота побічних дій була більшою при 14-денній терапії. 10-денна потрійна схема з левофлоксацином порівняно з 10-денною стандартною потрійною терапією мала
найменшу ефективність — 78 %. Проте при збільшенні тривалості терапії з левофлоксацином до 14 днів
ефективність її досягала рівня ефективності 14-денної потрійної терапії — 85 %. Найвищу ефективність
продемонструвала 10-денна та 14-денна терапія з фуразолідоном, який додавався до амоксициліну та ле
вофлоксацину: 95 і 97,8 % відповідно. Висновки. Ефективність схем з кларитроміцином в Україні залиша
ється високою. Прийом препаратів протягом 14 днів збільшує відсоток ерадикації Нр та частоту небажаних
ефектів. Додавання фуразолідону до левофлоксацину та амоксициліну збільшує успішність ерадикації Нр. Ключові слова: ерадикація; Helicobacter pylori; резистентність; фуразолідон © 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.
Для кореспонденції: Павловський Леонід Леонідович, доктор філософії, асистент кафедри внутрішньої медицини № 1, Національний медичний університет імені О.О. Богомольця, Вступ Тестування на
Нр було проведене пацієнтам з неускладненою пептич
ною виразкою ДПК або шлунка в стадії загострення або
тим, які мали виразку в анамнезі, пацієнтам з недослі
дженою диспепсією, пацієнтам, які приймають тривалий
час аналгетики та аспірин, пацієнтам, в анамнезі життя
яких були випадки раку шлунка. Як маркер контролю за
лікуванням визначався фекальний антиген або прово
дився швидкий уреазний тест через 1 місяць після закін
чення прийому антибіотиків і щонайменше через 2 тижні
після прийому інгібіторів протонної помпи. р
у
р
р
Лікування пацієнтів проводилося згідно з Маастрихт
ським консенсусом 5 і 6 перегляду. Тривалість лікування
в одних пацієнтів становила 10 днів (2020–2021 рік), в ін
ших — 14 днів (2022–2023 рік). 10-денна потрійна терапія
з кларитроміцином, амоксициліном та езомепразолом
була використана у 45 пацієнтів (20 жінок і 25 чоловіків)
та з лансопразолом — у 43 пацієнтів (19 жінок і 24 чоло
віки). 10-денна терапія з левофлоксацином, амоксици
ліном, езомепразолом використана у 33 пацієнтів (16 жі
нок і 17 чоловіків). 22 пацієнти (10 жінок і 12 чоловіків)
разом з 10-денною терапією з левофлоксацином також
приймали фуразолідон. 14-денна стандартна потрійна
терапія з езомепразолом була проведена у 35 пацієнтів
(20 жінок і 15 чоловіків), з лансопразолом — у 37 пацієн
тів (20 жінок і 17 чоловіків). 10 пацієнтів (4 жінки та 6 чо
ловіків) приймали потрійну терапію з левофлоксацином
протягом 14 днів. 17 пацієнтів (10 жінок і 7 чоловіків)
приймали 14-денну терапію з левофлоксацином, амокси
циліном, езомепразолом і фуразолідоном. Дозування
основних препаратів було таким: езомепразол 40 мг
(2 рази на день), лансопразол 30 мг (2 рази на день), кла
ритроміцин 500 мг (2 рази на день), амоксицилін 1000 мг
(2 рази на день), левофлоксацин 250 мг (2 рази на день),
фуразолідон 100 мг (3 рази на день). Кратність прийому
препаратів відповідала Маастрихтським рекомендаціям. У 2020–2021 роках у всіх схемах лікування був викорис
таний пробіотик на основі штаму B. clausii. У всіх випад
ках 14-денних схем додатково приймався комбінований
пробіотик, що містив комбінацію штамів: S. boullardii,
L. acidophilus, L. rhamnosus. На сьогодні ми маємо обмежені дані щодо популя
ційної кларитроміцин-резистентності в Україні, а до
сліджені протягом минулого десятиріччя схеми еради
кації в Україні цілком могли змінити свою ефективність
[11, 14, 15]. До того ж новий консенсус Маастрихт-6
закріпив рекомендований курс ерадикації тривалістю
14 днів, а на практиці в Україні до того часу перева
га віддавалась 10-денним схемам. Тому питання щодо
оптимальної стартової схеми ерадикації Hр в Україні
залишається досі не вирішеним. Вступ Це створює певні про
блеми у виборі правильної схеми лікування, що, у свою
чергу, призводить до зниження ефективності ерадикації
Hр та прогресування хронічного гастриту. Мета: ретроспективне дослідження ефективності
та безпечності 14-денних схем ерадикації Нр, що вико
ристовувались протягом 2022–2023 років, порівняно
з 10-денними схемами, що застосовувалися протягом
2020–2021 років в Україні. Вступ поширеності Hр в Україні є проблема несвоєчасної ді
агностики Hр-інфекції, а також неправильний вибір
оптимальної схеми лікування та резистентність самої
бактерії до антибіотиків. Helicobacter pylori (Hр) — це грамнегативна бактерія,
яка є однією з основних причин хронічного антрального
гастриту, пептичної виразки шлунка та дванадцятипалої
кишки (ДПК). Hр згідно з визначенням Міжнародного
агентства з дослідження раку є канцерогеном І класу,
становлячи основну частку серед усіх причин адено
карциноми та MALT-лімфоми шлунка [1–3]. Попри
те, що поширеність Hр-інфекції починаючи з 2011 і до
2022 року суттєво знизилась у всьому світі, питома вага
хронічного Hр-асоційованого гастриту в Україні зали
шається високою (23,3 %) [4, 5]. Серед причин значної Згідно з останніми рекомендаціями Маастрихт-6,
виділяють чотири лінії ерадикації Hр: потрійна терапія
з кларитроміцином, амоксициліном або метронідазо
лом, квадротерапія з вісмутом, терапія з левофлоксаци
ном та схема порятунку із застосуванням рифабутину [6]. Вибір початкового лікування залежить від резистент
ності Hр до кларитроміцину. На сьогодні поширеність
резистентності Hр до кларитроміцину в країнах Європи 1 Vol. 58, No. 1, 2024 www.gastro.org.ua, https://gastro.zaslavsky.com.ua Vol. 58, No. 1, 2024
www.gastro.org.ua, https://gastro.zaslavsky.com.ua Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract становить понад 15 % [7]. Схожа ситуація спостерігаєть
ся з резистентністю Hр і до інших антибіотиків, зокрема
до метронідазолу та левофлоксацину. Так, резистентність
до метронідазолу на 2016 рік становила 23–56 %, а до
левофлоксацину — понад 15 % [7, 8]. Окремою про
блемою також є одночасна мультирезистентність Hр до
кларитроміцину і метронідазолу, глобальна поширеність
якої становить 10–19 % [5, 9]. До факторів, що впли
вають на резистентність Hр до антибіотиків, відносять
безконтрольне та нераціональне використання анти
бактеріальних препаратів, а також вірулентність бактерії. Так, було виявлено, що первинна резистентність до кла
ритроміцину виявлялася частіше у пацієнтів зі штамами
цитотоксину, гена A (CagA), цитотоксину A (VacA) [10,
11]. Консенсус Маастрихт-5 засвідчив, що при обранні
схеми першої лінії ерадикаційної терапії факт вживання
будь-якого антибіотика в минулому у Нр-позитивного
пацієнта має трактуватися як резистентність до цього
препарату [12]. Пандемія COVID-19, особливо в перший
рік її поширення світом взагалі та Україною зокрема,
призвела до того, що масове і недостатньо обґрунтова
не призначення азитроміцину і фторхінолонів могло
суттєво змінити локальну резистентність до макролідів
і левофлоксацину в Україні [13]. носкопія включала взяття біопсії і дослідження згідно із
Сіднейським протоколом і оцінкою за системою OLGA,
OLGIM. Дослідження Hр залежно від клінічних завдань
(первинна діагностика чи оцінка успіху ерадикації) про
водилось при гістологічному дослідженні шлункових
біоптатів шляхом визначення фекального антигену або із
застосуванням швидкого уреазного тесту. Результати лікування цією схемою з 10 до 14 днів успішна ерадика
ція досягалася у 97,8 (95; 99) пацієнтів (p < 0,001). лікування цією схемою з 10 до 14 днів успішна ерадика
ція досягалася у 97,8 (95; 99) пацієнтів (p < 0,001). Схеми лікування та їх тривалість, а також розподіл
пацієнтів за кількістю та статтю в обох групах наведені
у табл. 1. Частота небажаних ефектів, що виникали в ході лі
кування, наведена у табл. 3. У ході нашого спостереження було виявлено, що на
фоні прийому ерадикаційних схем лікування Нр у па
цієнтів відмічалися різні побічні дії. Серед найбільш
частих були симптоми з боку ШКТ, а саме: нудота,
металевий або гіркий присмак у роті, діарея, блюван
ня (схеми з фуразолідоном) та кандидоз. Частота роз
витку небажаних реакцій у пацієнтів, що лікували Нр,
залежала від схеми лікування та тривалості прийому
препаратів. Схеми, тривалість яких була 14 днів, асоці
ювалися з більш частим розвитком побічних ефектів,
ніж 10-денні (p < 0,001). У всіх пацієнтів, які приймали
схеми з фуразолідоном як 10, так і 14 днів, виникали
декілька небажаних ефектів одночасно, серед яких най
частіше були нудота, блювання та гіркота у роті. Іноді це
ставало приводом скоротити 14-денну схему лікування
до 10 днів, тому в нашому аналізі більшою була загальна
кількість пацієнтів, що лікувалися 10-денною схемою. Ефективність різних схем лікування Нр залежно від
тривалості курсу наведена у табл. 2. При порівняльній характеристиці схем лікування
було виявлено, що ефективність 10-денної стандарт
ної потрійної терапії як з езомепразолом, так і з лансо
празолом суттєво не відрізнялася. Але при порівнян
ні 10-денного курсу з 14-денним курсом стандартної
потрійної терапії були виявлені вірогідні відмінності
(p < 0,001). Найнижча ефективність порівняно з іншими
10-денними схемами відмічалася у пацієнтів, які прий
мали левофлоксацин. Проте при збільшенні тривалості
лікування цією схемою до 14 днів ефективність її під
вищилась і була вірогідно вища порівняно з 10-денним
курсом лікування (p < 0,001). У результаті порівняння
також було виявлено, що найбільш ефективною схемою
лікування була лінія з додаванням фуразолідону до ле
вофлоксацину. Результати Крім цього, при збільшенні тривалості Таблиця 1 — Схеми лікування, їх тривалість та розподіл пацієнтів за кількістю і статтю
Схема
лікування
Тривалість та кількість пацієнтів (n = 242)
10 днів
14 днів
К+А+Е
45 пацієнтів (20 жінок і 25 чоловіків)*
35 пацієнтів (20 жінок і 15 чоловіків)*
К+А+Л
43 пацієнти (19 жінок і 24 чоловіки)*
37 пацієнтів (20 жінок і 17 чоловіків)*
Л+А+Е
33 пацієнти (16 жінок і 17 чоловіків)*
10 пацієнтів (4 жінки і 6 чоловіків)*
Л+А+Ф+Е
22 пацієнти (10 жінок і 12 чоловіків)*
17 пацієнтів (8 жінок і 9 чоловіків)*
Примітки: * — p > 0,05, різниця між 10-денними і 14-денними курсами; К — кларитроміцин; А — амоксици
лін; Л — левофлоксацин; Ф — фуразолідон; Е — езомепразол; Л — лансопразол. Таблиця 2 — Порівняльна характеристика ефективності різних схем лікування Нр
залежно від тривалості курсу, Mе (Q1; Q3)
Схема
лікування
Тривалість та ефективність (%)
10 днів
14 днів
К+А+Е
80 (77; 84)*
85 (81; 87)*
К+А+Л
81 (78; 83)*
86 (84; 87)*
Л+А+Е
78 (74; 80)*
85 (84; 86)*
Л+А+Ф+Е
95 (93; 97)*
97,8 (95; 99)*
П
і
*
0 001
і
і
10
і 14
К
і
А Таблиця 1 — Схеми лікування, їх тривалість та розподіл пацієнтів за кількістю і статтю
ма
ання
Тривалість та кількість пацієнтів (n = 242)
10 днів
14 днів Примітки: * — p < 0,001, різниця між 10-денними і 14-денними курсами; К — кларитроміцин; А — амоксици
лін; Л — левофлоксацин; Ф — фуразолідон; Е — езомепразол; Л — лансопразол. Примітки: * — p < 0,001, різниця між 10-денними і 14-денними курсами; К — кларитроміцин; А — амоксици
лін; Л — левофлоксацин; Ф — фуразолідон; Е — езомепразол; Л — лансопразол. Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Примітки: * — p < 0,001, різниця між 10-денними і 14-денними курсами; ** — наявність в одного пацієнта
кількох симптомів; К — кларитроміцин; А — амоксицилін; Л — левофлоксацин; Ф — фуразолідон; Е —
езомепразол; Л — лансопразол. Матеріали та методи Роботу виконано на базі кафедри внутрішньої ме
дицини № 1 НМУ ім. О.О. Богомольця з 2020 по 2023
рік. Протокол дослідження відповідав етичним вимогам
Гельсінської декларації 1975 р. Усі особисті дані ано
німізовано. Нами були отримані та ретроспективно
проаналізовані дані 242 пацієнтів (123 чоловіки та 119
жінок) віком від 18 до 65 років. Основними критеріями
включення у дослідження були пацієнти з хронічним га
стритом (OLGA 0–2, OLGIM 0–1), неускладненою пеп
тичною виразкою ДПК та шлунка, асоційованими з Hр. Діагнози «хронічний гастрит» та «пептична виразка»
встановлювалися за даними езофагогастродуоденоско
пії ендоскопічною системою Olympus Evis Х1 CV-1500,
відеогастроскопом Olympus GIF-EZ 1500 з високою
роздільною здатністю, хромоендоскопією і режимом
NBI (narrow-band imaging). Кожна езофагогастродуоде Статистичну обробку результатів було виконано за
допомогою Мicrosoft Office 2016, комп’ютерної програми
MedStat, версія 5.2 (НМУ ім. О.О. Богомольця, Київ). Для визначення характеру розподілу отриманих даних
використовували метод Шапіро — Уїлка. Для порівнян
ня показників, які не виявили відмінностей розподілу
значень від нормального, використовувався t-критерій
Стьюдента для незалежних вибірок. Для показників, які
відрізнялися від нормального розподілу, використовува
ли непараметричний критерій Вілкоксона (W). Кількісні
змінні описували медіаною (Ме), 25 і 75 процентилями
(Q1; Q3). Відмінності між групами вважались вірогідно
значимими при досягненні значення р < 0,05. Vol. 58, No. 1, 2024
Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online) 2 Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online) Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Висновки 1. Ефективність ерадикації схемами з кларитромі
цином в Україні залишається на рівні 85 та 86 % залежно
від препаратів групи інгібіторів протонної помпи. 2. Прийом препаратів протягом 14 днів збільшує
ефективність ерадикаційної терапії та повинен вико
ристовуватися в Україні навіть попри збільшення ри
зику розвитку побічних дій. 3. Додавання фуразолідону до 14-денних схем з ле
вофлоксацином і амоксициліном збільшує успішність
ерадикації Нр. Конфлікт інтересів. Автори заявляють про відсутність
конфлікту інтересів та власної фінансової зацікавлено
сті при підготовці даної статті. р
р
ц
д
Нами також було виявлено, що 10-денна схема з ле
вофлоксацином мала нижчу ефективність порівняно
зі схемами з кларитроміцином. Лише пролонгування
прийому левофлоксацину до 14 днів призводило до
порівнянної ефективності зі схемами з кларитроміци
ном. Також у результаті дослідження виявлено, що схе
ми з 14-денним прийомом антибіотиків асоціювалися
з високою частотою розвитку побічних дій, що додат
ково впливало на загальний комплаєнс. Певну надію на
зрештою ефективну ерадикацію Нр вселяє додавання
до стандартних схем фуразолідону [18]. Згідно з резуль
татами досліджень у світі, ефективність квадротерапії
з фуразолідоном в ерадикації Нр досягається в 93 та 95 %
випадків [19, 20]. Наше дослідження ці дані підтверджує. Проте серед недоліків схеми лікування з фуразолідоном
є більша частота розвитку побічних дій, а саме нудоти та
блювання. У більшості пацієнтів, що приймали фуразо
лідон, виникали одночасно декілька побічних дій. Не
зважаючи на це, користь схем лікування з фуразолідоном
переважає їх ризики, особливо у випадку їх застосування
як другої чи третьої лінії лікування, коли дуже хочеться
досягти успіху. Тому ця схема є одним з оптимальних
рішень при високій резистентності Нр до кларитромі
цину, левофлоксацину та метронідазолу. Однак через
те, що в нашому дослідженні було проаналізовано малу
кількість пацієнтів, що приймали фуразолідон, остаточні
висновки робити зарано. Також у нас наразі недостат
ньо даних, щоб дійти висновків про ефективність схем
з вісмутом і тетрацикліном, оскільки незручність форм
випуску останнього і необхідність прийому тетрациклі
ну по 5 таблеток (100 мг) 4 рази на добу значно обмежує
частоту використання цієї схеми. Також під питанням
у цій схемі є використання метронідазолу, резистент Інформація про фінансування. Не заявлено. Інформація про фінансування. Не заявлено. Внесок авторів. Чернявський В.В. — концепція і ди
зайн дослідження; збирання й обробка матеріалів, аналіз
отриманих даних, написання тексту; редагування тексту;
Павловський Л.Л., Решотько Д.О. — збирання й обробка
матеріалів, аналіз отриманих даних, написання тексту. Обговорення ність до якого очікується високою. І в перспективі ми
запланували дослідити ефективність схем зі зниженою
дозою тетрацикліну до 300 мг 4 рази на добу і заміною
в них метронідазолу на фуразолідон. Наявні обмежені
дані вказують, що в Україні саме ця схема частіше засто
совувалася як третя лінії лікування і «терапія порятунку». ність до якого очікується високою. І в перспективі ми
запланували дослідити ефективність схем зі зниженою
дозою тетрацикліну до 300 мг 4 рази на добу і заміною
в них метронідазолу на фуразолідон. Наявні обмежені
дані вказують, що в Україні саме ця схема частіше засто
совувалася як третя лінії лікування і «терапія порятунку». У ході нашого ретроспективного аналізу було ви
явлено, що більшість схем, які використовувалися для
лікування Нр, мали високу ефективність. Попри те, що
останнім часом резистентність до кларитроміцину зрос
ла, ефективність схем на основі кларитроміцину в Укра
їні залишається на високому рівні. Однак пандемія ко
ронавірусної інфекції та нераціональне використання
при ній макролідів (азитроміцину) та фторхінолонів
(левофлоксацину) вносить свої корективи. Цілком мож
на припустити, що особливо необґрунтований прийом
азитроміцину вплинув на крос-резистентність до інших
макролідів (кларитроміцину). Так, згідно з даними ран
домізованого клінічного дослідження, пацієнти, які
в минулому мали COVID-19, мали резистентність до
кларитроміцину і левофлоксацину на рівні 64 і 74 %
відповідно [16]. Тому якщо колись молекулярне та гене
тичне тестування щодо резистентності Нр до антибіоти
ків використовувалося після неефективності другої лінії
терапії, то згідно з Маастрихтом-6 ці тести виходять
на перший план [17]. У ході дослідження було виявле
но, що ефективність 10-денної та 14-денної ерадика
ційної терапії за період 2020–2021 та 2022–2023 роки
залишається відносно незмінною. Всупереч пандемії
COVID-19 в Україні і широкому використанню макро
лідів та фторхінолонів ефективність 14-денних схем
з кларитроміцином залишається достатньою. Серед потенційних питань, які залишаються без
відповіді, є питання впливу пандемії COVID-19 на ре
зистентність до основних препаратів, що застосовують
ся для лікування Нр в Україні. Нове ретроспективне
дослідження із залученням лише пацієнтів, які мали
COVID-19 в анамнезі та лікувалися макролідами або
фторхінолонами, дозволить з’ясувати це питання. Результати Таблиця 3 — Порівняльна характеристика частоти побічних ефектів, % Таблиця 3 — Порівняльна характеристика частоти побічних ефектів, %
Побічні дії
Схеми лікування та тривалість
К+А+Е
К+А+Л
Л+А+Е
Л+А+Ф+Е
10 днів
14 днів
10 днів
14 днів
10 днів
14 днів
10 днів
14 днів
Нудота
33,3
28,5
31
40,5*
15,2
20
81,9**
90,0**
Блювання
6,7
5,7
4,6
13,5*
9,0
–
50,0**
55,0**
Болі у животі
–
6,1
–
5,5
–
20,0
Діарея (понад 3 рази на добу)
8,8
14,2*
7,5
10,8*
60,6
60,0
35,0**
30,0**
Металевий (гіркий) присмак у роті
44,4
34,2
46,5
27,0
–
–
40,0**
60,0**
Кандидоз ротової порожнини
або урогенітальний
4,5
8,5*
3,2
2,7
15,2
–
–
–
Алергічні прояви
2,3
2,8
–
–
–
–
–
–
Примітки: * — p < 0,001, різниця між 10-денними і 14-денними курсами; ** — наявність в одного пацієнта
кількох симптомів; К
кларитроміцин; А
амоксицилін; Л
левофлоксацин; Ф
фуразолідон; Е Примітки: * — p < 0,001, різниця між 10-денними і 14-денними курсами; ** — наявність в одного пацієнта
кількох симптомів; К — кларитроміцин; А — амоксицилін; Л — левофлоксацин; Ф — фуразолідон; Е —
езомепразол; Л — лансопразол. www.gastro.org.ua, https://gastro.zaslavsky.com.ua Vol. 58, No. 1, 2024
www.gastro.org.ua, https://gastro.zaslavsky.com.ua 3 Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Relationship of helicobacter pylori caga and vaca status to
morphological changes of gastric mucosa and primary clarithromycin re
sistance rate in patients with chronic gastritis: a cross-sectional study. Wiad
Lek. 2023;76(4):709-714. doi: 10.36740/WLek202304103. 19. Zhuge L, Wang Y, Wu S, Zhao RL, Li Z, Xie Y. Furazolidone
treatment for Helicobacter Pylori infection: A systematic review and me
ta-analysis. Helicobacter. 2018 Apr;23(2):e12468. doi: 10.1111/hel.12468. 12. Malfertheiner P, Megraud F, O’Morain CA, et al.; European Heli
cobacter and Microbiota Study Group and Consensus panel. Management of
Helicobacter pylori infection-the Maastricht V/Florence Consensus Report. Gut. 2017 Jan;66(1):6-30. doi: 10.1136/gutjnl-2016-312288. 12. Malfertheiner P, Megraud F, O’Morain CA, et al.; European Heli
cobacter and Microbiota Study Group and Consensus panel. Management of
Helicobacter pylori infection-the Maastricht V/Florence Consensus Report. Gut. 2017 Jan;66(1):6-30. doi: 10.1136/gutjnl-2016-312288. 20. Song C, Qian X, Zhu Y, et al. Effectiveness and safety of furazo
lidone-containing quadruple regimens in patients with Helicobacter pylori
infection in real-world practice. Helicobacter. 2019 Aug;24(4):e12591. doi:
10.1111/hel.12591. 13. Langford BJ, Soucy JR, Leung V, et al. Antibiotic resistance asso
ciated with the COVID-19 pandemic: a systematic review and meta-anal
ysis. Clin Microbiol Infect. 2023 Mar;29(3):302-309. doi: 10.1016/j. cmi.2022.12.006. 13. Langford BJ, Soucy JR, Leung V, et al. Antibiotic resistance asso
ciated with the COVID-19 pandemic: a systematic review and meta-anal
ysis. Clin Microbiol Infect. 2023 Mar;29(3):302-309. doi: 10.1016/j. cmi.2022.12.006. Отримано/Received 08.02.2024
Рецензовано/Revised 19.02.2024
Прийнято до друку/Accepted 01.03.2024 14. Vdovychenko VI, Bodrevych BB, Demidova AL. Regional and 14. Vdovychenko VI, Bodrevych BB, Demidova AL. Regional and Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract Патологія верхніх відділів травного каналу / Pathology of Upper Gastrointestinal Tract individual resistance of Helicobacter pylori strains to antibiotics in the
Lviv region: state and prospects. Crimean journal of internal diseases. 2010;2(2):69-73. Ukrainain. individual resistance of Helicobacter pylori strains to antibiotics in the
Lviv region: state and prospects. Crimean journal of internal diseases. 2010;2(2):69-73. Ukrainain. 7. Megraud F, Bruyndonckx R, Coenen S, et al.; European Helico
bacter pylori Antimicrobial Susceptibility Testing Working Group. Helico
bacter pylori resistance to antibiotics in Europe in 2018 and its relationship
to antibiotic consumption in the community. Gut. 2021 Oct;70(10):1815-
1822. doi: 10.1136/gutjnl-2021-324032. 15. Vdovychenko VI, Demidova AL. The dynamics of Helіcobacter
pylorі strains resistance to antibiotics and efficiency of the treatment of du
odenal peptic ulcer. Modern Gastroenterology. 2006;(30):55-59. Ukrainian. 8. Boyanova L, Hadzhiyski P, Gergova R, Markovska R. Evolu
tion of Helicobacter pylori Resistance to Antibiotics: A Topic of Increasing
Concern. Antibiotics (Basel). 2023 Feb 4;12(2):332. doi: 10.3390/antibi
otics12020332. 16. Kamal A, Ghazy RM, Sherief D, Ismail A, Ellakany WI. Helico
bacter pylori eradication rates using clarithromycin and levofloxacin-based
regimens in patients with previous COVID-19 treatment: a randomized
clinical trial. BMC Infect Dis. 2023 Jan 20;23(1):36. doi: 10.1186/s12879-
023-07993-8. 9. Dascălu RI, Bolocan A, Păduaru DN, et al. Multidrug resistance in
Helicobacter pylori infection. Front Microbiol. 2023 Feb 27;14:1128497. doi: 10.3389/fmicb.2023.1128497. 17. Graham DY, Moss SF. Antimicrobial susceptibility testing for he
licobacter pylori is now widely available: when, how, why. Am J Gastroen
terol. 2022 Apr 1;117(4):524-528. doi: 10.14309/ajg.0000000000001659. 17. Graham DY, Moss SF. Antimicrobial susceptibility testing for he
licobacter pylori is now widely available: when, how, why. Am J Gastroen
terol. 2022 Apr 1;117(4):524-528. doi: 10.14309/ajg.0000000000001659. 10. Karbalaei M, Talebi Bezmin Abadi A, Keikha M. Clinical rele
vance of the cagA and vacA s1m1 status and antibiotic resistance in Heli
cobacter pylori: a systematic review and meta-analysis. BMC Infect Dis. 2022 Jun 25;22(1):573. doi: 10.1186/s12879-022-07546-5. 18. Resina E, Gisbert JP. Rescue Therapy with Furazolidone in Pa
tients with at Least Five Eradication Treatment Failures and Multi-Resis
tant H. pylori infection. Antibiotics (Basel). 2021 Aug 24;10(9):1028. doi:
10.3390/antibiotics10091028. 11. Nikulina LM, Solovyova GA, Svintsitskyi IA, Koliada AK,
Kovalova AV. Relationship of helicobacter pylori caga and vaca status to
morphological changes of gastric mucosa and primary clarithromycin re
sistance rate in patients with chronic gastritis: a cross-sectional study. Wiad
Lek. 2023;76(4):709-714. doi: 10.36740/WLek202304103. 11. Nikulina LM, Solovyova GA, Svintsitskyi IA, Koliada AK,
Kovalova AV. Experience of using different schemes of eradication therapy
for Helicobacter pylori infection and their effectiveness in Ukraine Abstract. Background. The resistance of Helicobacter pylori
(H.pylori) to antibacterial drugs has increased in recent years. This
is primarily due to the unwarranted use of antibiotics, as demonstra
ted by the recent COVID-19 pandemic. The choice of the optimal
regimen and duration of treatment are current issues today. The aim:
retrospective study on the effectiveness of 14-day H.pylori eradica
tion regimens used in 2022–2023 and comparison of their effective
ness and safety with those of 10-day regimens used in 2020–2021 in
Ukraine. Materials and methods. The data of 242 patients (123 men
and 119 women) aged 18 to 65 years with chronic gastritis, peptic
ulcer of the duodenum and stomach associated with H.pylori were
analyzed retrospectively. All patients were treated with standard
regimens according to the Maastricht V and VI Consensus. H.pylori
infection was confirmed by a rapid urease test, determination of
fecal antigen and histologically. Results. As a result of the study, it
was found that 10-day regimens with triple therapy had an efficien cy of 80–81 %. The effectiveness of a 14-day triple therapy with
esomeprazole and lansoprazole was significantly higher compared
to a 10-day regimen, 85 and 86 %, respectively (p < 0.05). Howe
ver, the frequency of side effects was higher with a 14-day therapy. A 10-day triple regimen with levofloxacin compared to a standard
10-day triple therapy had the lowest efficacy of 78 %. But when levo
floxacin therapy was increased to 14 days, its effectiveness became
comparable to that of a 14-day triple therapy, 85 %. The highest
efficiency was demonstrated by a 10-day and 14-day therapy with fu
razolidone, which was added to amoxicillin and levofloxacin: 95 and
97.8%, respectively. Conclusions. The effectiveness of schemes with
clarithromycin in Ukraine remains high. Taking drugs for 14 days
increases the percentage of H.pylori eradication and the frequency
of unwanted effects. Addition of furazolidone to levofloxacin and
amoxicillin increases the rate of successful H.pylori eradication. Keywords: eradication; Helicobacter pylori; resistance; furazolidone cy of 80–81 %. The effectiveness of a 14-day triple therapy with
esomeprazole and lansoprazole was significantly higher compared
to a 10-day regimen, 85 and 86 %, respectively (p < 0.05). Howe
ver, the frequency of side effects was higher with a 14-day therapy. A 10-day triple regimen with levofloxacin compared to a standard
10-day triple therapy had the lowest efficacy of 78 %. References 1. Ferlay J, Soerjomataram I, Ervik M, et al; International Agency
for Research on Cancer (IARC). GLOBOCAN 2012: Estimated Cancer
Incidence, Mortality and Prevalence Worldwide in 2012 v 1.0: IARC Can
cerBase № 11. Lyon, France: IARC; 2013. 2. Plummer M, Franceschi S, Vignat J, Forman D, de Martel C. Glob
al burden of gastric cancer attributable to Helicobacter pylori. Int J Cancer. 2015 Jan 15;136(2):487-490. doi: 10.1002/ijc.28999. 3. Salar A. Gastric MALT lymphoma and Helicobacter pylo
ri. Med Clin (Barc). 2019 Jan 18;152(2):65-71. doi: 10.1016/j.medc
li.2018.09.006. 3. Salar A. Gastric MALT lymphoma and Helicobacter pylo
ri. Med Clin (Barc). 2019 Jan 18;152(2):65-71. doi: 10.1016/j.medc
li.2018.09.006. 4. Osyodlo GV, Kotyk YY, Kalashnikov MA, Osyodlo VV. Preva
lence, clinical course and treatment of chronic gastritis at the present stage. Gastroenterologìa. 2021;55(2):74-80. Ukrainain. doi: 10.22141/2308-
2097.55.2.2021.233627. 4. Osyodlo GV, Kotyk YY, Kalashnikov MA, Osyodlo VV. Preva
lence, clinical course and treatment of chronic gastritis at the present stage. Gastroenterologìa. 2021;55(2):74-80. Ukrainain. doi: 10.22141/2308-
2097.55.2.2021.233627. 5. Savoldi A, Carrara E, Graham DY, Conti M, Tacconelli E. Preva
lence of Antibiotic Resistance in Helicobacter pylori: A Systematic Review
and Meta-analysis in World Health Organization Regions. Gastroenterol
ogy. 2018 Nov;155(5):1372-1382.e17. doi: 10.1053/j.gastro.2018.07.007. 6. Malfertheiner P, Megraud F, Rokkas T, et al.; European Helico
bacter and Microbiota Study group. Management of Helicobacter pylori in
fection: the Maastricht VI/Florence consensus report. Gut. 2022;71:1724–
1762. doi: 10.1136/gutjnl-2022-327745. 4 Vol. 58, No. 1, 2024 Vol. 58, No. 1, 2024
Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online) Gastroenterologìa, ISSN 2308-2097 (print), ISSN 2518-7880 (online) Information about authors Information about authors
Volodymyr Chernyavskyi, MD, PhD, Professor, Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: vvch1979@gmail.com; https://orcid.org/
0000-0001-5831-8810
Leonid Pavlovskyi, PhD, Assistant at the Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: Leonya545@gmail.com; phone: +380 (50) 216-02-71;
https //orcid org/0000 0001 7121 5867 Volodymyr Chernyavskyi, MD, PhD, Professor, Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: vvch1979@gmail.com; https://orcid.org/
0000-0001-5831-8810 PhD, Assistant at the Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: Leonya545@gmail.com; phone: +380 (50) 216-02
0-0001-7121-5867 Leonid Pavlovskyi, PhD, Assistant at the Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: Leonya545@gmail.com; ph
https://orcid.org/0000-0001-7121-5867 p
g
Dmytro Reshotko, PhD, Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: ordinator2010@gmail.com; https://orcid.org Dmytro Reshotko, PhD, Department of Internal Medicine № 1, Bogomolets National Medical University, Kyiv, Ukraine; e-mail: ordinator2010@gmail.com; https://orcid.org/0000-0003-4492-3336
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 onflicts 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 t
formation about funding. Not stated. Authors’ contribution. Chernyavskyi V.V. — the concept and design of the study, collection and processing of materials, analysis of the received data, writing the text, text editing; Pavlovskyi L.L.,
Reshotko D.O. — collection and processing of materials, analysis of the received data, writing the text. V.V. Chernyavskyi, L.L. Pavlovskyi, D.O. Reshotko
Bogomolets National Medical University, Kyiv, Ukraine V.V. Chernyavskyi, L.L. Pavlovskyi, D.O. Reshotko
Bogomolets National Medical University, Kyiv, Ukraine V.V. Chernyavskyi, L.L. Pavlovskyi, D.O. Reshotko
Bogomolets National Medical University, Kyiv, Ukraine Experience of using different schemes of eradication therapy
for Helicobacter pylori infection and their effectiveness in Ukraine Vol. 58, No. 1, 2024
www.gastro.org.ua, https://gastro.zaslavsky.com.ua Experience of using different schemes of eradication therapy
for Helicobacter pylori infection and their effectiveness in Ukraine But when levo
floxacin therapy was increased to 14 days, its effectiveness became
comparable to that of a 14-day triple therapy, 85 %. The highest
efficiency was demonstrated by a 10-day and 14-day therapy with fu
razolidone, which was added to amoxicillin and levofloxacin: 95 and
97.8%, respectively. Conclusions. The effectiveness of schemes with
clarithromycin in Ukraine remains high. Taking drugs for 14 days
increases the percentage of H.pylori eradication and the frequency
of unwanted effects. Addition of furazolidone to levofloxacin and
amoxicillin increases the rate of successful H.pylori eradication. Keywords: eradication; Helicobacter pylori; resistance; furazolidone Keywords: eradication; Helicobacter pylori; resistance; furazolidone Vol. 58, No. 1, 2024 5 Vol. 58, No. 1, 2024
www.gastro.org.ua, https://gastro.zaslavsky.com.ua www.gastro.org.ua, https://gastro.zaslavsky.com.ua
|
https://openalex.org/W4309498452
|
https://zenodo.org/records/7339837/files/649.pdf
|
English
| null |
Teaching and Learning during and post COVID-19 pandemic in Nigeria
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,022
|
cc-by
| 8,532
|
ABSTRACT Coronavirus broke out in China in 2019 and has since spread across the world to become a
pandemic that affected all the facets of the world economy, health, education and other social
services. To curtail the spread of the disease various measures such as lockdown, closure of
schools, international bothers among others. While learning was taking place remotely in
developed countries during the school closures, such was not the case in Nigeria. The need to
ensure resilient education in Nigeria arises by adopting online teaching and learning to
complement face-to-face teaching. This paper discussed two major online learning methods
namely synchronous and asynchronous; the means of delivering online sessions such as
webinar, web-based, live stream. It further examined the various online applications and
electronic media used to facilitate learning, benefits, challenges and sustainability is. The
paper recommends that ministries of education should imbibe new normal in teaching and
learning and ensure sustainability by accessibility to all students, build capacity of
teachers/lecturers and students, and provide relevant online infrastructure, provide means of
affordability, among others. International Journal of Research
(IJR)
e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022
Teaching and Learning during and post COVID-19
pandemic in Nigeria
Dr. A. D. Shofoyeke
National Institute for Educational Planning and Administration, (N.I.E.P.A,
Nigeria), Ondo
Omale Monday
National Institute for Educational Planning and Administration, (N.I.E.P.A,
Nigeria), Ondo
Egbeja Mathew Monday
National Institute for Educational Planning and Administration, (N.I.E.P.A,
Nigeria), Ondo
ABSTRACT e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 Nigeria), Ondo INTRODUCTION History reveals that outbreaks of diseases had been affecting human beings at one time or the
other before medical cure were found. Diseases such as cholera bubonic plague, smallpox,
HIV and influenza were infectious and contagious diseases that spread across international
boundaries (pandemic) that killed people in millions before cure and vaccines were discovered
while some such as HIV have no permanent cure yet. Henderson (2009) posited that smallpox
killed at least half a billion people in the last hundred years of its existence. According to
UNAIDS (2020) globally, since the beginning of the HIV epidemic, around 75.7 million people
has become infected and 32.7 million people have died from AIDS (acquired
immunodeficiency syndrome) related illnesses while 38.0 million people are living with HIV. This indicates that no permanent cure for HIV has been discovered. The 1918 influenza
pandemic was the most severe pandemic in recent history then spread between 1918 and 1919,
affected an estimated 500 million people or one-third of the world’s population became
infected with this virus. The number of deaths was estimated to be at least 50 million worldwide
(Centers for Disease Control and Prevention, 2009). Stellino (2020) stated that 3-5 million
people and 20-50 million people died of Spanish flu in the first and second wave respectively. Cholera is another disease that affected the world which spread in across the globe in seven
pandemics 1817, 1829, 1852, 1863, 1881, 1899 and 1961 respectively and killed millions of
people across all continents (Claeso and Waldman, 2019). Zika, Ebola and Severe Acute
Respiratory Syndrome (SARS) are other diseases in recent years that have their toll on human
health and socio-economic activities but eventually faded out. The most recent and trending public health disease that cuts across the world is Coronavirus
COVID-19. The pandemic was first discovered in December 2019 in Wuhan, China and has
since spread across the world, resulting in the on-going health and economic crisis. As of 30th
January, 2020 COVID-19 was declared to be public Health emergency of international concern
and recognized as a pandemic on 11 March, 2020 by World Health Organisation (WHO, 2020). As at May 03, 2020, Worldometer recorded a total of 3,562,426 confirmed cases of coronavirus
and a death toll of 248,101 in 212 countries and Territories around the world. Key words Teaching and learning, pandemic, online learning, synchronous and asynchronous. Teaching and learning, pandemic, online learning, synchronous and asynchronous. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 44 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Impact of COVID-19 pandemic on education COVID-19 pandemic affects all facets of the economy including health and education across
the world. African Union (2020) stated that because of the continent’s openness to international
trade and migration, it is not immune to the harmful effects of COVID-19, which are of two
kinds: endogenous and exogenous. Accordingly, the exogenous effects come from direct trade
links between affected partner continents such as Asia, Europe and the United States; tourism;
the decline in remittances from African Diaspora; Foreign Direct Investment and Official
Development Assistance; illicit financing flows and domestic financial market tightening, etc. It describes the endogenous effects as those that occur as a result of the rapid spread of the
virus in many African countries. On one hand, they are linked to morbidity and mortality. On
the other hand, they lead to a disruption of economic activities. This may cause a decrease in
domestic demand in tax revenue due to the loss of oil and commodity prices coupled with an
increase in public expenditure to safeguard human health and support economic activities. Nigeria as an African country is affected be the endogenous and exogenous impacts. On education, the scale of the COVID-19 pandemic’s impact on education systems and on
children and young people’s learning and wellbeing increased daily. This is a global crisis
which is preventing children and adolescents in every country, including those affected by
conflict and displacement, from fulfilling their right to quality, safe and inclusive education. With Sustainable Development Goal 4 (SDG4), the global community committed to realising
the right to quality education for all children and adolescents by 2030. The COVID-19 crisis
puts this promise into jeopardy more than ever before (Inter-agency Network for Education in
Emergency). As of early April, 2020, most countries have introduced nation-wide early
childhood care, school and university closures affecting nearly 91% of the world’s student
population – more than 1.5 billion students (UNESCO, 2020). This was an attempt to reduce
the spread of COVID-19. In other words, at the outbreak of the COVID-19 pandemic in
Nigeria, schools and all learning facilities were closed in order to safeguard the health and
general wellbeing of children, youths, teachers, and educational personnel. INTRODUCTION By February 13,
2021, the world has recorded increase in the incidence of the pandemic thus: 109, 061,773
Coronavirus cases, 2,403,138 deaths and 81,049,697 recoveries (Worldometer, 2021). According to the Nigeria Centre for Disease Control (NCDC) number of confirmed cases rose
from one in February 27, 2020 when the first index case was reported to 131 confirmed cases
with 2 deaths on 30th March, 2020 and by February 13, 2021, it rose to 145,664 confirmed
cases, 120,339 discharged and 1,747 deaths (Africanews, 2021 February 13). Thus, the spread
of the pandemic has effects on global and national health, economy and social life which have
led to putting various measures such as lockdown, partial lockdown, closure of schools, remote
working, teaching and learning. In view of these measures, the paper examined impact of
COVID-19 on education, teaching methods adopted, the benefits and challenges of the teaching
methods, sustainability issue in the teaching methods offered conclusion and
recommendations. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 45 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 Impact of COVID-19 pandemic on education Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 causing interrupted learning, compromised nutrition, childcare problems and consequent
economic cost to families who could not work (Bjork Lund and Salvanes, 2011). The effects
of COVID-19 might also lead to conflict as a result of resource limitations and protracted
exposure to stressful circumstances. In these circumstances, causing interrupted learning, compromised nutrition, childcare problems and consequent
economic cost to families who could not work (Bjork Lund and Salvanes, 2011). The effects
of COVID-19 might also lead to conflict as a result of resource limitations and protracted
exposure to stressful circumstances. In these circumstances, In response to school closure, UNESCO recommended the use of distance learning
programmes and open education applications and platforms that schools and teachers can use
to reach students remotely and limit the disruption of education. Colleges have scrambled to
find creative solutions to teaching students online, in person but socially distant or in a hybrid
format (UNESCO, 2020). According to Federal Ministry of Education (2020) The Guidelines
for Schools and Learning Facilities Reopening after COVID-19 Pandemic Closures outline key
strategies for safe and equitable plans for school reopening and operation which include
attendance, social distancing, hygiene, cleaning, and non-pharmaceutical interventions for safe
and healthy school activities and programs. In this regards, the education response needs to be
innovative while adhering to standards that support impactful programming. Accordingly,
education sector specialists need to work with their existing skill sets for crisis-responsive
programming but also need to develop new skills since we are all working under new
conditions - specifically driven by social distancing parameters. In line with this, the Federal
Ministry of Education is working to promote distance learning for schools based on the existing
curricular by establishing ICT base in 16 focal states. Thus, the new knowledge and skills for
teachers to teach in crisis-sensitive condition and ensure sustainable education delivery are
discussed in the subsequent section. Impact of COVID-19 pandemic on education By August 2020,
COVID-19 curves began to flatten in most countries leading to remover of restrictions
culminating into gradual return of normalcy in economy as well as education, However, the
breakout of second wave led to increase in confirmed cases and deaths, a scenario that again
led to closure of schools in many countries including Nigeria that could not resume in January
as planned. Thus, as of 12 January, 2021, approximately 825 million students were affected
due to school closures in response to the pandemic. According to the UNESCO Monitoring, 23
countries are currently implementing local closures, impacting about 47 percent of world’s
student population. 112 countries schools are currently open. School closures impact not only on students, teachers and families but have far-reaching
economic and societal consequences. Schools closure in response to the pandemic have shed
light on various social and economic issues, including student debt, digital learning, food
insecurity and homelessness as well as access childcare, health care, housing, internet and
disability services. The impact was more severe for disadvantaged children and their families 46 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Synchronous learning Kokoulina (2020) define synchronous learning as any learning activity in which all learners are
simultaneously participating. It can happen either online or offline; in both cases, it is highly
time-related, highly interactive, and very social. Traditional synchronous learning typically
has learners gathering, participating, and getting hands-on experience in a face-to-face setting
with an instructor. Two of the most common synchronous formats are: live classroom sessions
and on-the-job coaching. The main difference between online and traditional learning is the
setting. With synchronous online learning, instructors (teachers/lecturers) and students are in
different locations and meet in the virtual environment with the help of computers, mobile
devices, and specific software tools. Online sessions can be hosted as a webinar, a web-based
class, or a live stream. It has a less flexible learning plan because the classes are conducted on
a set schedule using videoconferencing or live online webinars. Fixed-time online courses: These are the most common type of distance education. Students
sign into their online educational portal to access distance learning resources, including live
class video streams. Using this method, students and instructors make use of live chats and
discussion boards for communication. Video conferencing: This takes advantage of tools and platforms, like Zoom, that have
expansive capabilities and can be used globally. Video conferencing provides learning
opportunities for students by allowing them to see their instructors and peers in real time,
creating a sense of community in the virtual classroom. Web-based classes: This format is almost the same as the classroom training, with the
exception that the participants are not physically present in the same room. Still, remote
teachers and students can freely interact with each other by asking and answering questions,
sharing learning materials and demonstrating how to do various tasks. Webinars: In a typical webinar, only an instructor (teacher/lecturer) has the right to speak;
learners use text chat to send their questions and give feedback. Therefore, it is often more of
a talking-head lecture in which screencasts, slides, polls, and chat feedback might be included. Live streams: This is the most informal approach of this list, since learning occurs outside of
class time and enters the arena where people communicate and have fun – social media. For
instance, think of Facebook Live, Instagram Stories, YouTube Live streaming, or Twitch. With the support of these services, students can watch experts doing their job in real-time. Teaching methods (e-learning) approach Conventional face-to-face method of teaching had been in vogue for long and later distance
learning became another learning mode. Distance learning is the education students who may
not always be physically present at a school receives. In the past, this involved correspondence
between an individual and an academic institution by mail. At present, due to improvement
and application of technology, distance learning involves learning through online tools and
platforms. TopHat (2020) asserted that a distance learning program can take place entirely in
online learning environments, or a combination of distance learning and traditional classroom
instruction (called blended or hybrid). Massive open online courses (MOOCs), offering large-
scale interactive participation and learning resources, are more recent developments in distance
learning. During the COVID-19 pandemic and subsequent closures of schools, face-to-face
teaching could not be used and as a result educators and institutions relied heavily on distance
learning methods to complete education programmes and maintain sustainability of teaching
and learning. Two most common types of online learning (distance learning) are synchronous
and asynchronous (Hrastinski, 2008; Er and Arifoglu, 2009; Simonson, Smaldino, Albright and
Zvacek, 2012 in Higley, 2013). Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 47 Synchronous learning Apart from watching, students can also comment, like, or even donate some money. International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 This combined network of learners and the electronic network in which they communicate are
referred to as an asynchronous learning network. It uses resources that facilitate information
sharing outside the constraints of time and place among a network of people.. According to Bourne (1998) Online learning resources that can be used to support
asynchronous learning include email, electronic mailing lists, online discussion boards, blogs,
threaded conferencing systems and wikis. Course management systems have been developed
to support online interaction, allowing users to organize discussions, post and reply to
messages, and upload and access multimedia. Bourne (1998) stated that asynchronous forms
of communication are sometimes supplemented with synchronous components, including text
and video chat, videoconferencing, telephone conversations and even meetings in virtual
spaces such as Second Life, where discussions can be facilitated among groups of students. Asynchronous learning allows the students to work at their own pace, and normally has a very
distinct syllabus, with weekly deadlines for homework and other assignments. Students have
regular access to their peers and their instructors, although this is typically managed through
email and discussion boards. ▪
Open schedule online courses: These give students the greatest amount of freedom. All
deadlines are pre-set and students are encouraged to be self-sufficient and complete their
assignments on their own timelines. Without dedicated class time, students complete their
coursework whenever they choose to allot the time to do so. Final exams normally occur at
the end of the semester, and are open for several days to provide students with some
flexibility as to when they choose to take it. ▪
Hybrid distance education: This combines synchronous and asynchronous methods of
online learning. Students must adhere to specific predetermined deadlines for assignment
completion. The majority of the coursework is completed online, but in some cases, the
student can physically speak with an instructor (teacher/lecturer) in person through live
chats or video conferencing. Hybrid distance education may also include attending a
physical classroom for certain periods of time. Conversely, it may involve covering specific
modules and then returning to distance learning to complete additional modules and
assignments. (1) Online teaching and learning (Teaching through internet services) tools. Online
learning is an educational medium that allows students to participate in online courses
via internet. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Asynchronous learning Mayadas (1997) referred to asynchronous learning as a general term used to describe forms of
education, instruction, and learning that do not occur in the same place or at the same time. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 48 International Journal of Research
(IJR)
e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022
This combined network of learners and the electronic network in which they communicate are
referred to as an asynchronous learning network. It uses resources that facilitate information
sharing outside the constraints of time and place among a network of people.. International Journal of Research
(IJR) (d) Virtual education: It refers to instructions in a learning environment where
teachers and students are separated by time or space or both, and the teacher
provides course content through course management applications, multimedia
resources, the internet, video conferencing etc. students receive the content and
communicate with the teacher via the same technologies. (d) Virtual education: It refers to instructions in a learning environment where
teachers and students are separated by time or space or both, and the teacher
provides course content through course management applications, multimedia
resources, the internet, video conferencing etc. students receive the content and
communicate with the teacher via the same technologies. (d) Virtual education: It refers to instructions in a learning environment where
teachers and students are separated by time or space or both, and the teacher
provides course content through course management applications, multimedia
resources, the internet, video conferencing etc. students receive the content and
communicate with the teacher via the same technologies. (e) Skype: Skype is a free web-based communication tool which allows people to
video conference, make calls and instant messaging. Skype provides a variety of
educational opportunities for classrooms. Students can connect with other students,
increase their knowledge and interact with other cultures. It can be used to share
projects, polish their language skills, exchange information about particular books
with students who read the same books. Skype provides students and teachers with
opportunity to participate in virtual tours of historical places, communicate with
authors and researchers and engage in conversations with classrooms around the
world. (e) Skype: Skype is a free web-based communication tool which allows people to
video conference, make calls and instant messaging. Skype provides a variety of
educational opportunities for classrooms. Students can connect with other students,
increase their knowledge and interact with other cultures. It can be used to share
projects, polish their language skills, exchange information about particular books
with students who read the same books. Skype provides students and teachers with
opportunity to participate in virtual tours of historical places, communicate with
authors and researchers and engage in conversations with classrooms around the
world. Angelo State University (2021) states that Skype is very useful when verbal
interaction is required between student and instructor. Activities for Skype include
hosting virtual office hours and one-on-one tutoring. Students can Skype with each
other sharing their experiences and collaborate in project work. International Journal of Research
(IJR) They do not need to visit lecture halls or class rooms, and they can choose
to learn whatever they want from the comfort of their own houses. The following
applications can be used for online teaching and learning (Synchronous Asynchronous
learning) via the internet services: (a) Zoom: With this feature, teachers can easily conduct online classes hassle-free. It
is equipped with features like screen sharing, whiteboard for presenters, video
recording and more, which makes it an ideal companion to your offline learning
online. Teachers can set up a class room across the batches with just a click and
student can view the recorded lectures. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 49 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 (b) WhatsApp: The use of whatsApp for educational purpose is to facilitate
communication and at its basic level, education is nothing but communication. WhatsApp can provide a channel through which teachers can achieve faster and
more seamless communication with their students. It can also increase level of
communication between students and create another venue for learning. Education
strategies for whatsApp: use the group chart feature to create learning and study
groups, create audio lessons that can be sent directly to students, send videos to
students and teacher stays in contact with parents. It is very cost effective (b) WhatsApp: The use of whatsApp for educational purpose is to facilitate
communication and at its basic level, education is nothing but communication. WhatsApp can provide a channel through which teachers can achieve faster and
more seamless communication with their students. It can also increase level of
communication between students and create another venue for learning. Education
strategies for whatsApp: use the group chart feature to create learning and study
groups, create audio lessons that can be sent directly to students, send videos to
students and teacher stays in contact with parents. It is very cost effective (b) WhatsApp: The use of whatsApp for educational purpose is to facilitate
communication and at its basic level, education is nothing but communication. WhatsApp can provide a channel through which teachers can achieve faster and
more seamless communication with their students. It can also increase level of
communication between students and create another venue for learning. Education
strategies for whatsApp: use the group chart feature to create learning and study
groups, create audio lessons that can be sent directly to students, send videos to
students and teacher stays in contact with parents. It is very cost effective (c) Telegram: Telegram is a free-ware, cross-platform, cloud-based instant messaging
software and application service. The service also provides end-to-end encrypted
video calling, file sharing and several other features. Teachers can use the Telegram
program to serve his/her teaching materials to his/her students. (c) Telegram: Telegram is a free-ware, cross-platform, cloud-based instant messaging
software and application service. The service also provides end-to-end encrypted
video calling, file sharing and several other features. Teachers can use the Telegram
program to serve his/her teaching materials to his/her students. International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 (a) Television: To support formal education, television normally functions as a
supportive and reinforcement tool. Learning television is the use of television
programs in the field of distance education. It may be in form of individual
television programs or dedicated specialty channel that is often associated with
cable television in the United States as public education and government access
(PEG) channel providers. (a) Television: To support formal education, television normally functions as a
supportive and reinforcement tool. Learning television is the use of television
programs in the field of distance education. It may be in form of individual
television programs or dedicated specialty channel that is often associated with
cable television in the United States as public education and government access
(PEG) channel providers. (a) Television: To support formal education, television normally functions as a
supportive and reinforcement tool. Learning television is the use of television
programs in the field of distance education. It may be in form of individual
television programs or dedicated specialty channel that is often associated with
cable television in the United States as public education and government access
(PEG) channel providers. (b) Radio: A radio-based approach to learning during Covid-19 may be the best way
to reach students during the pandemic and perhaps beyond. With an estimated 90
percent of all students unable to attend school in person because of the Covid-19
pandemic, many countries are using distance learning methodology to reach all
their students. Interactive audio instructions (IAI), a technology pioneered by EDC,
is one of such method. In remote communities around the world, IAI has been used
to deliver high-quality education to students. In an IAI classroom, children interact
with each other and with their “audio” teacher during each broadcast. Pauses are
also built into the recordings so children can have time to answer questions and
perform tasks. This stands in contrast to classic radio education where students are
expected to simply sit and listen to lectures. (b) Radio: A radio-based approach to learning during Covid-19 may be the best way
to reach students during the pandemic and perhaps beyond. With an estimated 90
percent of all students unable to attend school in person because of the Covid-19
pandemic, many countries are using distance learning methodology to reach all
their students. International Journal of Research
(IJR) Interactive audio instructions (IAI), a technology pioneered by EDC,
is one of such method. In remote communities around the world, IAI has been used
to deliver high-quality education to students. In an IAI classroom, children interact
with each other and with their “audio” teacher during each broadcast. Pauses are
also built into the recordings so children can have time to answer questions and
perform tasks. This stands in contrast to classic radio education where students are
expected to simply sit and listen to lectures. (c) Mobile phone: One of the original distance learning media still in widespread use. Mobile short messaging services (SMS) along with live telecast can be used to
create almost an ideal classroom situation. Mobile phone increases access for those
who are mobile or cannot physically attend learning institutions. The portability of
mobile technology means that mobile learning is not bound by fixed class times, it
enables learning at all times and all places during pandemic, breaks, before or after,
at home or on the go. Even on transit, one can utilize the time spent to learn
(Christine 2002). (c) Mobile phone: One of the original distance learning media still in widespread use. Mobile short messaging services (SMS) along with live telecast can be used to
create almost an ideal classroom situation. Mobile phone increases access for those
who are mobile or cannot physically attend learning institutions. The portability of
mobile technology means that mobile learning is not bound by fixed class times, it
enables learning at all times and all places during pandemic, breaks, before or after,
at home or on the go. Even on transit, one can utilize the time spent to learn
(Christine 2002). International Journal of Research
(IJR) Instructors can
instant message to colleagues and students. YouTube: It is a video-sharing website that provides good quality education that
has been developed a lot during the past years. By using it, a student from one country can have access to education from another
country. Learning on YouTube has gained tremendous importance. If a student is YouTube: It is a video-sharing website that provides good quality education that
has been developed a lot during the past years. By using it, a student from one country can have access to education from another
country. Learning on YouTube has gained tremendous importance. If a student is
not able to understand a concept, he has the option to watch it again. There are no
requirements for any classroom, benches or other school materials to learn
anything. Only a robust and reliable internet connection and a smartphone are
needed. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022
50
(2) Electronic media (E-media): Any equipment used in the electronic
communication process (e.g. television, radio mobile phone) may also be
considered electronic media. They are media that use electronic or
electromechanical audiences to access the content. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
50
(2) Electronic media (E-media): Any equipment used in the electronic
communication process (e.g. television, radio mobile phone) may also be
considered electronic media. They are media that use electronic or
electromechanical audiences to access the content. Received: 22 Oct 2022
50
(2) Electronic media (E-media): Any equipment used in the electronic
communication process (e.g. television, radio mobile phone) may also be
considered electronic media. They are media that use electronic or
electromechanical audiences to access the content. 50 Received: 22 Oct. 2022
Revised: 7 Nov. 2022 Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 (9)
Synchronous provides better chances to build a community. Synchronous learning
assists students avoid the feeling of isolation that may occur in Asynchronous activities (9)
Synchronous provides better chances to build a community. Synchronous learning
assists students avoid the feeling of isolation that may occur in Asynchronous activities (10) Synchronous learning provides learning from anywhere or location. Students and
teachers can meet in a single virtual environment regardless of where they are and all that
they need is a computer or a mobile device with internet access. Challenges OF online (internet) teaching and learning methods Online teaching and learning methods have a number of challenges that need be overcoe. These
include but not limited to the followings: (1)
Some students without reliable internet access and technology struggle to participate in
digital or online learning (Oreopoulos et al 2006). (1)
Some students without reliable internet access and technology struggle to participate in
digital or online learning (Oreopoulos et al 2006). (2)
Prone to sense of isolation. Students can learn a lot from being in the company of their
peers. However, in an online class, there are minimal physical interaction between
students and teachers. (3)
Online learning requires teachers to have a basic understanding of using digital forms of
learning. However, this is not the case always. Very often, teachers have a basic
understanding of technology. Sometimes, they do not even have the necessary resources
and tools to conduct online classes. (4)
Asynchronous learning environments pose several challenges for instructors, institutions,
and students. Course development and initial setup can be costly. Institutions must
provide a computer network infrastructure, including servers, audio/visual equipment,
software, and the technical support needed to develop and maintain asynchronous
learning environments. Technical support includes initial training and setup, user
management, data storage and recovery, as well as hardware repairs and updates (Palmer,
Holt and Bray, 2008). (5) (5)
Synchronous is time-consuming. It requires being online or in class at a certain time
which depends on the availability of the internet and electricity that are epileptic in
nature. The highly interactive nature of live classes requires spending more time on self-
preparation. (6)
Synchronous learning requires accurate scheduling. Where scheduling is overlooked,
there will be a risk that just a few students present which tends to limit participation. (6)
Synchronous learning requires accurate scheduling. Where scheduling is overlooked,
there will be a risk that just a few students present which tends to limit participation. (7)
Synchronous learning is often more expensive than asynchronous learning. The more
time a person spends on conducting a course, the more expense is incurred. (7)
Synchronous learning is often more expensive than asynchronous learning. The more
time a person spends on conducting a course, the more expense is incurred. Benefits of online (internet) teaching and learning methods (1)
Classes can be taken from any place and at the time which students or tutors pref (2)
It ensures quick delivery of lessons. Traditional classroom involves some or the kind of
delay whereas, e-learning provides expeditious and exclusive delivery of lessons. (2)
It ensures quick delivery of lessons. Traditional classroom involves some or the kind of
delay whereas, e-learning provides expeditious and exclusive delivery of lessons. (3)
It promotes active and independent learning. (4)
E-learning is based upon convenience and flexibility. All the resources for the students
as well as teachers are available in one place. (5)
Global level education: Tutors (Teachers/Lecturers) can provide online education in
multiple languages and people from different time zones. (5)
Global level education: Tutors (Teachers/Lecturers) can provide online education in
multiple languages and people from different time zones. (6)
All students can receive the same type of syllables, study material and train through E-
learning. (7)
Online learning saves time as a student does not need to travel to the training venue. A
student can learn at the comfort of your own place. (8)
A better feedback flop. The facilitator can share feedback on how students are doing in
real-time. This allows them to correct mistakes right on the spot and get positive
reinforcement of the desired behaviour, performance, or using a ne new skill. Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022
51 Received: 22 Oct. 2022
Revised: 7 Nov. 2022 51 Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 International Journal of Research
(IJR) i. A learning design involving information and communications technology has been
developed and implemented within a course or sources of study. It has been through a
proof of concept stage and has been judged, on the basis of evidence produced, to be
beneficial to teaching and learning. i. A learning design involving information and communications technology has been
developed and implemented within a course or sources of study. It has been through a
proof of concept stage and has been judged, on the basis of evidence produced, to be
beneficial to teaching and learning. ii. The e-learning concept, design, system or resources have proven potential to be adopted
and possibly adopted for use beyond the original development environment. ii. The e-learning concept, design, system or resources have proven potential to be adopted
and possibly adopted for use beyond the original development environment. p
y
p
y
g
p
iii. Maintenance, use and further development of the e-learning concept, design, system or
resources do not remain dependent on one or few individuals who created them, to the
extent that if their involvement ceased, future respect would not be compromised. While other definitions are acknowledged, this one reflects a common scenario that
challenges the aim to disseminate effective e-learning practice beyond the development
context. Many institutions and nationally founded projects fall into this category, along
with valuable early adopters (Taylor 1998). The overall aim of sustainable development is
to transform teaching, learning and higher education organizations in the way described
by Nicol and Draper (2009).). Nigeria does not fall into the category of early adopters of
online teaching and learning otherwise during school closures and lockdown teaching and
learning would be taking place remotely and academic loss time would not arise, similarly,
education sector workers and planners would be working at home remotely and delivering
on service. Now that Nigeria is adopting online to deliver education in most universities
and blended approach in other levels of education, the sustainability will depend on the
following issues in line with the three conditions I the foregoing: iii. Maintenance, use and further development of the e-learning concept, design, system or
resources do not remain dependent on one or few individuals who created them, to the
extent that if their involvement ceased, future respect would not be compromised. International Journal of Research
(IJR) While other definitions are acknowledged, this one reflects a common scenario that
challenges the aim to disseminate effective e-learning practice beyond the development
context. Many institutions and nationally founded projects fall into this category, along
with valuable early adopters (Taylor 1998). The overall aim of sustainable development is
to transform teaching, learning and higher education organizations in the way described
by Nicol and Draper (2009).). Nigeria does not fall into the category of early adopters of
online teaching and learning otherwise during school closures and lockdown teaching and
learning would be taking place remotely and academic loss time would not arise, similarly,
education sector workers and planners would be working at home remotely and delivering
on service. Now that Nigeria is adopting online to deliver education in most universities
and blended approach in other levels of education, the sustainability will depend on the
following issues in line with the three conditions I the foregoing: iii. Maintenance, use and further development of the e-learning concept, design, system or
resources do not remain dependent on one or few individuals who created them, to the
extent that if their involvement ceased, future respect would not be compromised. Affordability Issue: to participate in synchronous and asynchronous learning
environments, students must have access to computers, Android or smart phone and
access to internet. Although personal computers and web access are becoming more and
more prevalent every day, this requirement can be a barrier to entry for many students
yet many students cannot afford it. Where they have computers or tablets of phone,
subscription to data for internet services may become a problem due to poverty. Capacity issue: Students must also have the computer/technology skills required to
participate in the online learning programme. Similarly, teachers/lecturers /instructors
need to develop learning materials using relevant tools but how many have had their
capacity built and prepared to continue using such skills? To what extent are
lecturers/teachers capacity enhanced to deliver online lectures/lessons and how effective
is this? To what extent are Ministries of Education, State Universal Basic Education
Boards, Teaching Service Boards of/Commissions, the regulatory agencies (National
Universities Commission, National Board for Technical Education and National
Commission for Colleges of Education) and Tertiary Education Trust Fund prepare to
encourage, fund and support teachers’/lecturers’ capacity on online teaching? Sustainability issues in online teaching and learning Sustainability refers to the ability to be maintained at a certain rate or level. Thus, within this
context, sustainability is the ability of Nigeria education system to maintain and improve the
online teaching approaches when they are fully put in place at all levels of education even when
the current COVID-19 pandemic vanishes. An e-learning initiative is considered sustainable
when all three of these conditions are met: Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 52 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 challenged households, physically or mentally challenged and out-of-school children Thus, the extent to which no one is left behind in the learning process is germane. Thus, the extent to which no one is left behind in the learning process is germane. Infrastructure issues: The extent to which Federal and states provide and maintain online
platforms, support infrastructures and relevant software, technical support. Stable
source of power is very significant in online learning both at the source of delivery and
the receiving end. How prepared are the states and institutions in providing the enabling
infrastructure? Infrastructure issues: The extent to which Federal and states provide and maintain online
platforms, support infrastructures and relevant software, technical support. Stable
source of power is very significant in online learning both at the source of delivery and
the receiving end. How prepared are the states and institutions in providing the enabling
infrastructure? Policy issue: Most programmes/projects are abandoned when solutions to challenges or
government changes hand whereas good practice requires sustainability and
improvement. Pandemic or crisis is bound to occur unnoticed anytime hence the need for
resilience of the education. The questions therefore are: Will education policy review
integrate online teaching in in the schools? Will the education ministries and institutions
monitor policy implementation? Policy issue: Most programmes/projects are abandoned when solutions to challenges or
government changes hand whereas good practice requires sustainability and
improvement. Pandemic or crisis is bound to occur unnoticed anytime hence the need for
resilience of the education. The questions therefore are: Will education policy review
integrate online teaching in in the schools? Will the education ministries and institutions
monitor policy implementation? Conclusion There is no doubt that the outbreak of COVID-19 has had its impact on school calendar
disruption, reduction in economic development of the country which has affected the education
finance as well. The pandemic lockdown has led to shortage of funds for the educational
system, parents as well are being faced with the reality of having to pay extra cost on their
children academic whenever they resume to school. It was envisaged that the pandemic could
lead to increase in number of out-of-school children but the Federal Ministry of Education
reported reduction in the number from 10.5 million to 6.95 million. Teaching and learning were
taking place in more developed countries during lockdown and school closure because of use
of deployment and use of functional learning management system via online. For Nigeria
education system to be functional during and after the COVID-19 pandemic, governments and
stakeholders need to adopt online learning just as the universities are currently doing. The use
of synchronous and asynchronous learning has been found useful and should be adopted by all
levels of education. During normalcy period, blended learning could be taking place wherein
the face-to-face teaching is supplemented with synchronous and asynchronous learning. However, in the course of implementing online teaching and learning mode, sustainability
issues such as capacity building of teachers/lecturers, students and support staff need be built,
affordability of online learning on the part of governments, instructions and students/parents,
accessibility by students across the country including rural areas, difficult terrain, special needs
children and out-of-school children, infrastructure support and policy. International Journal of Research
(IJR) Accessibility issue: Accessibility of students in rural areas to internet and media is difficult
and will continue to get excluded from participation in online learning. g. Specifically,
one of the fundamental issues is how the education sector will to promote distance
learning solutions that will also cater for those in marginalised communities, financially Accessibility issue: Accessibility of students in rural areas to internet and media is difficult
and will continue to get excluded from participation in online learning. g. Specifically,
one of the fundamental issues is how the education sector will to promote distance
learning solutions that will also cater for those in marginalised communities, financially Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 53 International Journal of Research
(IJR) e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 It has been observed that there are no proper plans in place to curb the influence of corona
virus on the education system, it is highly recommended for the government and Concerned
education personnel should ensure there are futuristic plans in case of another similar
experience. This is Covid-19, nobody knows what other occurrences will happen in the future
and will lead to interruption of the activities of the educational system in Nigeria, therefore
plans are to be made in ensuring the future of the education system is secured and not being
interrupted with emergence of diseases. There is need for the Ministries of Education to imbibe the new normal in teaching and learning
by deploying online learning/learning management system in order to make education
functional always and as well compete with the outside world even in the period of global
pandemic lockdown. In this regard, ICT experts should be engaged to design synchronous and
asynchronous online teaching activities through social media platforms such as Google
classroom which is a free web service that is developed by Google for schools that aims to
simply creating, distributing and grading assignment in a paperless way with the purpose of
streamlining the process of sharing files between lecturers and students. Other platforms like
WhatsApp, Zoom, Blog etc are recommended for learning. Promoting and adopting online teaching and learning mode requires building of capacity of
teachers/lecturers in relevant learning management system by Ministries of Education and
relevant agencies as well as institutions of learning. Government should provide relevant infrastructure to support online learning as well as
technical skills. Similarly, provision of staple electricity and solar energy is desirable for
functional online learning platforms. Accessibility is germane in online learning. Strategies need to be put in place to reach those in
difficult terrain and rural areas as well as the out-of-school children including special needs. In
the same token, provision of taps to students to aid learning including means of connecting to
internet services are necessary for successful digital learning. This approach will ensure no
learner is left behind. Recommendations Since COVID-19 still remains with us and has no permanent cure yet, observing the new
normal becomes imperative. Besides, the county’s education should be sustainable and
functional in case of future pandemic or emergencies. In the light of these, the following
recommendations are made: Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 54 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Africanews
(2021,
February
13).
Coronavirus
–
Nigeria
COVID-19
update.
https://www.africanews.com/2021/02/14/coronavirus-nigeria-covid-19-update-13-
february-2021
African Union (2020). Impact of the Coronavirus (COVID-19) on the African Economy.
https://au.int/sites/default/files/documents/38326-doc-covid-
19_impact_on_african_economy.pdf Africanews
(2021,
February
13). Coronavirus
–
Nigeria
COVID-19
update. https://www.africanews.com/2021/02/14/coronavirus-nigeria-covid-19-update-13-
february-2021 African Union (2020). Impact of the Coronavirus (COVID-19) on the African Economy. https://au.int/sites/default/files/documents/38326-doc-covid-
19_impact_on_african_economy.pdf 55 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 e-ISSN: 2348-6848
p-ISSN: 2348-795X
Vol. 9 Issue 11
November 2022 International Journal of Research
(IJR) Angelo State University (2021). Interaction and Collaboration. https://www.angelo.edu/live/profiles/8256-interaction-collaboration Bjorklund, A and Salvanes, K. (2011).” Education and Family Background: Mechanisms and
Policies”, in Hanushek, E; Machin, S. and Woessmann, L. (eds), Handbook of the
Economics of Education, Vol. 3 Bourne, J. R. (1998), "Net-learning: strategies for on-campus and off-campus network-enabled
learning", Journal of Asynchronous Learning Networks, 2 (2). Centers for Disease Control and Prevention (2009). 1918 pandemic (H1N1 virus). http://www.cdc.gov//flu/pandemic-resources/1918-pandemic-h1n1.html Claeso, M. and Waldman, R. (2019). Cholera – The Seventh Pandemic in the 21st Century. Britannica. https://www.britannica.com/science/cholera/The-seventh-pandemic-in-the-
21st-century Federal Ministry of Education (2020, July 13). Guidelines for schools and learning facilities,
reopening after Covid-19 Pandemic closures. Abuja: Federal Ministry of Education. Henderson, D. A. (2009). Smallpox: The Death of a Disease – The Inside story of Eradicating
a Worldwide Killer. Prometheus Books. Higley, M. (2013). Benefits of Synchronous and Asynchronous e-Learning. https://elearningindustry.com/benefits-of-synchronous-and-asynchronous-e-learning Inter-agency Network for Education in Emergencies (INEE). (2020). Technical Note:
Education during the COVID-19 Pandemic. New York, NY. https://inee.org/resources/technical-note-education-during-covid-19-pandemic Kokoulina, O. (2020). Synchronous Learning Simply Put: Definition, Benefits, and Tools. https://www.ispringsolutions.com/blog/what-is-synchronous-learning Mayadas, F (1997), "Asynchronous learning networks: a Sloan Foundation
perspective", Journal of Asynchronous Learning Networks, Nicol, D. and Drapers, S. (2009). A blueprint for transformational organisational change in
higher education: REAP as a case study. https://www.psy.gla.ac.uk/~steve/rap/docs/NicolDraperTransf4.pdf Nigeria Centre for Disease Control (2020, March 3). Outbreak of Covid-19 case in Nigeria:
Situation report,
https://m.facebook.com/NCDCgov/photos/a.1045178838878472/3029182443811425 Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022 56 International Journal of Research
(IJR) Oreopulos, P; Page, M. E. and Stevens, A.H. (2006). The Intergenerational Effects of
Compulsory Schooling. https://oreopoulos.faculty.economics.utoronto.ca/wp-
content/uploads/2014/03/The-Intergenerational-Effects-of-Compulsory-Schooling.pdf Palmer, S; Holt, D; and Bray, S (2008), "Does the discussion help? the impact of a formally
assessed online discussion on final student results", British Journal of Educational
Technology, 39 (5): 847–58,https://dio.org/10.1111/j.1467-8535.2007.00780.x Stellino, M. (2020, April 25). Fack Check: Was second wave of Spanish flu worse? Did it kill
at least 20 million people? USA Today. https://www.usatoday.com/story/news/factcheck/2020/04/25/fact-check-total-deaths-
eas-spanish-flu-wave-unknown/3024648001 Taylor, P. (1998). Institutional Change in uncertain times: Lone ranging is not enough in
studies in Higher Education. Studies in higher Education, 23(3) 269-279711
https://doi.org/10.1080/03075079812331380246 TopHat (2020). What is Distance? The Benefits of Students Studying Remotely. https://tophat.com.blog/distance-learning Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022
57
Worldometer (2020, May 3). Coronavirus Pandemic Cases. https://www.worldometers.info/coronavirus
Worldometer (2021, February 13). Coronavirus Pandemic Cases. https://www.worldometers.info/coronavirus
World Health Organisation (2020, March, 11). Virtual press conference on COVID-19. Who-
audio-press-conference-full-and-final-11mar2020.pdf
UNAIDS (2020, July). Global HIV/AIDS statistics fact sheet. https://www.unaids.org/en/resources/fact-sheet
UNESCO (2020, March, 24). Covid-19 Educational Disruption and Response. https://en.unesco.org/news/covid-19-educational-disruption-and-response
UNESCO (2020). Distance learning solutions. https://en.unesco.org/covid19/educationresponse/solutions
UNICEF Nigeria (2020): Nigeria Education Emergencies Working Group. Nigeria Education
Sector Covid-19 response strategy in North East. Worldometer (2020, May 3). Coronavirus Pandemic Cases. https://www.worldometers.info/coronavirus Worldometer (2021, February 13). Coronavirus Pandemic Cases. https://www.worldometers.info/coronavirus Worldometer (2021, February 13). Coronavirus Pandemic Cases. https://www.worldometers.info/coronavirus World Health Organisation (2020, March, 11). Virtual press conference on COVID-19. Who-
audio-press-conference-full-and-final-11mar2020.pdf UNAIDS (2020, July). Global HIV/AIDS statistics fact sheet. https://www.unaids.org/en/resources/fact-sheet Received: 22 Oct. 2022
Revised: 7 Nov. 2022
Final Accepted for publication: 14 Nov 2022
Copyright © authors 2022
57
UNAIDS (2020, July). Global HIV/AIDS statistics fact sheet. https://www.unaids.org/en/resources/fact-sheet
UNESCO (2020, March, 24). Covid-19 Educational Disruption and Response. https://en.unesco.org/news/covid-19-educational-disruption-and-response
UNESCO (2020). Distance learning solutions. https://en.unesco.org/covid19/educationresponse/solutions
UNICEF Nigeria (2020): Nigeria Education Emergencies Working Group. Nigeria Education
Sector Covid-19 response strategy in North East. 57 57
|
https://openalex.org/W2373390550
|
https://cris.unibo.it/bitstream/11585/562042/1/Violante_Scand%20J%20Work%20Environ%20Health%202016.pdf
|
English
| null |
Carpal tunnel syndrome and manual work: the OCTOPUS cohort, results of a ten-year longitudinal study
|
Scandinavian journal of work, environment & health
| 2,016
|
cc-by
| 8,923
|
Refers to the following texts of the Journal: 2014;40(6):539-654
2013;39(2):121-216 2009;35(1):1-80 Refers to the following texts of the Journal: 2014;40(6):539-654
2013;39(2):121-216 2009;35(1):1-80 Key terms: carpal tunnel syndrome; Italy; longitudinal study; manual
work; musculoskeletal disorder; occupational exposure; occupational
exposure; occupational mononeuropathy; OCTOPUS cohort; threshold
limit value; upper-limb disorder This article in PubMed: www.ncbi.nlm.nih.gov/pubmed/27159901 doi:10.5271/sjweh.3566 doi:10.5271/sjweh.3566 Carpal tunnel syndrome and manual work: the OCTOPUS
cohort, results of a ten-year longitudinal study Carpal tunnel syndrome and manual work: the OCTOPUS
cohort, results of a ten-year longitudinal study
by Violante FS, Farioli A, Graziosi F, Marinelli F, Curti S, Armstrong TJ,
Mattioli S, Bonfiglioli R Carpal tunnel syndrome and manual work: the OCTOPUS
cohort, results of a ten-year longitudinal study
by Violante FS, Farioli A, Graziosi F, Marinelli F, Curti S, Armstrong TJ,
Mattioli S, Bonfiglioli R This large longitudinal cohort study provides a prospective validation
of the ACGIH TLV® method for the assessment of biomechanical
exposures at work. It confirmed that “forceful hand exertions” more
than “any exertion” significantly increase the risk of CTS. This study
suggests that the current limits (AL and TLV) might not be sufficiently
protective for some workers. Affiliation: Unità Operativa di Medicina del Lavoro, Policlinico
S.Orsola-Malpighi, via Pelagio Palagi 9, I-40138 Bologna, Italy. s.mattioli@unibo.it Original article Scand J Work Environ Health 2016;42(4):280-290 doi:10.5271/sjweh.3566 Downloaded from www.sjweh.fi on September 09, 2016 Downloaded from www.sjweh.fi on September 09, 2016 Downloaded from www.sjweh.fi on September 09, 2016 Downloaded from www.sjweh.fi on September 09, 2016 1 Department of Medical and Surgical Sciences, University of Bologna, Italy.
2 Center for Ergonomics, Department of Industrial and Operations Engineering, University of Michigan, Ann Arbor, USA.
Correspondence to: Prof Stefano Mattioli, Unità Operativa di Medicina del Lavoro, Policlinico S.Orsola-Malpighi, via Pelagio Palagi 9,
I-40138 Bologna, Italy. [E-mail: s.mattioli@unibo.it] Original article Scand J Work Environ Health. 2016;42(4):280–290. doi:10.5271/sjweh.3566 cand J Work Environ Health. 2016;42(4):280–290. doi:10.5271/sjweh.3566 Carpal tunnel syndrome and manual work: the OCTOPUS cohort, results of a
ten-year longitudinal study by Francesco S Violante, MD,1 Andrea Farioli, MD,1 Francesca Graziosi, PhD,1 Francesco Marinelli, PhD,1
Stefania Curti, PhD,1 Thomas J Armstrong, PhD,2 Stefano Mattioli, MD,1 Roberta Bonfiglioli, MD 1 by Francesco S Violante, MD,1 Andrea Farioli, MD,1 Francesca Graziosi, PhD,1 Francesco Marinelli, PhD,1
Stefania Curti, PhD,1 Thomas J Armstrong, PhD,2 Stefano Mattioli, MD,1 Roberta Bonfiglioli, MD 1 Violante FS, Farioli A, Graziosi F, Marinelli F, Curti S, Armstrong TJ, Mattioli S, Bonfiglioli R. Carpal tunnel syndrome
and manual work: the OCTOPUS cohort, results of a ten-year longitudinal study. Scand J Work Environ Health. 2016;42(4):280–290. doi:10.5271/sjweh.3566 Objective The American Conference of Governmental Industrial Hygienists (ACGIH) proposed a method to
assess the hand, wrist and forearm biomechanical overload based on exertions frequency (hand-activity level)
and force use (normalized peak force). We applied the ACGIH threshold limit value (TLV)® method to a large
occupational cohort to assess its ability to predict carpal tunnel syndrome (CTS) onset. Methods A cohort of industrial and service workers was followed-up between 2000 and 2011. We investigated
the incidence of CTS symptoms and CTS confirmed by nerve conduction studies (NCS). We then classified
exposure with respect to action limit (AL) and TLV. Cox regression models including age, gender, body mass
index, and presence of predisposing pathologies were conducted to estimate hazard ratios (HR) of CTS and
population attributable fractions. Results We analyzed data from 3131 workers [females, N=2032 (65%); mean age at baseline 39.3, standard
deviation (SD) 9.4 years]. We observed 431 incident cases of CTS symptoms in 8000 person-years and 126 cases
of CTS confirmed by NCS in 8883 person-years. The ACGIH TLV® method predicted both CTS symptoms [HR
between AL and TLV 2.18, 95% confidence interval (95% CI) 1.86–2.56; above TLV 2.07, 95% CI 1.52–2.81]
and CTS confirmed by NCS (HR between AL and TLV 1.93, 95% CI 1.38–2.71; above TLV 1.95, 95% CI
1.27–3.00). About one third of CTS cases were attributable to exposure levels above the AL. Results We analyzed data from 3131 workers [females, N=2032 (65%); mean age at baseline 39.3, standard
deviation (SD) 9.4 years]. We observed 431 incident cases of CTS symptoms in 8000 person-years and 126 cases
of CTS confirmed by NCS in 8883 person-years. Additional material Please note that there is additional material available belonging to
this article on the Scandinavian Journal of Work, Environment & Health
-website. Print ISSN: 0355-3140 Electronic ISSN: 1795-990X Copyright (c) Scandinavian Journal of Work, Environment & Health Exposure assessment proportion of cases attributable to these factors is prob-
ably limited as they are rare (eg, systemic amyloidosis,
sarcoidosis) or only weakly associated with CTS (eg,
diabetes, hypothyroidism) (13–16). During the eighties,
a landmark study posited an association between fast
and forceful manual work and CTS (17): since then,
the issue of occupationally induced CTS has been the
subject of a number of epidemiological studies, although
the majority of them were cross-sectional in design (18). Recently, two large longitudinal studies on CTS and fast
and forceful manual work were published, from Italy
(19) and the USA (20). To date, these are the largest and
most informative studies published on the issue, mainly
because of the design, the number of subjects observed,
the rigorous case definition used, and the detailed assess-
ment of the exposure. Exposure to fast and forceful manual work was assessed
according to the American Conference of Governmental
Industrial Hygienists (ACGIH) threshold limit value
(TLV)® method (22) by a team of trained profession-
als (ergonomists and industrial hygienists) who rated
all suitable jobs in each company. Assessment was
performed at task level, based mainly on observation
(with videotapes whenever possible) and was comple-
mented, where available, by standard production times
and data. Many tasks involved the use of both hands and
the exposure level of each hand was assessed. For each
task, the ergonomists evaluated the exposure level of the
hand-wrist system in terms of hand activity level (HAL)
and normalized peak force (nPF). Then, we linked each
worker to the estimated exposure values based on the
assigned tasks. In presence of job rotation, we assigned
to each worker the average value of HAL calculated over
all performed jobs and the highest PF estimated for the
pool of jobs under consideration. Finally, we combined
HAL and nPF values using the formula nPF/(10-HAL)
and we compared the estimated value against the action
limit (AL) and the TLV proposed by the ACGIH (22). Exposure to hand–arm vibrations (HAV) was assessed
in terms of presence or absence; workers performing
jobs in which any vibratory tools were used for any
work-cycle, independently of total time of usage, were
considered exposed to HAV. Here, we report the last follow-up of the Italian
cohort (the OCTOPUS study), presenting data on indus-
trial/service workers followed-up for up to ten years. Carpal tunnel syndrome and manual work: the OCTOPUS cohort, results of a
ten-year longitudinal study The ACGIH TLV® method predicted both CTS symptoms [HR
between AL and TLV 2.18, 95% confidence interval (95% CI) 1.86–2.56; above TLV 2.07, 95% CI 1.52–2.81]
and CTS confirmed by NCS (HR between AL and TLV 1.93, 95% CI 1.38–2.71; above TLV 1.95, 95% CI
1.27–3.00). About one third of CTS cases were attributable to exposure levels above the AL. Conclusions The ACGIH TLV® method predicted the risk of CTS, but the dose–response was flat above the
AL; a fine-tuning of the proposed thresholds should be considered. Key terms musculoskeletal disorder; Italy; occupational exposure; occupational mononeuropathy; threshold
limit value; upper-limb disorder. Key terms musculoskeletal disorder; Italy; occupational exposure; occupational mononeuropathy; threshold
limit value; upper-limb disorder. Carpal tunnel syndrome (CTS), determined by the com-
pression of the median nerve within the carpal canal at
the wrist, is broadly recognized as the most common
mononeuropathy (1). It may adversely affect the quality
of life of the patient (sleep disruption as the symptoms
– tingling, burning and pain in the first three fingers of
the hand – typically present during the night, and/or
loss of sensitivity and force, which may affect manual
dexterity) (2). CTS incidence in the general population
has been reported to be as high as 3 cases per 1000
person-years, and CTS surgery is very common as well
(1 per 1000 person-years) (3, 4). Surgically-treated CTS was three to seven times more common (depending on
age/gender) among manual compared to non-manual
workers (5). Thus, CTS is a common cause of work
disability and determines high healthcare expenditures
(6, 7). A large amount of literature documented that
CTS is associated with several personal characteristics,
including increasing age, overweight/obesity, female
gender, and musculoskeletal comorbidities (eg, trigger
finger, rotator cuff syndrome, and cervical radiculopa-
thy – double crush syndrome) (8–12). Many clinical
investigations have focused on the role of systemic
diseases in increasing the risk of CTS. However, the 280 Scand J Work Environ Health 2016, vol 42, no 4 Violante et al Violante et al Outcome assessment Each worker underwent a clinical examination by a
trained physician to collect symptoms of CTS and other
personal variables using a structured questionnaire. Dur-
ing the first three follow-up assessments, each worker
who was symptomatic for CTS underwent median
nerve conduction study of both hands, according to a
standard method (21). During the last follow-up assess-
ment (2008–2011), all the subjects underwent nerve
conduction studies (NCS), irrespective of the presence
of symptoms. Physicians performing clinical assess-
ment were unaware of exposure data collected for the
company and NCS were performed without knowledge
of the clinical data of the subject.i Methods The OCTOPUS study has been described in detail
elsewhere (19, 21): here we briefly summarize the main
features of the study population and methods used for
exposure assessment, outcome assessment and statisti-
cal analyses. Exposure assessment In
this setting, representing the largest prospective cohort
study ever published, we aimed at investigating the role
of ergonomic and personal risk factors in the genesis of
CTS, as well as their relative contribution to the burden
of the disease. Scand J Work Environ Health 2016, vol 42, no 4 Figure 1. Flowchart of the study (Panel A) and participation of workers to follow-up assessments (Panel B). The OCTOPUS cohort, Italy, 2000–2011. [ACGIH=American Conference of Government Industrial Hygienists; BMI=body mass index; CTS=carpal tunnel syndrome; TLV=threshold limit value] from the full model, which included all the pre-selected
baseline covariates alongside the variable for the ACGIH
TLV® method; then, we performed a backward dele-
tion, retaining the variables that reached the statistical
significance level of P<0.1 at the likelihood ratio test. After preliminary analysis, gender, age, body mass index
(BMI), and presence of pathologies predisposing to CTS
onset were retained as covariates in multivariate models. Of note, exposure to HAV did not reach the predefined
significance level for the selection of covariates (P<0.1)
for any of the outcome variables. Age, ACGIH TLV®
categories, HAL, and nPF were treated as time-varying
covariates; for each person-year, the exposure was classi-
fied independently. To assess the presence of linear trends
across levels of ordinal variables, we evaluated the Wald
chi-square statistic after fitting regression models includ-
ing a linear term for the covariate of interest.i who reported numbness, tingling, burning, or pain in
one or more of first three fingers of the hand (“possible”
or “classic/probable” symptoms of CTS) were classified
as symptomatic cases. We also performed a sensitivity
analysis based only on subjects who reported classic/
probable symptoms. Experienced electro-diagnostic technicians per-
formed NCS, as described elsewhere (21). The short-
segment (8 cm) sensory conduction velocity of the
median nerve from wrist to palm was classified as
“slowed” if it fell below the lower 99% confidence limit
(43.8 m/s) of the electro-diagnostic reference values
described by Kimura (25). The Institutional Review
Board of the University Hospital of Bologna approved
the study protocol (n. 054/2000/O). Study population The OCTOPUS cohort was established in 2000–2001
(first examination): workers enrolled in the study were
full-time employees of seven industrial (tiles, small
appliance, large appliances, garment and shoes – two
companies – manufacturing) and service (nursery and
early childhood centers) organizations. At each subse-
quent examination (2001–2002, 2002–2003, 2008–2011)
newly hired workers in each company where invited
to participate in the study (open cohort design). Four
companies (tiles, small appliance, large appliances,
and one of the shoes manufacturing) participated to
all follow-up assessments. One of the shoes industries
and the nursery and early childhood centers voluntary
exited the study after the third follow-up assessment. The garment manufactory closed down after the second
follow-up assessment. We used two different case definitions: (i) presence
of CTS symptoms in the 30 days before the interview
(hereinafter referred to as “CTS symptoms”); and (ii)
presence of CTS symptoms and slowing of sensory con-
duction velocity of the median nerve from wrist to palm
(referred to as “CTS confirmed by NCS”). Symptoms
were classified according to the consensus criteria for
the classification of CTS in epidemiologic studies (23). Information was collected with a structured question-
naire including the Katz hand diagram (24); subjects More details about the number of workers enrolled
in the study are reported in figure 1. 281 Scand J Work Environ Health 2016, vol 42, no 4 Longitudinal study of carpal tunnel syndrome and manual work Longitudinal study of carpal tunnel syndrome and manual work Longitudinal study of carpal tunnel syndrome and manual work Longitudinal study of carpal tunnel syndrome and manual work Figure 1. Flowchart of the study (Panel A) and participation of workers to follow-up assessments (Panel B). The OCTOPUS cohort, Italy, 2000–2011. [ACGIH=American Conference of Government Industrial Hygienists; BMI=body mass index; CTS=carpal tunnel syndrome; TLV=threshold limit value] Scand J Work Environ Health 2016, vol 42, no 4 Statistical analysis Population attributable fractions (PAF), defined as the
percentage of cases attributable to the exposure of inter-
est, were estimated after fitting regression models through
maximum likelihood methods (26). To give a perspective
on the role of the studied risk factors in the genesis of
each observed CTS case, we applied the method proposed
by McElduff and colleagues to estimate the proportional
contribution of multiple risk factors to the development
of a disease in an individual (27). This method allows
partitioning the excess risk so that the contribution of each
risk factor plus an additional component for “unknown”
risk factors sum up to 100%. The proportional contribu-
tion may be interpreted as a lower bound value for the
probability of causation (27). Only subjects with complete information on selected
covariates entered the main analyses (list wise deletion). Summary statistics were expressed as numbers (percent-
ages), means [standard deviation (SD)], or medians
[interquartile range (IQR)] as appropriate. The correla-
tion between variables was studied using Spearman’s
rank correlation coefficient (Spearman’s rho). i We fitted Cox proportional hazards regression mod-
els with robust standard errors clustered on the seven
companies to estimate hazard ratios (HR) and relative
95% confidence intervals (95% CI). Covariates to be
included in the multivariable models were selected with
a “significance-test-of-the-covariate” strategy. We started 282 Scand J Work Environ Health 2016, vol 42, no 4 Violante et al All the analyses were performed using Stata 14.1
SE (Stata Corp, College Station, TX, US). All the tests
were two-sided and we defined as statistically significant
a P-value <0.05. definition based on solely symptoms). Supplemental
table A (www.sjweh.fi/data_repository.php) presents
a summary of baseline characteristics by participation
to NCS: we observed minor differences only for the
distribution of gender and ACGIH TLV® categories. When investigating the incidence of CTS symptoms,
we excluded 451 prevalent cases at their baseline and we
analyzed data on 431 incident cases in 8000 person-years
with an overall incidence rate of 5.4 per 100 person-years. Of these, 119 cases (28%) and 3286 person-years (41%)
represented the incremental contribution of the forth
follow-up assessment. When investigating CTS confirmed
by NCS, after the exclusion of 108 prevalent cases, we
observed 126 incident cases in 8883 person-years (over-
all incidence rate, 1.4 per 100 person-years). The fourth
follow-up assessment accounted for 55 cases (44%)
and 3813 person-years (43%). Results Panel A of figure 1 reports the flow diagram of the study
population. Out of the 4734 invited workers, there were
4505 workers eligible for the cohort: of these, 273 (6%)
were non-responders. As shown by Panel B of figure
1, four outcome assessments were performed in this
dynamic cohort. A total of 1101 subjects were lost to
follow-up after their baseline assessment; 693 of them
had participated only to the first cohort assessment, 231 to
the second, and 177 to the third one. The dynamic cohort
includes 860 workers entered after the first follow-up
assessment and 825 subjects were still under follow-up
at the end of the study. The dropout rates due to personal
factors (ie, subjects who quitted the study while their
company was still under follow-up) were: 16.4% after the
first follow-up assessment; 16.5% after the second follow-
up assessment; 20.3% after the third follow-up assess-
ment. Also, 191 workers (4.5%) left the study after the
second follow-up assessment and 967 (22.8%) after the
third one because their companies abandoned the study. Among the 3131 subjects with complete information
at their baseline, the mean age was 37.9 (SD 9.5) years,
the mean BMI was 24.0 (SD 4.0) kg/m2, and median
current job seniority was 7 (IQR 2–15) years. The All subjects symptomatic at their baseline or during
follow-up (N=882) were invited to undergo NCS: 245
(28%) of them refused to undergo the tests (of these,
122 were classified as incident cases according to the Table 1. Incidence rates (per 100 person-years) of CTS symptoms in the studied population by ACGIH TLV® method and personal
characteristics. The OCTOPUS cohort, Italy, 2000–2011. [ACGIH=American Conference of Government Industrial Hygienists; AL=action
limit; CTS=carpal tunnel syndrome; IR=incidence rate; Pyrs=person-years; TLV=threshold limit value; 95% CI=95% confidence intervals]. Statistical analysis Incidence rates (per 100
person-years) of CTS symptoms in the studied population
by ACGIH TLV® method and personal characteristics
are presented in table 1. As expected, we documented
higher rates among females and with increasing age and
BMI. Remarkably, no linear trend across ACGIH TLV®
categories was observed; indeed, subjects in the inter-
mediate category (above AL and below TLV) presented
higher rates compared to those in the upper category
(above the TLV). Incidence rates of CTS confirmed with
NCS are presented in supplemental table B (www.sjweh. fi/data repository.php). Scand J Work Environ Health 2016, vol 42, no 4 Results Biomechanical exposure classified according to ACGIH TLV® method
Below AL
Between AL and TLV
Above TLV
Cases
Pyrs
IR
95% CI
Cases
Pyrs
IR
95% CI
Cases
Pyrs
IR
95%CI
Entire cohort
187
4946.0
3.8
3.3–4.4
107
1080.5
9.9
8.2–12.0
137
1973.5
6.9
5.9–8.2
Sex
Male
55
2692.0
2.0
1.6–2.7
33
402.5
8.2
5.8–11.5
28
822.0
3.4
2.4–4.9
Female
132
2254.0
5.9
4.9–6.9
74
678.0
10.9
8.7–13.7
109
1151.5
9.5
7.8–11.4
Age (years)
Up to 35
41
2194.5
1.9
1.4–2.5
34
483.5
7.8
5.5–10.9
42
959.0
4.4
3.2–5.9
36–45
81
1758.5
4.6
3.7–5.7
42
410.0
10.2
7.6–13.9
47
584.5
8.0
6.0–10.7
46–55
59
925.0
6.4
4.9–8.2
28
224.0
12.5
8.6–18.1
41
409.0
10.0
7.4–13.6
>55
6
68.0
8.8
4.0–19.6
3
8.0
37.5
12.1–116
7
21.0
33.3
15.9–70
Body mass index (kg/m2)
<25
115
3363.5
3.4
2.8–4.1
70
751.5
9.3
7.4–11.8
76
1335.0
5.7
4.5–7.1
25.0–29.9
53
1234.5
4.3
3.3–5.6
26
251.0
10.4
7.1–15.2
48
510.5
9.4
7.1–12.5
≥30.0
19
348.0
5.5
3.5–8.6
11
78.0
14.1
7.8–25.5
13
128.0
10.2
5.9–17.5
Predisposing diseases a
0
163
4717.5
3.5
3.0–4.0
99
1030.5
9.6
7.9–11.7
116
1864.0
6.2
5.2–7.5
≥1
24
228.5
10.5
7.0–15.7
8
50
16.0
8.0–32.0
21
109.5
19.2
12.5–29.4
a This group includes: diabetes, amyloidosis, gout, thyroid disorders, scleroderma, rheumatoid arthritis, systemic lupus erythematosus. Biomechanical exposure classified according to ACGIH TLV® method 283 Scand J Work Environ Health 2016, vol 42, no 4 Longitudinal study of carpal tunnel syndrome and manual work Table 2. Results from Multivariate Proportional Hazards Regression Models including exposure classified according to American Confer-
ence of Government Industrial Hygienists (ACGIH) threshold limit value (TLV)® method alongside selected covariates. The OCTOPUS
cohort, Italy, 2000–2011. [AL=action limit; CTS=carpal tunnel syndrome; HR=hazard ratio; NCS=nerve conduction studies; Ref=reference;
95%CI=95% confidence interval]. Characteristics
CTS symptoms
CTS confirmed by NCS
Univariate analysis
Multivariate model a
Univariate analysis
Multivariate model a
Cases
(N=431)
HR
95% CI
P for
trend
HR
95% CI
P for
trend
Cases
(N=126)
HR
95% CI
P for
trend
HR
95% CI
P for
trend
Sex
Male
116
1.00
Ref. 1.00
Ref. 31
1.00
Ref. 1.00
Ref. Female
315
2.23 1.31–3.80
1.98
1.53–2.56
95
2.15
1.65–2.80
1.91
1.26–2.90
Age (years)
<0.001
<0.001
<0.001
<0.001
Up to 35
117
1.00
Ref. 1.00
Ref. 20
1.00
Ref. 1.00
Ref. Results 36–45
170
1.76 1.46–2.12
1.65
1.42–1.92
51
2.47
1.62–3.77
2.20
1.32–3.67
46–55
128
2.26 1.67–3.07
1.89
1.62–2.21
51
4.90
2.91–8.25
3.85
2.10–7.08
>55
16
3.89 1.71–8.88
3.17
1.86–5.40
4
6.63
3.14–14.0
4.89
2.86–8.37
Body mass index (kg/m2)
<0.001
0.017
<0.001
<0.001
<25
261
1.00
Ref. 1.00
Ref. 60
1.00
Ref. 1.00
Ref. 25.0–29.9
127
1.39 1.13–1.71
1.42
1.19–1.70
45
2.16
1.56–2.99
2.04
1.48–2.81
≥30.0
43
1.60 1.04–2.47
1.47
0.85–2.53
21
3.63
2.05–6.43
3.04
1.48–6.23
Predisposing diseases b
0
378
1.00
Ref. 1.00
Ref. 107
1.00
Ref. 1.00
Ref. ≥1
53
2.47 2.02–3.02
1.65
1.21–2.24
19
2.96
1.63–5.37
1.52
0.82–2.83
ACGIH TLV® categories
<0.001
<0.001
0.005
0.001
Below the AL
187
1.00
Ref. 1.00
Ref. 51
1.00
Ref. 1.00
Ref. Between AL and TLV
107
2.37 1.59–3.54
2.18
1.86–2.56
36
2.24
1.22–4.10
1.93
1.38–2.71
Above TLV
137
2.11 1.35–3.28
2.07
1.52–2.81
39
2.02
1.17–3.49
1.95
1.27–3.00
a Multivariate models included sex, age, body mass index, predisposing diseases and ACGIH categories. b This group includes: diabetes, amyloidosis, gout, thyroid disorders, scleroderma, rheumatoid arthritis, systemic lupus erythematosus. Table 2. Results from Multivariate Proportional Hazards Regression Models including exposure classified according to American Confer-
ence of Government Industrial Hygienists (ACGIH) threshold limit value (TLV)® method alongside selected covariates. The OCTOPUS
cohort, Italy, 2000–2011. [AL=action limit; CTS=carpal tunnel syndrome; HR=hazard ratio; NCS=nerve conduction studies; Ref=reference;
95%CI 95% confidence interval] Multivariate models included sex, age, body mass index, predisposing diseases and ACGIH categories. This group includes: diabetes, amyloidosis, gout, thyroid disorders, scleroderma, rheumatoid arthritis, systemic lupus erythematosus cohort consisted of 2032 (65%) women [mean age 39.3
(SD 9.4) years, mean BMI of 23.4 (SD 4.1) kg/m2, and
median current job seniority of 8 (IQR 2–18) years] and
1099 (35%) men [mean age 35.3 (SD 9.1) years, mean
BMI of 25.0 (SD 3.4) kg/m2, and median current job
seniority of 6 (IQR 2–11) years]. Baseline characteristics
according to follow-up status are presented in supple-
mental table C (www.sjweh.fi/data_repository.php). times higher than those observed for CTS symptoms. Sim-
ilarly, BMI was strongly associated with CTS confirmed
by NCS, but not with CTS symptoms. On the opposite,
estimates for gender and presence of predisposing dis-
eases were similar when investigating CTS symptoms or
CTS confirmed by NCS. Supplemental table D (www. sjweh.fi/data_repository.php) presents the estimates
obtained when considering a more stringent definition
for symptoms (only subjects reporting “classic/probable”
symptoms). Results The change of the case definition determined
only a minor loss of cases [60 (13.9%) for CTS symptoms
and 9 (7.1%) for CTS confirmed by NCS]. All the esti-
mates obtained from this sensitivity analysis were in line
with those obtained when including in the case definition
both “classic/probable” and “possible” symptoms. The distribution of the study population according
to HAL, nPF and gender is presented in supplemental
figure A (www.sjweh.fi/data_repository.php). Overall,
a moderate correlation (Spearman’s rho 0.42) was found
between the values of HAL and nPF at the baseline; the
correlation was stronger among women (Spearman’s rho
0.51) than men (Spearman’s rho 0.28). p
p
y p
Based on the reported multivariate risk estimates
(table 2), we calculated the fractions of cases attributable
in our study population to each risk factor (table 3). For
both case definitions, about one third of the cases could
be attributed to biomechanical exposure levels above
the AL or female gender. Age >35 years contributed to
more than 50% of CTS confirmed by NCS and one third
of CTS symptoms. BMI ≥25 kg/m2 determined one third
of CTS confirmed by NCS, but was implied only in the
genesis of the 12% of CTS symptoms. The presence of
predisposing pathologies was responsible for a limited
proportion of cases. We further estimated for each case
the relative contribution of each risk factor to the occur- Table 2 presents the estimates of the multivariate
Cox regression models selected to study the relation-
ship between ACGIH TLV® categories and risk of CTS. The HR of CTS symptoms and CTS confirmed by NCS
showed a similar pattern in relation to the category of
ACGIH TLV®: compared to subjects exposed below
the AL, both workers with exposure levels between the
AL and the TLV or above the TLV showed a two-fold
increase in risk. The magnitude of the risks associated
with personal characteristics varied depending on the case
definition. Advanced age was the most important predic-
tor for both case definitions, but the HR estimated when
investigating CTS confirmed by NCS were almost two 284 Scand J Work Environ Health 2016, vol 42, no 4 Violante et al tional hazards regression models where nPF and HAL
were introduced separately or jointly. Whereas the HR
for HAL were comparable for both outcomes, nPF was
clearly associated only with the occurrence of CTS
confirmed by NCS. Results Of note, we tested for multiplica-
tive interaction between HAL and nPF, but the product
terms did not reach the statistical significance threshold
(P<0.05) for the inclusion in the final regression models
(P-value of the interaction term in multivariable models:
CTS symptoms, 0.513; CTS confirmed by NCS, 0.428,
data not shown). Table 3. Population attributable fractions (PAF) of CTS. Max-
imum likelihood estimates from the multivariate models pre-
sented in table 2. [ACGIH=American Conference of Government
Industrial Hygienists; AL=action limit; CTS=carpal tunnel syn-
drome; NCS=nerve conduction studies;TLV=threshold limit value;
95%CI=95% confidence interval]. Characteristics
CTS symptoms
CTS confirmed by NCS
PAF %
95% CI
PAF %
95% CI
Female gender
34.5
21.8–45.1
33.7
11.8–50.1
Age >35 years
31.1
23.1–38.3
53.7
29.6–69.5
Body mass index of
≥25 kg/m2
12.0
0.0–18.6
29.9
15.6–41.7
Presence of predisposing
diseases a
4.4
1.4–7.4
4.8
-3.9–12.7
ACGIH TLV® method,
exposure above the AL
30.6
21.8–38.5
28.2
16.3–38.4
a This group includes: diabetes, amyloidosis, gout, thyroid disorders,
scleroderma, rheumatoid arthritis, systemic lupus erythematosus. Discussion That may well be the case with CTS, a disease
in which the relevant period of exposure may be rela-
tively short (as demonstrated by the pregnancy-associ-
ated CTS) (31). Of note, in a supplementary analysis of
the incidence of CTS symptoms in our cohort between
2003 and 2011, we found an incidence rate (IR) of 4.7
per 100 person-years among workers who decreased
their exposure level (ie, ACGIH TLV® category) over
the study period and an IR of 2.7 per 100 person-years
among those who kept a stable level of exposure (data
not shown). This observation seems to support the
hypothesis of a healthy worker survivor effect. Were
this the case, we might have slightly underestimated the
incidence of CTS. Indeed, some workers might have
quit the job during the study period due to CTS; in our
analysis, these subjects would be included among the
lost-to-follow-up. Of note, our cohort included many
workers with relatively long job tenures (≥7 years). This
fact increases the possibility that one of the components factors (28). However, in this highly informative study
conducted among industrial and service workers we
found that no more than one third of the cases could
be attributed to exposure levels above the ACGIH AL. Moreover, biomechanical risk factors captured by the
ACGIH TLV® method seldom were the most impor-
tant contributing factors to CTS cases (although in
many patients it appeared to be the most important one
for the onset of CTS symptoms with negative NCS). On the one hand, our findings suggest that preventive
efforts targeted at reducing workload on upper limbs
may substantially reduce the burden of CTS. On the
other hand, the major role of personal risk factors in the
genesis of CTS hampers the retrospective assessment of
the occupational origin of this disease; indeed, the pro-
portional contribution – that represents a lower bound
estimate for the probability of causation (27) – is often
well below 50% also in a population mainly composed
by blue-collar workers. Compared to our previous report on the first two
years of observation of the cohort (19), this extended
follow-up documented a previously non-observed result,
that is a small reduction in the incidence of CTS with
the highest level of exposure (above the TLV) when
compared to the intermediate level of exposure (between
the AL and the TLV). Discussion With this report on the complete follow-up of the
OCTOPUS cohort, we were able to confirm the main
findings of our previous accounts, ie, that the ACGIH
TLV® metrics correctly predicts that, when the TLV is
exceeded, a sizable increase (roughly two-fold) in the
risk of CTS can be expected. Our result is stable and
robust, being based on a follow-up extended up to ten
years, 8883 total person-years of observation and 126
cases defined according to a state-of-the art clinical
definition of the outcome based on NCS (8000 person/
years and 431 cases if considering the case definition
based on symptoms only). rence of the disease (figure 2). Biomechanical exposure
above the AL established by the ACGIH TLV® method
was the most important contributing factor (54% of
cases) when investigating CTS symptoms; however,
the proportional contribution decreased dramatically
when investigating CTS confirmed by NCS and this
exposure was the main contributing factor only in 6%
of subjects affected by CTS (data not shown). Indeed,
CTS confirmed by NCS was mainly related to age >35
years (main contributor in the 78.6% of the cases) and
BMI ≥25 kg/m2 (11.9% of the cases) (data not shown). Concerning the study of biomechanical exposure
indexes, table 4 presents the estimates from propor- Past investigations on CTS suggested that more that
50% of the cases might be attributable to occupational 285
Figure 2. Proportional contribution to subjects affected by CTS symptoms and subjects affected by CTS confirmed by NCS for selected risk
factors. Estimates derived from the multivariate hazard ratios reported in table 2. [CTS=carpal tunnel syndrome; NCS=nerve conduction studies] Figure 2. Proportional contribution to subjects affected by CTS symptoms and subjects affected by CTS confirmed by NCS for selected risk
factors. Estimates derived from the multivariate hazard ratios reported in table 2. [CTS=carpal tunnel syndrome; NCS=nerve conduction studies] 285 Scand J Work Environ Health 2016, vol 42, no 4 Longitudinal study of carpal tunnel syndrome and manual work on CTS (20). Of note, the relative magnitude of the risk
estimates in both our study and the US one is compa-
rable, giving additional solidity to both results. These
observations might be explained by the well-known
“healthy worker survivor effect”, that is the tendency
for subjects more susceptible to a certain disease to exit
a cohort, especially if the observation period is long
enough compared to the natural history of the disease
(29, 30). 286
Scand J Work Environ Health 2016, vol 42, no 4 ards regression model adjusted by sex, age, body mass index and presence of predisposing pathologies. Discussion 286 286
Scand J Work Environ Health 2016, vol 42, no 4 Violante et al Violante et al ACGIH AL, confirmed by both our and US study, calls
for a fine-tuning of the TLV, which might not be protec-
tive enough for the most susceptible workers (those who
may develop the disease at the lower levels of exposure,
mostly female workers older than 40 years of age) (20). of the healthy worker effect – namely the continuing
employment of healthy individuals – might affect our
estimates (32). Of course, other alternative explanations
still held validity, such as a possible misclassification of
exposure, or an issue with the predictive power of the
ACGIH TLV® method (but this would not explain why
in the previous report, based on the same exposure data,
the effect was not observed), and, of course, a chance
effect should not even be overlooked: all considered,
however, based on our present knowledge, a “healthy
worker survivor effect” seems to be a likely explanation
for the pattern observed. ACGIH AL, confirmed by both our and US study, calls
for a fine-tuning of the TLV, which might not be protec-
tive enough for the most susceptible workers (those who
may develop the disease at the lower levels of exposure,
mostly female workers older than 40 years of age) (20). Lastly, it should be noted that the incidence of CTS
confirmed by NCS reported in our study was consider-
ably higher than in population studies, as the one con-
ducted in Tuscany (3). This can be related to two factors. Firstly, we were actively seeking cases, asking for symp-
tomatic subjects to undergo a NCS, whereas patients
who did not seek medical care were not included in the
passively collected rates based on cases seen by neurolo-
gists in nerve conduction laboratories. Secondly, we did
not study the general population, but a population of
manual workers, where the CTS incidence is >4 times
higher compared to non-manual workers (5). p
Another finding which needs to be mentioned is
that, differently from the US study, nPF did not seem
to correlate with the incidence of CTS better than HAL
(20). Study strengths and limitations Our study is one of the very few prospective validations
of observational methods for the assessment of biome-
chanical exposures at work (35). To our knowledge, the
OCTOPUS cohort is larger than any other previously
published occupational cohort designed to study the
association between CTS and biomechanical risk factors
and as large as the pooled US study (20). The exposure
assessment was performed using a standardized and
fast observational method (22). We analyzed a sample
of representative workers to estimate the HAL and nPF
associated with each task. A drawback of our approach is
the potential for exposure misclassification because we
did not account directly for personal and anthropometric
characteristics that may influence the intensity of bio-
mechanical exposures (eg, postural changes). Also, on
the opposite of the US study, we did not apply objective
methods to measure nPF (20). However, while our expo-
sure assessment method does not ensure the absence
of exposure misclassification, it does represent a real
world setting. In fact, the observational ACGIH TLV®
method can be applied on a large scale because it is fast,
inexpensive and easily implemented. This fact increases
the external validity of our study; our risk stratification
based on ACGIH categories could be replicated in many
industrial settings. A recent overview of systematic reviews and a meta-
analysis of current research concluded that there is high
evidence for an increased risk of CTS in activities requir-
ing a high degree of repetition and forceful exertion while
the evidence for vibration and non-neutral wrist postures
appears moderate and low respectively (33). This study and the pooled US analysis (20) provide
independent validations of the ACGIH TLV® method
by means of the only effective method, that is, a longi-
tudinal cohort study. Some prospective evidence is also
available for the Strain Index method (34). Whereas all
the other methods to assess workload on upper limbs
have been tested, at best, only in cross-sectional stud-
ies, which do not provide a suitable validation strategy
because they are unable to assess causality (35). On
balance, the ACGIH TLV® method for manual work
may be considered the one for which the largest body
of evidence of predictive value is available, although
it seems to need some minor adjustments. Discussion However, by definition, HAL is computed taking
into consideration “exertion frequency and duty cycle
(% of work cycle where force is >5% of maximum)”,
whereas nPF is irrespective of the duration of the exer-
tion in relation to that of the work cycle (22). So, it
may be that in work cycles characterized by a relatively
elevated HAL, but where the duration of the most force-
ful exertion (nPF) is relatively short, HAL may capture
the total “load” received by the hand better than nPF. Another issue to be considered is that the distribution of
exposure in our study seems to be more skewed towards
groups with elevated HAL and relatively lower PF than
the US study, including fewer subjects with relatively
high PF and low HAL. Everything else considered, our
study confirms that hand activity, performed with some
amount of forceful exertions, significantly increases the
risk of the disease. Risk assessment methods based on
the frequency of “any exertion” might overestimate the
risk in work cycles where movements are frequent but
the force used is slight. Discussion Whereas this finding is new for
our cohort, it has already been reported in the US study Table 4. Estimates from eight proportional hazards regression models adopting different metrics for occupational exposure to biomechani-
cal risk factors. The OCTOPUS cohort, Italy, 2000-2011. [ACGIH=American Conference of Industrial Hygienists; AL=action limit; HAL=hand
activity level; HR=hazard ratio; NCS=nerve conduction studies; nPF=normalized peak force; Pyrs=person-years; Ref=reference category;
TLV=threshold limit value; 95% CI=95% confidence interval]. Exposure metric
CTS symptoms
CTS confirmed by NCS
Crude estimates
Adjusted estimates
Crude estimates
Adjusted estimates
Cases
(N=431)
HR
95%CI
P for
trend
HR a
95%CI a
P for
trend
Cases
(N=126)
HR
95%CI
P for
trend
HR a
95%CI a
P for
trend
MODEL A, ACGIH TLV® categories
<0.001
<0.001
0.005
0.001
Below AL
187
1.00
Ref. 1.00
Ref. 51
1.00
Ref. 1.00
Ref. Between AL and TLV
107
2.37 1.59–3.54
2.18 1.88–2.56
36
2.24
1.22–4.10
1.93 1.38–2.71
Above TLV
137
2.11 1.35–3.28
2.07 1.52–2.81
39
2.02
1.17–3.49
1.95 1.27–3.00
MODEL B (HAL)
<0.001
<0.001
0.006
<0.001
1.0–3.0
170
1.00
Ref. 1.00
Ref. 44
1.00
Ref. 1.00
Ref. 3.1–5.0
213
2.03 1.38–3.01
2.10 1.61–2.73
60
2.17
1.43–3.31
2.06 1.61–2.65
5.1–8.5
48
2.41 1.50–3.87
2.05 1.54–2.74
22
2.38
1.15–4.95
2.06 1.37–3.09
MODEL C (nPF)
0.004
0.014
0.001
<0.001
1.0–3.0
69
1.00
Ref. 1.00
Ref. 18
1.00
Ref. 1.00
Ref. 3.1–5.0
293
1.55 1.28–1.89
1.24 0.93–1.66
86
1.93
1.19–3.12
1.68 0.87–3.23
5.1–7.0
69
1.62 1.13–2.32
1.54 1.10–2.16
22
2.64
1.54–4.51
2.62 1.63–4.21
MODEL D
<0.001
<0.001
0.071
0.030
HAL
1.0–3.0
170
1.00
Ref. 1.00
Ref. 44
1.00
Ref. 1.00
Ref. 3.1–5.0
213
2.29 1.54–3.39
2.24 1.80–2.79
60
2.15
1.40–3.31
1.97 1.63–2.38
5.1–8.5
48
2.72 1.56–4.74
2.31 1.80–2.96
22
2.18
0.91–5.25
1.79 1.06–3.03
nPF
0.980
0.625
0.048
0.007
1.0–3.0
69
1.00
Ref. 1.00
Ref. 18
1.00
Ref. 1.00
Ref. 3.1–5.0
293
1.50 1.26–1.78
1.19 0.98–1.44
86
1.76
1.09–2.86
1.60 0.94–2.71
5.1–7.0
69
0.93 0.56–1.52
0.89 0.58–1.38
22
1.53
0.85–2.77
1.70 1.08–2.69
a Estimates from proportional hazards regression model adjusted by sex, age, body mass index and presence of predisposing pathologies. Scand J Work Environ Health 2016, vol 42, no 4 Study strengths and limitations been mainly determined by workers who changed their
employment status, were from factories that closed down
(the underwear factory), or were temporarily absent from
work (eg, due to illness, parental leave, or other employee
benefits). All the aforementioned conditions seem to be
weakly related to CTS: hence, we hypothesize that loss
to follow-up should not substantially bias our results. Another possible limitation of our study is that the assess-
ment of biomechanical risk factors was performed only
at baseline and at the end of the study, or when we were
notified of significant changes in the workplaces followed
for the entire period of the study. However, there were
no changes in production technologies or volumes in the
seven participating enterprises during the study period. Therefore, misclassification of biomechanical expo-
sure due to changes in jobs should be nearly negligible,
although we cannot completely rule out the presence
of residual confounding due to exposure misclassifica-
tion for unknown causes. Since exposure assessment
was not feasible for multi-task jobs, only activities that
included regular or predictable patterns of exertions over
the course of each work shift were assessed. Thus, our
study is not able to provide information for multi-task
manual jobs (eg, maintenance workers, cleaners, jani-
tors). In the OCTOPUS cohort, HAV were registered as a
dichotomous exposure; no measurements of frequency or
acceleration were collected. Hence, we were not able to
study HAV properly as a risk factor for CTS and we only
explored the exposure to HAV as a possible confounder
of the relationship between the ACGIH classification and
the risk of CTS. Concluding remarks Based on the measure of the HAL and nPF, the ACGIH
TLV® method enables the classification of workers in
three categories (below the AL, between the AL and
the TLV, above the TLV). The HAL is based on the
frequency of hand exertions and the duty cycle (distri-
bution of work and recovery periods), while the nPF is
the peak effort exerted by the hand during each regular
work cycle. According to the ACGIH, the TLV should
not be exceeded; also, the AL is considered the level
that triggers general controls, including surveillance. As expected, we found an increased incidence of CTS
for workers exposed above the TLV. However, we also
observed a non-negligible risk for workers exposed
between the AL and the TLV; hence, the current limits
– AL and TLV – might not be sufficiently protective for
some workers. The growing body of evidence suggests
a fine-tuning of the proposed thresholds. Acknowledgments During the ten years of this study, many people contrib-
uted in various way to it: we want to thank all of them,
particularly Patrizia Mussoni MSc, for the contribution
offered. We would also like to thank the workers and
management of the companies who participated in the
study for their generous cooperation and support. Institutional funding was used for this study, with
additional support from the Department of Health of the
Emilia Romagna Region and (for the first three years
of the study) from the National Institute for Workers
Compensation (INAIL). We estimated the attributable fractions of CTS. When interpreting these figures, it should be kept in
mind that the values for ACGIH TLV® categories might
be underestimated due to exposure misclassification –
that is more likely to attenuate rather than inflate our
estimates. Also, we did not measure some personal (eg,
cardiovascular risk factors) and occupational (eg, psy-
chosocial risk factors) characteristics that were recently
hypothesized as risk factors for CTS (36, 37). On the
one hand, the absence of information on these variables
could substantially affect the calculation of attributable
fractions only under the unlikely hypothesis that these
factors were strong confounders of the association
between CTS risk and biomechanical exposures. On the
other hand, the proportional contributions determined
by each risk factor might be overestimated. Indeed,
the proportional contributions are rescaled so that their
total always sums up to 100%. Hence, the inclusion
of any further risk factors in the multivariable models
determines a decrease of the proportional contributions
estimated for the other covariates. None of the authors have any financial interest
related to this study. FSV, SM and RB have acted as
expert witnesses for courts or private parties in litiga-
tions involving musculoskeletal disorders, including
carpal tunnel syndrome. Study strengths and limitations Indeed, the
excess risk of CTS in workers exposed just above the Another strength of our study is that information on
possible confounders was collected prospectively by
direct interviews. Regarding participation in the study,
on the one hand, the initial response proportion was
satisfactory (>94%), on the other hand, loss to follow-up
was not negligible and attrition bias could represent a
concern. A comparison of baseline characteristics in the
OCTOPUS cohort highlighted that women tended to be
lost to follow-up more than men. However, we should
consider that loss of subjects to follow-up could have 287 Scand J Work Environ Health 2016, vol 42, no 4 Longitudinal study of carpal tunnel syndrome and manual work been mainly determined by workers who changed their
employment status, were from factories that closed down
(the underwear factory), or were temporarily absent from
work (eg, due to illness, parental leave, or other employee
benefits). All the aforementioned conditions seem to be
weakly related to CTS: hence, we hypothesize that loss
to follow-up should not substantially bias our results. Another possible limitation of our study is that the assess-
ment of biomechanical risk factors was performed only
at baseline and at the end of the study, or when we were
notified of significant changes in the workplaces followed
for the entire period of the study. However, there were
no changes in production technologies or volumes in the
seven participating enterprises during the study period. Therefore, misclassification of biomechanical expo-
sure due to changes in jobs should be nearly negligible,
although we cannot completely rule out the presence
of residual confounding due to exposure misclassifica-
tion for unknown causes. Since exposure assessment
was not feasible for multi-task jobs, only activities that
included regular or predictable patterns of exertions over
the course of each work shift were assessed. Thus, our
study is not able to provide information for multi-task
manual jobs (eg, maintenance workers, cleaners, jani-
tors). In the OCTOPUS cohort, HAV were registered as a
dichotomous exposure; no measurements of frequency or
acceleration were collected. Hence, we were not able to
study HAV properly as a risk factor for CTS and we only
explored the exposure to HAV as a possible confounder
of the relationship between the ACGIH classification and
the risk of CTS. 3.
Mondelli M, Giannini F, Giacchi M. Carpal tunnel syndrome References 1. Atroshi I, Gummesson C, Johnsson R, Ornstein E, Ranstam
J, Rosen I. Prevalence of carpal tunnel syndrome in a
general population. JAMA. 1999;282:153–8. http://dx.doi. org/10.1001/jama.282.2.153. 2. Giannini F, Cioni R, Mondelli M, Padua R, Gregori B, D’Amico
P, et al. A new clinical scale of carpal tunnel syndrome:
validation of the measurement and clinical-neurophysiological
assessment. Clin Neurophysiol. 2002;113:71–7. http://dx.doi. org/10.1016/S1388-2457(01)00704-0. 3. Mondelli M, Giannini F, Giacchi M. Carpal tunnel syndrome 288 Scand J Work Environ Health 2016, vol 42, no 4 Violante et al trauma disorders in industry. Br J Ind Med. 1986;43(11):779–
84. http://dx.doi.org/10.1136/oem.43.11.779. incidence in a general population. Neurology. 2002;58(2):289-
94. http://dx.doi.org/10.1212/WNL.58.2.289. 18. van Rijn RM, Huisstede BM, Koes BW, Burdorf A. Associations between work-related factors and the carpal
tunnel syndrome--a systematic review. Scand J Work Environ
Health. 2009;35(1):19–36. http://dx.doi.org/10.5271/
sjweh.1306. 4. Mattioli S, Baldasseroni A, Curti S, Cooke RM, Bena A, de
Giacomi G, et al. Incidence rates of in-hospital carpal tunnel
syndrome in the general population and possible associations
with marital status. BMC Public Health. 2008;8:374. http://
dx.doi.org/10.1186/1471-2458-8-374. 5. Mattioli S, Baldasseroni A, Curti S, Cooke RM, Mandes
A, Zanardi F, et al. Incidence rates of surgically treated
idiopathic carpal tunnel syndrome in blue- and white-collar
workers and housewives in Tuscany, Italy. Occup Environ
Med. 2009;66(5):299–304. http://dx.doi.org/10.1136/
oem.2008.040212. 19. Bonfiglioli R, Mattioli S, Armstrong TJ, Graziosi F, Marinelli
F, Farioli A, et al. Validation of the ACGIH TLV for hand
activity level in the OCTOPUS cohort: a two-year longitudinal
study of carpal tunnel syndrome. Scand J Work Environ
Health. 2013;39(2):155–63. http://dx.doi.org/10.5271/
sjweh.3312. 6. Feuerstein M, Miller VL, Burrell LM, Berger R. Occupational
upper extremity disorders in the federal workforce: prevalence,
health care expenditures, and patterns of work disability. J Occup Environ Med. 1998;40:546–55. http://dx.doi. org/10.1097/00043764-199806000-00007. 20. Kapellusch JM, Gerr FE, Malloy EJ, Garg A, Harris-Adamson
C, Bao SS, et al. Exposure-response relationships for the
ACGIH threshold limit value for hand-activity level: results
from a pooled data study of carpal tunnel syndrome. Scand
J Work Environ Health. 2014;40(6):610–20. http://dx.doi. org/10.5271/sjweh.3456. 7. Foley M, Silverstein B, Polissar N. The economic burden of
carpal tunnel syndrome: long-term earnings of CTS claimants
in Washington State. Am J Ind Med. 2007;50:155–72. http://
dx.doi.org/10.1002/ajim.20430. 21. Violante FS, Armstrong TJ, Fiorentini C, Graziosi F, Risi A,
Venturi S, et al. Carpal tunnel syndrome and manual work: a
longitudinal study. J Occup Environ Med. 2007;49(11):1189–
96. http://dx.doi.org/10.1097/JOM.0b013e3181594873. 8. References Mattioli S, Baldasseroni A, Bovenzi M, Curti S, Cooke
RM, Campo G, et al. Risk factors for operated carpal tunnel
syndrome: a multicenter population-based case-control
study. BMC Public Health. 2009;9:343. http://dx.doi. org/10.1186/1471-2458-9-343. 22. American Conference of Governmental Industrial Hygienists
(ACGIH). TLVs and BEIs. Cincinnati: American Conference
of Governmental Industrial Hygienists; 2000. 23. Rempel D, Evanoff B, Amadio PC, de Krom M, Franklin G,
Franzblau A, et al. Consensus criteria for the classification
of carpal tunnel syndrome in epidemiologic studies. Am J
Public Health. 1998;88:1447–51. http://dx.doi.org/10.2105/
AJPH.88.10.1447. 9. Shiri R, Pourmemari MH, Falah-Hassani K, Viikari-Juntura
E. The effect of excess body mass on the risk of carpal
tunnel syndrome: a meta-analysis of 58 studies. Obes Rev. 2015;16(12):1094–104. http://dx.doi.org/10.1111/obr.12324. 10. Rottgers SA, Lewis D, Wollstein RA. Concomitant
presentation of carpal tunnel syndrome and trigger finger. J
Brachial Plex Peripher Nerve Inj. 2009;4:13. 24. Katz JN, Stirrat CR, Larson MG, Fossel AH, Eaton HM,
Liang MH. A self-administered hand symptom diagram for the
diagnosis and epidemiologic study of carpal tunnel syndrome. J Rheumatol. 1990;17:1495–8. 11. Titchener AG, White JJ, Hinchliffe SR, Tambe AA, Hubbard
RB, Clark DI. Comorbidities in rotator cuff disease: a case-
control study. J Shoulder Elbow Surg. 2014;23(9):1282–8. http://dx.doi.org/10.1016/j.jse.2013.12.019. 25. Kimura J. The carpal tunnel syndrome. Localization of
conduction abnormalities within the distal segment of the
median nerve. Brain. 1979;102:619–35. http://dx.doi. org/10.1093/brain/102.3.619. 12. Molinari WJ 3rd, Elfar JC. The double crush syndrome. J Hand
Surg Am. 2013;38(4):799–80. http://dx.doi.org/10.1016/j. jhsa.2012.12.038. 26. Newson RB. Attributable and unattributable risks and fractions
and other scenario comparisons. Stata J. 2013;13:672–98. 13. Niemer GW, Bolster MB, Buxbaum L, Judson MA. Carpal
tunnel syndrome in sarcoidosis. Sarcoidosis Vasc Diffuse Lung
Dis. 2001;18(3):296–300. 27. McElduff P, Attia J, Ewald B, Cockburn J, Heller R. Estimating
the contribution of individual risk factors to disease in a
person with more than one risk factor. J Clin Epidemiol. 2002;55(6):588–92. http://dx.doi.org/10.1016/S0895-
4356(02)00388-8. 14. M’bappé P, Grateau G. Osteo-articular manifestations of
amyloidosis. Best Pract Res Clin Rheumatol. 2012;26(4):459–
75. http://dx.doi.org/10.1016/j.berh.2012.07.003. 28. Rossignol M, Stock S, Patry L, Armstrong B. Carpal tunnel
syndrome: what is attributable to work? The Montreal study. Occup Environ Med. 1997;54(7):519–23. http://dx.doi. org/10.1136/oem.54.7.519. 15. Pourmemari MH, Shiri R. Diabetes as a risk factor for carpal
tunnel syndrome: a systematic review and meta-analysis. Diabet Med. 2016;33(1):10–6. http://dx.doi.org/10.1111/
dme.12855. 29. Arrighi HM, Hertz-Picciotto I. The evolving concept of the
healthy worker survivor effect. Epidemiology. 1994;5(2):189–
96. http://dx.doi.org/10.1097/00001648-199403000-00009. 16. Shiri R. References Hypothyroidism and carpal tunnel syndrome: a meta-
analysis. Muscle Nerve. 2014;50(6):879–83. http://dx.doi. org/10.1002/mus.24453. 30. Buckley JP, Keil AP, McGrath LJ, Edwards JK. Evolving
methods for inference in the presence of healthy worker 17. Silverstein BA, Fine LJ, Armstrong TJ. Hand wrist cumulative 289 Scand J Work Environ Health 2016, vol 42, no 4 Longitudinal study of carpal tunnel syndrome and manual work survivor bias. Epidemiology. 2015;26(2):204–12. http://
dx.doi.org/10.1097/EDE.0000000000000217. 31. Padua L, Di Pasquale A, Pazzaglia C, Liotta GA, Librante A,
Mondelli M. Systematic review of pregnancy-related carpal
tunnel syndrome. Muscle Nerve. 2010;42(5):697–702. http://
dx.doi.org/10.1002/mus.21910. 32. Howe GR, Chiarelli AM, Lindsay JP. Components and
modifiers of the healthy worker effect: evidence from
three occupational cohorts and implications for industrial
compensation. Am J Epidemiol. 1988;128(6):1364–75. 33. Kozak A, Schedlbauer G, Wirth T, Euler U, Westermann C,
Nienhaus A. Association between work-related biomechanical
risk factors and the occurrence of carpal tunnel syndrome: an
overview of systematic reviews and a meta-analysis of current
research. BMC Musculoskelet Disord. 2015;16:231. http://
dx.doi.org/10.1186/s12891-015-0685-0. 34. Moore JS, Garg A. The Strain Index: a proposed method
to analyze jobs for risk of distal upper extremity disorders. Am Ind Hyg Assoc J. 1995;56(5):443–5. http://dx.doi. org/10.1080/15428119591016863. 35. Takala EP, Pehkonen I, Forsman M, Hansson GA,
Mathiassen SE, Neumann WP, et al. Systematic evaluation of
observational methods assessing biomechanical exposures at
work. Scand J Work Environ Health. 2010;36(1):3–24. http://
dx.doi.org/10.5271/sjweh.2876. 36. Rigouin P, Ha C, Bodin J, Le Manac’h AP, Descatha A,
Goldberg M, et al. Organizational and psychosocial risk factors
for carpal tunnel syndrome: a cross-sectional study of French
workers. Int Arch Occup Environ Health. 2014;87(2):147–54. http://dx.doi.org/10.1007/s00420-013-0846-0. 37. Hegmann KT, Thiese MS, Kapellusch J, Merryweather AS,
Bao S, Silverstein B, et al. Association Between Cardiovascular
Risk Factors and Carpal Tunnel Syndrome in Pooled
Occupational Cohorts. J Occup Environ Med. 2016;58(1):87–
93. http://dx.doi.org/10.1097/JOM.0000000000000573. Received for publication: 13 January 2016 survivor bias. Epidemiology. 2015;26(2):204–12. http://
dx.doi.org/10.1097/EDE.0000000000000217. 35. Takala EP, Pehkonen I, Forsman M, Hansson GA,
Mathiassen SE, Neumann WP, et al. Systematic evaluation of
observational methods assessing biomechanical exposures at
work. Scand J Work Environ Health. 2010;36(1):3–24. http://
dx.doi.org/10.5271/sjweh.2876. 31. Padua L, Di Pasquale A, Pazzaglia C, Liotta GA, Librante A,
Mondelli M. Systematic review of pregnancy-related carpal
tunnel syndrome. Muscle Nerve. 2010;42(5):697–702. http://
dx.doi.org/10.1002/mus.21910. 36. Rigouin P, Ha C, Bodin J, Le Manac’h AP, Descatha A,
Goldberg M, et al. Scand J Work Environ Health 2016, vol 42, no 4 References Organizational and psychosocial risk factors
for carpal tunnel syndrome: a cross-sectional study of French
workers. Int Arch Occup Environ Health. 2014;87(2):147–54. http://dx.doi.org/10.1007/s00420-013-0846-0. 32. Howe GR, Chiarelli AM, Lindsay JP. Components and
modifiers of the healthy worker effect: evidence from
three occupational cohorts and implications for industrial
compensation. Am J Epidemiol. 1988;128(6):1364–75. 37. Hegmann KT, Thiese MS, Kapellusch J, Merryweather AS,
Bao S, Silverstein B, et al. Association Between Cardiovascular
Risk Factors and Carpal Tunnel Syndrome in Pooled
Occupational Cohorts. J Occup Environ Med. 2016;58(1):87–
93. http://dx.doi.org/10.1097/JOM.0000000000000573. 33. Kozak A, Schedlbauer G, Wirth T, Euler U, Westermann C,
Nienhaus A. Association between work-related biomechanical
risk factors and the occurrence of carpal tunnel syndrome: an
overview of systematic reviews and a meta-analysis of current
research. BMC Musculoskelet Disord. 2015;16:231. http://
dx.doi.org/10.1186/s12891-015-0685-0. 34. Moore JS, Garg A. The Strain Index: a proposed method
to analyze jobs for risk of distal upper extremity disorders. Am Ind Hyg Assoc J. 1995;56(5):443–5. http://dx.doi. org/10.1080/15428119591016863. Received for publication: 13 January 2016 290 Scand J Work Environ Health 2016, vol 42, no 4
|
https://openalex.org/W4362531319
|
https://figshare.com/articles/journal_contribution/Supplementary_Figure_S1_from_Selective_Estrogen_Receptor_Modulators_and_Pharmacogenomic_Variation_in_ZNF423_Regulation_of_BRCA1_Expression_Individualized_Breast_Cancer_Prevention/22529574/1/files/39992688.pdf
|
unk
| null |
Supplementary Figure S1 from Selective Estrogen Receptor Modulators and Pharmacogenomic Variation in ZNF423 Regulation of BRCA1 Expression: Individualized Breast Cancer Prevention
| null | 2,023
|
cc-by
| 7
|
Fig. S1 Fig. S1 Fig. S1 1
|
https://openalex.org/W1830943933
|
https://europepmc.org/articles/pmc4636531?pdf=render
|
English
| null |
A systematic literature review of the quality of evidence for injury and rehabilitation interventions in humanitarian crises
|
International journal of public health
| 2,015
|
cc-by
| 5,606
|
Abstract Introduction
Humanitarian crises continue to pose a sig-
nificant threat to health; the United Nations estimates that
144 million people are directly affected by conflict or
environmental disasters. During most humanitarian crises,
surgical and rehabilitative interventions remain a priority. Objectives
This review assessed the quality of evidence
that informs injury and physical rehabilitation interventions
in humanitarian crises. Keywords
Injury Rehabilitation Global surgery
Humanitarianism Disasters Conflict Methods
Peer-reviewed and grey literature sources were
assessed in a systematic manner. Selected papers were
evaluated using quality criteria based on a modified version
of the STROBE protocol. Results
46 papers met the inclusion criteria. 63 % of the
papers referred to situations of armed conflict, of which the
Yugoslav Wars were the most studied crisis context. 59 % A systematic literature review of the quality of evidence for injury
and rehabilitation interventions in humanitarian crises James Smith
. Bayard Roberts . Abigail Knight . Richard Gosselin . James Smith
. Bayard Roberts . Abigail Knight . Richard Gosselin . Karl Blanchet Received: 10 March 2015 / Revised: 3 July 2015 / Accepted: 8 July 2015 / Published online: 23 August 2015
The Author(s) 2015. This article is published with open access at Springerlink.com of the studies were published since the year 2000. How-
ever, only two studies were considered of a high quality. Conclusions
While there is now a greater emphasis on
research in this sector, the volume of evidence remains
inadequate given the growing number of humanitarian
programmes worldwide. Further research is needed to
ensure a greater breadth and depth of understanding of the
most appropriate interventions in different settings. REVIEW
A systematic literature review of the quality of evidence for injury
and rehabilitation interventions in humanitarian crises
J
S
ith
B
d R b
t
Abi
il K i ht
Ri h
d G
li REVIEW REVIEW Int J Public Health (2015) 60:865–872
DOI 10.1007/s00038-015-0723-6 Int J Public Health (2015) 60:865–872
DOI 10.1007/s00038-015-0723-6 Introduction Humanitarian crises continue to pose a significant threat to
health. In 2012, the United Nations identified 144 million
people directly affected by conflict or environmental dis-
asters (OCHA 2013). During the acute phase of most
humanitarian crises, the provision of surgical support
remains a priority (Sphere Project 2011). A surge in the
number of traumatic injuries in the acute phase can over-
whelm pre-existing health services; for example, over a
10-week period following the 2010 Haiti earthquake,
Me´decins sans Frontie`res/Doctors without Borders (MSF)
alone performed more than 4000 surgical procedures (Chu
et al. 2011). At the same time, there is often a need to
supplement routine surgical activities in the wake of
widespread infrastructural damage and disruption to the
local medical human resource pool. Rehabilitation inter-
ventions play an equally important role as efforts are made
to support patients during their longer term recovery. This review is part of the special issue ‘‘Driving the Best Science to
Meet Global Health Challenges’’ edited on the occasion of the 9th
European Congress on Tropical Medicine and International Health
2015. Electronic supplementary material
The online version of this
article (doi:10.1007/s00038-015-0723-6) contains supplementary
material, which is available to authorized users. J. Smith (&) A. Knight K. Blanchet
Public Health in Humanitarian Crises Group, London School of
Hygiene and Tropical Medicine, London, UK
e-mail: james.dominic.smith@gmail.com J. Smith (&) A. Knight K. Blanchet
Public Health in Humanitarian Crises Group, London School of
Hygiene and Tropical Medicine, London, UK
e-mail: james.dominic.smith@gmail.com B. Roberts
ECOHOST-The Centre for Health and Social Change, The
London School of Hygiene and Tropical Medicine, London, UK
R. Gosselin
Department of Orthopaedic Surgery, University of California,
San Francisco, USA B. Roberts
ECOHOST-The Centre for Health and Social Change, The
London School of Hygiene and Tropical Medicine, London, UK B. Roberts
ECOHOST-The Centre for Health and Social Change, The
London School of Hygiene and Tropical Medicine, London, UK In recent years, increased scrutiny of the humanitarian
sector has encouraged a drive towards professionalism and
accountability, and has prompted humanitarian agencies to R. Gosselin
Department of Orthopaedic Surgery, University of California,
San Francisco, USA 12 3 866 J. Smith et al. better demonstrate the impact and effectiveness of their
programmes (Bradt 2009; Bantavala and Zwi 2000). Introduction Despite the fact that the treatment of injuries and the
provision of rehabilitative programmes represent a key
component of the health response during most humanitar-
ian crises, the evidence base for these interventions is not
well understood. inclusion if they documented that an intervention had taken
place, and that subsequently either primary or secondary
health outcomes had been measured. Any studies that
documented outputs, but that did not draw an association
between outputs and outcomes were excluded. Data sources In an effort to better inform policy makers, donors, and
other humanitarian stakeholders, the Wellcome Trust and
the UK. Department for International Development (DfID)
launched the Research for Health in Humanitarian Crises
(R2HC) initiative in 2013. A systematic review was com-
missioned by R2HC to examine the quality and quantity of
evidence for a range of contextual factors and the following
health topics: communicable disease control; mental health
and psychosocial support; sexual and reproductive health
and gender-based violence; nutrition; water, sanitation and
hygiene; non-communicable diseases; and injury and
physical rehabilitation (Blanchet et al. 2013). Both peer-reviewed and grey literature sources were eval-
uated. Peer-reviewed
databases
included:
Embase,
Medline, PsycInfo, International Bibliography of the Social
Sciences (IBSS), and Global Health. The grey literature
sources were chosen following consultation with specialists
in the field of injury and rehabilitation, and included:
SourceInfo, the International Disability and Development
Consortium (IDDC), Leonard Cheshire Disability (LCD),
ELDIS, European Disability Forum (EDF), Christoffel
Blinded Mission (CBM), the Center for International
Rehabilitation Research Information and Exchange (CIR-
RIE), Research for Development (R4D), MSF (Me´decins
Sans Frontie`res/Doctors Without Borders) France and
Belgium, the Active Learning Network for Accountability
and Performance (ALNAP), the World Health Organisa-
tion Library Database (WHOLIS), the Centre for Research
on the Epidemiology of Disasters (CRED), and the Inter-
national Society of Physical and Rehabilitation Medicine
(ISPRM). Inclusion and exclusion criteria Studies were selected or excluded based on the seven
categories listed in Table 1. For the purpose of this review,
we were concerned with health interventions in low- and
middle-income countries only, as crises in these countries
often present unique challenges that are not reproducible in
high-income contexts. Similarly, interventions led by mil-
itary contingents deployed from high-income countries for
the treatment of injured combatants were not included in
this review. While acknowledging that military medicine
has advanced our understanding of the treatment of con-
flict-related injuries, the resources and facilities available
to the military invariably create a unique environment that
is unrepresentative of the broader crisis context. Methods Staff at the London School of Hygiene and Tropical
Medicine (LSHTM) performed a systematic review of the
available evidence for injury and physical rehabilitation
interventions in crisis contexts. This review offers a thor-
ough assessment of the quantity and quality of published
evidence that informs humanitarian health programming in
this field. The search structure was prepared with the support of
experienced librarians based at LSHTM and consisted of:
(1) terms related to humanitarian crises/early recovery; and
(2) terms related to public health interventions; and (3)
terms related to low- and middle-income countries; and (4)
terms related to injury and physical rehabilitation (see
Electronic Supplemental Material). The reference lists for
each of the selected articles were also reviewed in full to
identify other relevant papers. Similarly, other reviewers
participating in the R2HC-commissioned review were
encouraged to recommend additional papers that were
better suited to an alternative health topic (e.g. crush-re-
lated injuries captured during the non-communicable
diseases search, which were more appropriately listed as
injury and physical rehabilitation interventions). Inclusion and exclusion criteria Paper selection and data extraction Papers were selected as part of a five-stage process, with
one reviewer assessing the papers at each stage. The pro-
cess was as follows: (1) the electronic database searches
were
performed
and
amalgamated;
the
results
were
imported into EndNote X6 reference software, and dupli-
cate entries were removed; (2) papers were reviewed by
title and abstract; (2a) manuscripts were reviewed in the
event of ambiguity regarding the justification for inclusion This review sought to investigate outcomes attributed to
interventions performed in acute and prolonged crises, the
early recovery or stabilisation phases, or studies that
examined a link between pre-emptive interventions and
their effect on health outcomes following the onset of a
crisis. For this reason, interventions in stable contexts were
excluded. Studies were
only
considered eligible
for 123 123 A systematic literature review of the quality of evidence for injury and rehabilitation… 867 Table 1 Inclusion and exclusion criteria
Category
Included
Excluded
Populations of interest
Populations affected by humanitarian crises and
receiving humanitarian assistance (including
refugees and internally displaced persons), in
low- and middle-income countries (based
upon World Bank country classification of
2012 (World Bank 2015)
Studies related to health interventions in high-
income countries; studies pertaining to
military operations involving combatants from
high-income countries
Humanitarian crises
Studies that occurred during the acute, chronic,
early recovery, or stabilisation phases of
humanitarian crises including those that
measured the impact of preparedness and
resilience on public health outcomes during a
humanitarian crisis
Studies that occurred before a humanitarian
crisis (i.e. focused on preparedness or
resilience measures), or that measured an
outcome or intervention of interest in a post-
crisis context
Intervention type
Public health interventions in which the outcome
was measured before and after the
intervention, or an intervention was studied
against another intervention or control group
Studies with no specific health intervention (i.e. studies examining only health needs,
prevalence, health risk factors, and
coordination)
Health outcomes and outputs
of interest
Primary outcomes (e.g. morbidity, mortality,
vaccination status), secondary outcomes (e.g. attendance at health clinics, adherence to
treatment)
Primary outputs (e.g. number of operations
performed, number of surgical kits distributed,
etc.)
Study design
Primary quantitative studies including:
randomised and non-randomised controlled
trials, longitudinal, cross-sectional, and
economic studies
Qualitative studies (i.e. focused on processes and
the perception of interventions); quantitative
studies that did not measure a change in health
outcomes; review papers
Intervention/publication date
January 1, 1980–April 30, 2013. Paper selection and data extraction Studies published before 1980
Publication language
English, French
Any other language
y
q
y
j
y Studies related to health interventions in high-
income countries; studies pertaining to
military operations involving combatants from
high-income countries Qualitative studies (i.e. focused on processes and
the perception of interventions); quantitative
studies that did not measure a change in health
outcomes; review papers Primary quantitative studies including:
randomised and non-randomised controlled
trials, longitudinal, cross-sectional, and
economic studies Studies published before 1980 January 1, 1980–April 30, 2013. the inclusion and exclusion criteria; (4) the reference lists
of selected papers were reviewed (‘references of refer-
ences’)
and
additional
papers
captured
during
the
concurrent health topic reviews were also assessed; (5) a
final list of eligible papers was assembled, and data
extraction and a quality assessment were performed in full
(see Fig. 1). For quality assurance, a second reviewer
corroborated study selection, data extraction, and study
quality assessment. Stage 1: peer
reviewed literature
search (N=4798)
Stage 2a:
title & abstract review
of peer-reviewed
literature (N=4596)
Stage 2b: peer
reviewed literature
(N=20)
4575 excluded
(non-topic)
Stage 5: peer reviewed
and grey literature (N=46)
202 duplicates
excluded
Stage 3: grey
literature
(N=1)
Stage 4: ‘references of
references’ and other health
topics (NCDs = 2; health
systems = 23) (N=25)
Fig. 1 Screening process for the selection of papers Stage 1: peer
reviewed literature
search (N=4798) 4575 excluded
(non-topic) Once selected, data from each of the papers were
inputted into a Microsoft Excel database. The data captured
included: study characteristics (i.e. author, year, study
country, crisis setting); the study population (e.g. refugee,
internally displaced, or general population); the nature of
the humanitarian crisis (armed conflict or environmental
disaster); the health outcome(s) assessed; the intervention
evaluated; and the study methodology (e.g. study design). Search results 4798 studies were identified following a search of the peer-
reviewed literature published between 1980 and 2013. A
final total of 46 studies met the criteria for inclusion in this
review (see Fig. 1). One paper was chosen from the grey
literature search, while a further 24 papers were included
following the non-communicable disease and health sys-
tems searches conducted during the commissioned, multi-
topic evidence review. 4798 studies were identified following a search of the peer-
reviewed literature published between 1980 and 2013. A
final total of 46 studies met the criteria for inclusion in this
review (see Fig. 1). One paper was chosen from the grey
literature search, while a further 24 papers were included
following the non-communicable disease and health sys-
tems searches conducted during the commissioned, multi-
topic evidence review. Crisis context The majority of studies described programmes imple-
mented during the acute phase of a humanitarian crisis. A
total of eight studies assessed health outcomes during
either the early recovery phase (Ebrahimzadeh and Rajabi
2007; Li et al. 2012; Motamedi et al. 1999; Roy et al. 2005), or the stabilisation phase (Tajsic and Husum 2008;
Xiao et al. 2011; Zhang et al. 2012, 2013) (see Table 2). No papers were identified that examined the relationship
between preparedness and health outcomes. p p
63 % of the studies documented health outcomes in sit-
uations of armed conflict, while the remaining 37 %
assessed interventions implemented following an environ-
mental disaster. The Yugoslav Wars of 1991–1999 were
the most studied crisis setting, followed by the Sichuan
Earthquake that devastated Wenchuan County, China in
May 2008. A further three studies were published follow-
ing each of the following crises: the Iran–Iraq war of
1980–1988 (Ebrahimzadeh and Rajabi 2007; Amirjamshidi
et al. 2003; Gousheh 1995), the Soviet War in Afghanistan
of 1979–1989 (Gosselin et al. 1993; Rautio and Paavolai-
nen 1987; Strada et al. 1993), and the Iraq War of
2003–2011 (Fakri et al. 2012; Leininger et al. 2006; Zan-
gana 2007). short, emergency orthopaedic programmes following the
Haitian Earthquake in 2010, against a non-governmental
organisation’s (NGO’s) established elective missions in
neighbouring Dominican Republic (Gosselin et al. 2011). A single multi-region study examined the effect of traction
versus external fixation for patients with high-velocity
missile injuries treated at International Committee of the
Red Cross hospitals in northern Kenya and Afghanistan
(Rowley 1996). Asia represents the most studied continent, of which the
majority of studies originated from China. Eastern Europe
and the Middle East were the source of 14 and 12 papers,
respectively, while Africa and Latin America were partic-
ularly understudied geographical regions. A multi-country
study in the Caribbean evaluated the cost-effectiveness of Study quality Study quality was assessed using criteria distilled from an
adapted version of the Strengthening the Reporting of
Observational Studies in Epidemiology (STROBE) proto-
col (see Electronic Supplemental Material) (von Elm et al. 2007). The score range for the protocol was 0–8, with
scores of 0–3 rated as low quality, 4–6 as moderate quality, Fig. 1 Screening process for the selection of papers or exclusion; (2b) studies were removed based on one or
more of the exclusion criteria (see Table 1); (3) the grey
literature sources were explored and again assessed against 12 3 3 868 J. Smith et al. Table 2 Crisis context, population type, and study methodology
Study characteristics
%
n
Geographical region
Asia
34.8
16
Eastern Europe
30.4
14
Middle East
26.1
12
Africa
4.3
2
Caribbean/Latin America
2.2
1
Multi-region
2.2
1
Crisis context
Yugoslav wars (1991–1999)
30.4
14
Sichuan earthquake, China (2008)
21.7
10
Iran–Iraq war (1980–1988)
6.5
3
Iraq war (2003–2011)
6.5
3
Soviet war in Afghanistan (1979–1989)
6.5
3
Other
28.3
13
Crisis type
Armed conflict
63.0
29
Environmental disaster
37.0
17
Population type
General population
97.8
45
Refugee
2.2
1
Crisis location
Urban
8.7
4
Rural
19.6
9
Mixed
71.7
33
Crisis phase
Acute crisis
82.6
38
Early recovery
8.7
4
Stabilisation
8.7
4
Study type
Cross-sectional
67.4
31
Longitudinal
21.7
10
Non-random trial
8.7
4
Economic
2.2
1 and 7–8 as high quality. Studies were further categorised
based on whether or not they reported a measure of sta-
tistical association. Those papers that quoted a statistical
measure were graded A, while those papers that described
the relationship between an intervention and a health out-
come, but that did not quote a statistical measure were
graded B. Results Search results Trends in publication quantity and quality Trends in publication quantity and quality Both the quantity and the quality of papers have increased
over the course of the last 33 years. Of the 46 studies,
58.7 % (n = 27) were published between the year 2000
and 2015. 79 % (n = 15) of the higher quality studies were
published in the same time period (see Fig. 2). 0
0.5
1
1.5
2
2.5
3
3.5
4
1985
1990
1995
2000
2005
2010
2015
LOW
MODERATE
HIGH
Fig. 2 Number of studies published by year (1980–2013), disaggre-
gated by quality Study type The majority of papers published adhered to a cross-sec-
tional study design (n = 31), followed by a much smaller
proportion of uncontrolled longitudinal studies (n = 10). 123 123 A systematic literature review of the quality of evidence for injury and rehabilitation… 869 Four studies utilised a non-randomised trial methodology:
a joint Cambodian Ministry of Health and MSF study
evaluated health outcomes following primary repair or
colostomy for 102 war injured patients with penetrating
intraperitoneal colon injuries (Moreels et al. 1994); one
study by collaborating clinician-researchers at the Tehran
University of Medical Sciences and Yale University School
of Medicine examined the impact of a standardised rehy-
dration protocol against existing hydration guidelines on
the number of cases of acute renal failure, mortality, and
the rate of fasciotomy among patients treated at three inner
city hospitals following an earthquake in northwestern Iran
in June 1990 (Nadjafiet al. 1997). A further two non-
random trials, led by the same author, were published
following the Sichuan Earthquake in 2008. The first study
evaluated the functional health outcomes of 390 patients
who had suffered fractures and subsequently received
early, late, or no institutional rehabilitation (Zhang et al. 2012). The second study, published the following year,
evaluated the physical functioning of patients who had
received either early or late institutional and community
rehabilitation against a control group that received neither
institutional nor community rehabilitation (Zhang et al. 2013). Measured against the modified STROBE criteria, only 2
papers were considered of a high quality (Zhang et al. 2012, 2013). A further 17 papers were of a moderate
quality, while the remaining 27 papers were deemed of a
low quality. None of the papers met the full quality criteria,
as sample size calculations were consistently absent. Seventeen of the 46 papers evaluated health outcomes
and quoted some form of significance test (category A). The remaining 29 articles described health outcomes fol-
lowing some form of surgical, medical, or rehabilitative
intervention, but did not quote a statistical association
(category B). Health outcomes and interventions Orthopaedic injuries (n = 14), of which the repair of
fractures featured prominently, were the most studied
health outcome. Following orthopaedic outcomes, multi-
ple or non-specific injuries featured frequently (n = 9), as
did the medical and/or surgical response to crush injuries
or renal failure (n = 7). Craniofacial injuries, and the
repair of abdominal and thoracic injuries, were each the
subject of five papers. A further three studies examined
nerve or spinal cord injuries (Gousheh 1995; Li et al. 2012; Splavski et al. 1996), and three studies assessed
revascularisation techniques or the repair of major blood
vessels (Gosselin et al. 1993; Lovric et al. 1994; Roostar
1995). A single economic study compared the cost-effective-
ness of the emergency relief operations of a small non-
governmental organisation in Haiti and the Dominican
Republic following the Haitian Earthquake of January
2010, against the organisation’s elective programmes in
neighbouring Dominican Republic and Nicaragua in a
similar time period (Gosselin et al. 2011). Twenty-one studies described a range of non-specific
surgical interventions. Seven papers looked at surgical
external and internal fixation techniques in particular. This
type of operation was the focal point of published research
more frequently than any other complex surgical technique. Seven papers described different forms of renal therapy,
and/or fasciotomy. A further four papers looked at health
outcomes following limb amputation specifically (Ebra-
himzadeh and Rajabi 2007; Fakri et al. 2012; Gosselin
et al. 1993; Roostar 1995), while only four studies, all of
which were carried out in China, evaluated different forms
of rehabilitation (Li et al. 2012; Xiao et al. 2011; Zhang
et al. 2012, 2013). Three papers examined pre-hospital care
and triage (Bazardzanovic´ et al. 1998; Jevtic´ et al. 1996;
Roy et al. 2005). Discussion This systematic review yielded 46 papers that assessed
injury and physical rehabilitation interventions in human-
itarian crises. Given that the review covered a 33-year
catchment period, and in light of the heavy financial and
human resource investment in emergency humanitarian
operations during that period, these findings suggest that Fig. 2 Number of studies published by year (1980–2013), disaggre-
gated by quality 12 3 870 J. Smith et al. hindered by the unpredictable and rapidly changing nature
of any given crisis context, recent calls for ‘off-the-shelf’
studies with full prior ethical approval may now help
humanitarians to better integrate research alongside their
existing programmes (Gerdin et al. 2014). operational research remains the exception, rather than the
norm. From the available evidence, it is clear that the injury
and physical rehabilitation sector is characterised by a
strong focus on surgical and medical care; the rehabilitative
needs of patients are markedly understudied. This is
indicative of a preserved tendency toward short-term pro-
gramming, with minimal or no follow-up in the post-acute
phase of many humanitarian crises. These findings are not
unique to injury and physical rehabilitation programmes; a
lack of evidence for health interventions remains a cross-
sectorial problem (Blanchet et al. 2013; Clarke et al. 2014). From the available evidence, it is clear that the injury
and physical rehabilitation sector is characterised by a
strong focus on surgical and medical care; the rehabilitative
needs of patients are markedly understudied. This is
indicative of a preserved tendency toward short-term pro-
gramming, with minimal or no follow-up in the post-acute
phase of many humanitarian crises. These findings are not
unique to injury and physical rehabilitation programmes; a
lack of evidence for health interventions remains a cross-
sectorial problem (Blanchet et al. 2013; Clarke et al. 2014). The quality of studies remains highly variable. While
there is arguably a trend towards an increased quantity and
quality of research in recent years, many studies remain
subject to methodological flaws; enrolment of a compar-
ison group, adjustment for potential confounding factors,
and justification of the study sample size were repeatedly
absent from study methodologies. Such omissions are
understandable given the rapidly developing nature of
many humanitarian crises, and the reactive approach of
many relief agencies. With this in mind, the available
studies are representative of an opportunistic approach
towards health research in humanitarian crises in recent
decades. Limitations A number of limitations have affected this review. Fore-
most is the fact that the review looks specifically at
research in humanitarian contexts. This is not to say that
research conducted in stable settings does not carry value. While delivery mechanisms and variation in health needs
are necessary considerations when comparing research
generated in crisis and non-crisis settings, the well-studied
benefit of certain interventions (e.g. fixation of fractured
limbs) should not be overlooked. The quality of studies remains highly variable. While
there is arguably a trend towards an increased quantity and
quality of research in recent years, many studies remain
subject to methodological flaws; enrolment of a compar-
ison group, adjustment for potential confounding factors,
and justification of the study sample size were repeatedly
absent from study methodologies. Such omissions are
understandable given the rapidly developing nature of
many humanitarian crises, and the reactive approach of
many relief agencies. With this in mind, the available
studies are representative of an opportunistic approach
towards health research in humanitarian crises in recent
decades. Only English and French publications were selected for
inclusion in this review. Given that a number of studies
have emerged from China and the Middle East, it is pos-
sible that papers published in Mandarin and Arabic in
national and regional journals have been overlooked. Similarly, as we did not capture Spanish or Portuguese
publications, or search the LILACS database, our findings
related to Latin America and the Caribbean should be
viewed with caution. Evidence gathering in the humanitarian sector remains a
relatively new phenomenon for a number of reasons. Humanitarian programmes, particularly emergency surgi-
cal missions, during much of the twentieth century were
short term and reactionary, with little or no prior planning
or preparation beyond the need to provide immediate,
lifesaving assistance. Insufficient population data in many
crisis contexts also make it difficult for humanitarian
agencies to identify target populations, and to situate
research projects within a broader understanding of popu-
lation health in any given context. References NadjafiI, Atef MR, Broumand B et al (1997) Suggested guidelines for
the treatment of acute renal failure in earthquake victims. Ren
Fail 19(5):655–664 Amirjamshidi A, Abbassioun K, Rahmat H (2003) Minimal debride-
ment or simple wound closure as the only surgical treatment in
war victims with low-velocity penetrating head injuries. Indica-
tions and management protocol based upon more than 8 years
follow-up of 99 cases from Iran–Iraq conflict. Surg Neurol
60(2):105–111 OCHA (2013) World humanitarian data and trends 2013. Policy
Development and Studies Branch (PDSB), UN Office for the
Coordination of Humanitarian Affairs, New York ODI (2009) Humanitarian diagnostics: the use of information and
analysis in crisis response decisions. Paper prepared for FAO. Overseas Development Institute, London Bantavala N, Zwi AB (2000) Public health and humanitarian
interventions: developing the evidence base. BMJ 22:101–105 Bazardzanovic´ M, Brkic´ H, Korkut D et al (1998) Craniocerebral
injuries in combat soldiers treated at the Sapna war hospital,
Bosnia and Herzegovina. Croat Med J 39(4):446–449 R2HC (2015) Research projects [online]. Available from: www.elrha. org/r2hc/projects/. Accessed 2 Jul 2015 Rautio J, Paavolainen P (1987) Delayed treatment of complicated
fractures in war wounded. Injury. 18(4):238–240 Blanchet K, Sistenich V, Ramesh A et al (2013) An evidence review
on health interventions in humanitarian crises. London School of
Hygiene and Tropical Medicine, London Roostar L (1995) Treatment plan used for vascular injuries in the
Afghanistan war. Cardiovasc Surg 3(1):42–45 Bradt D (2009) Evidence-based decision-making in humanitarian
assistance. Overseas Development Institute, London Rowley D (1996) The management of war wounds involving bone. J Bone Jt Surg 78(5):706–709 Chu K, Stokes C, Trelles M, et al (2011) Improving effective surgical
delivery in humanitarian disasters: lessons from Haiti. PLoS
Med 8(4):e1001025 Roy N, Shah H, Patel V et al (2005) Surgical and psychosocial
outcomes in the rural injured—a follow-up study of the 2001
earthquake victims. Injury. 36(8):927–934 Clarke M, Allen C, Archer F et al (2014) What evidence is available
and what is required, in humanitarian assistance?. International
Initiative for Impact Evaluation, London Sphere Project (2011) The sphere handbook: humanitarian charter and
minimum standards in humanitarian response. Practical Action
Publishing, Rugby Darcy J, Stoubaugh H, Walker P et al (2013) The use of evidence in
humanitarian decision making. ACAPS operational learning
paper. Feinstein
International
Center,
Tufts
University,
Somerville Splavski B, Vrankovic´ D, Saric´ G et al (1996) Early management of
war missile spine and spinal cord injuries: experience with 21
cases. Conclusion J Hand Surg. 20(3):S68–S76 Jevtic´ M, Petrovic´ M, Ignjatovic´ D et al (1996) Treatment of wounded
in the combat zone. J Trauma Inj Infect Crit Care 40(3
supp):s173–s176 Funding
The Wellcome Trust and the UK. Department for Inter-
national Development (DfID) funded a multi-topic evidence review
as a preliminary to the Research for Health in Humanitarian Crises
Initiative. This review featured in a condensed format alongside a
number of other health topic reviews. The study funders had no role in
the design of this study, or the production of the final review. Leininger BE, Rasmussen TE, Smith DL et al (2006) Experience with
wound VAC and delayed primary closure of contaminated soft
tissue
injuries
in
Iraq. J
Trauma
Inj
Infect
Crit
Care
61(5):1207–1211 Li Y, Reinhardt JD, Gosney JE et al (2012) Evaluation of functional
outcomes of physical rehabilitation and medical complications in
spinal cord injury victims of the Sichuan earthquake. J Rehabil
Med 44:534–540 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. Lovric Z, Wertheimer B, Candrlic K et al (1994) War injuries of
major extremity vessels. J Trauma-Injury Infect Crit Care
36(2):248–251 Moreels R, Pont M, Ean S et al (1994) Wartime colon injuries:
primary repair or colostomy? J R Soc Med 87(5):265–267 Motamedi MH, Hashemi HM, Shams MG (1999) Rehabilitation of
war-injured patients with implants: analysis of 442 implants
placed during a 6-year period. J Oral Maxillofacc Surg
57(8):907–915 Conclusion This review is the first of its kind to examine the quantity
and quality of evidence for injury and physical rehabilita-
tion interventions in humanitarian crises. While the
evidence base has increased in recent years, inadequate
attention has been paid to research in humanitarian settings
as the number of humanitarian actors, and the budget allo-
cated to humanitarian operations, continues to grow. The trade-off between the need to act quickly and the
need to act effectively presents a unique challenge for
humanitarians. Humanitarian action can only benefit from
the improved application of rigorously tested and context-
appropriate research that identifies not only what works,
but why. It is important not only to improve the quality of
available evidence, but also to bridge the gap between the
academic and operational communities. This will require a
long-term vision, an iterative research process that is firmly
embedded within new and existing systems for monitoring
and evaluation, and a continuous dialogue between multi-
ple stakeholders invested in the humanitarian endeavour. A promising drive towards population and donor
accountability in humanitarian action, and the overarching
moral obligation to provide the most effective and appro-
priate interventions in different crisis settings, has re-
centred evidence-based care as an important programmatic
objective. Recent studies suggest that decision-making in
the humanitarian sector has been driven by organisational
strategic priorities, established practice, and inter-agency
relationships (ODI 2009; Darcy et al. 2013). The pursuit of
evidence-based decision-making challenges this embedded
behaviour, and encourages humanitarian agencies to reflect
on the available evidence during the design and imple-
mentation of health programmes in crisis settings. Acknowledgments
We would like to thank the Wellcome Trust and
the UK. Department for International Development for funding the
original evidence review, which featured a review of injury and
physical rehabilitation interventions alongside the review of a number
of other health topics and contextual factors. We would also like to Following the launch of the R2HC programme in 2013,
a number of studies have been funded in direct response to
gaps identified by the health topic reviews (R2HC 2015). In
light of the fact that research in crisis settings is often 123 123 A systematic literature review of the quality of evidence for injury and rehabilitation… 871 thank Enhancing Learning and Research in Humanitarian Assistance
(ELHRA) for its managerial oversight of the original review. Gousheh J (1995) The treatment of war injuries of the brachial plexus. Zhang X, Reinhardt JD, Gosney JE et al (2013) The NHV
rehabilitation services program improves long-term physical
functioning in survivors of the 2008 Sichuan earthquake: a
longitudinal quasi experiment. PLoS One 8(1):e53995 Zangana AM (2007) Penetrating liver war injury: a report on 676
cases, after Baghdad invasion and Iraqi civilian war April 2003.
Adv Med Dent Sci 1(1):10–14 Zhang X, Hu XR, Reinhardt JD et al (2012) Functional outcomes and
health-related quality of life in fracture victims 27 months after
the Sichuan earthquake. J Rehabil Med 44(3):206–209 References Injury 27(10):699–702 Strada G, Raad L, Belloni G et al (1993) Large bowel perforations in
war surgery: one-stage treatment in a field hospital. Int J
Colorectal Dis 8(4):213–216 Ebrahimzadeh MH, Rajabi MT (2007) Long-term outcomes of
patients undergoing war-related amputations of the foot and
ankle. J Foot Ankle Surg 46(6):429–433 Tajsic NB, Husum H (2008) Reconstructive surgery including free
flap transfers can be performed in low-resource settings:
experiences from a wartime scenario. J Trauma Inj Infect Crit
Care. 65(6):1463–1467 Fakri RM, Al Ani AMK, Rose AMC et al (2012) Reconstruction of
nonunion
tibial
fractures
in
war-wounded
Iraqi
civilians,
2006–2008:
better
late
than
never. J
Orthop
Trauma
26(7):e76–e82 von Elm E, Altman DG, Egger M et al (2007) The strengthening the
reporting of observational studies in epidemiology (strobe)
statement: guidelines for reporting observational studies. Lancet. 370(9596):1453–1457 Gerdin M, Clarke M, Allan C et al (2014) Optimal evidence in
difficult settings: improving health interventions and decision
making in disasters. PLoS Med. 11(4):e1001632 Gosselin RA, Siegberg CJ, Coupland R et al (1993) Outcome of
arterial repairs in 23 consecutive patients at the ICRC-Peshawar
hospital for war wounded. J Trauma Inj Infect Crit Care
34(3):373–376 World Bank (2015) Country and lending groups [online]. Available
from:
http://data.worldbank.org/about/country-and-lending-
groups. Accessed 15 Jun 2015 Xiao M, Li J, Zhang X et al (2011) Factors affecting functional
outcome of Sichuan-earthquake survivors with tibial shaft
fractures: a follow-up study. J Rehabil Med 43(6):515–520 Gosselin RA, Gialamas G, Atkin DM (2011) Comparing the cost-
effectiveness of short orthopedic missions in elective and relief
situations in developing countries. World J Surg 35(5):951–955 12 3 872 J. Smith et al. 123 123
|
https://openalex.org/W3153501660
|
https://bmcpublichealth.biomedcentral.com/counter/pdf/10.1186/s12889-020-09522-7
|
English
| null |
Multicausal analysis on psychosocial and lifestyle factors among patients undergoing assisted reproductive therapy – with special regard to self-reported and objective measures of pre-treatment habitual physical activity
|
BMC public health
| 2,021
|
cc-by
| 11,599
|
Multicausal analysis on psychosocial and
lifestyle factors among patients undergoing
assisted reproductive therapy – with special
regard to self-reported and objective
measures of pre-treatment habitual
physical activity Multicausal analysis on psychosocial and
lifestyle factors among patients undergoing
assisted reproductive therapy – with special
regard to self-reported and objective
measures of pre-treatment habitual
physical activity Viktória Prémusz1,2*
, Alexandra Makai1
, Beatrix Perjés1
, Orsolya Máté1
, Márta Hock1
, Pongrác Ács1
,
Miklós Koppán3
, József Bódis2,3
, Ákos Várnagy2,3†
and Kinga Lampek1† Viktória Prémusz1,2*
, Alexandra Makai1
, Beatrix Perjés1
, Orsolya Máté1
, Márta Hock1
, Pongrác Ács1
,
Miklós Koppán3
, József Bódis2,3
, Ákos Várnagy2,3†
and Kinga Lampek1† Viktória Prémusz1,2*
, Alexandra Makai1
, Beatrix Perjés1
, Orsolya Máté1
, Márta Hock1
Miklós Koppán3
, József Bódis2,3
, Ákos Várnagy2,3†
and Kinga Lampek1† Abstract Background: National, regional and global trends in prevalence of infertility indicate its public health importance,
however it effects various life dimensions of individuals and couples as well. Lifestyle habits may counteract with
these factors. The aim of the study was the multicausal analysis of psychosocial and lifestyle factors undergoing
assisted reproductive therapy (ART) with special regard to pre-treatment habitual physical activity (PA). Methods: In a cross-sectional, observational cohort study on ART patients (N = 60, age 34.6 ± 5.2 years, BMI 24.2 ±
4.9 kg/m2) with follow up on outcome measures a detailed description was given on PA patterns (ActriGraph GT3X,
GPAQ-H) and on general and infertility related distress (BDI-13, FPI). Results: Respondents reported normal mood state (BDI-13) but moderately high infertility-related distress (FPI) in
Social- and very high distress in Sexual Concern. It was revealed that time spent with recreational PA (RPA) could
counteract with infertility-related distress (Social Concern R = -0.378, p = 0.013; Relationship Concern R = -0.365, p = 0.019). In the presence of clinical pregnancy GPAQ-H RPA MET was significantly higher (p = 0.048), in the non-pregnant group
cumulative values and work-related PA were higher. Correlations could be found between RPA time and the number of
oocytes (R = 0.315, p = 0.045), matured oocytes (R = 0.339, p = 0.030) and embryos (R = 0.294, p = 0.062) by women who
reached at least 150 min RPA (GPAQ-H). Multivariate linear regression revealed that the number of oocytes was positively
influenced by the GPAQ-H recreation MET (R2 = 0.367; F = 10.994, p = 0.004; B = 0.005, p = 0.004, B Constant = 4.604). (Continued on next page) * Correspondence: premusz.viktoria@pte.hu * Correspondence: premusz.viktoria@pte.hu p
p
p
†Ákos Várnagy and Kinga Lampek contributed equally to this work. 1Faculty of Health Sciences, University of Pécs, Vorosmarty u. 4, Pécs 7621,
Hungary p
p
p
†Ákos Várnagy and Kinga Lampek contributed equally to this work. 1Faculty of Health Sciences, University of Pécs, Vorosmarty u. 4, Pécs 7621,
Hungary g y
2MTA-PTE Human Reproduction Scientific Research Group, University of
Édesanyák u. 17, Pécs H-7624, Hungary
Full list of author information is available at the end of the article 2MTA-PTE Human Reproduction Scientific Research Group, Univers
Édesanyák u. 17, Pécs H-7624, Hungary
Full list of author information is available at the end of the article RESEARCH Open Access Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480
https://doi.org/10.1186/s12889-020-09522-7 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480
https://doi.org/10.1186/s12889-020-09522-7 Study design and sample
Study design A cross-sectional, observational cohort study was con-
ducted
with
consecutive
sampling
at
the
Assisted
Reproduction Unit, Department of Obstetrics and Gy-
naecology, University of Pécs, Hungary. All female pa-
tients with both female and male factors of infertility
who were indicated for fertility treatment (IVF/ICSI)
were consecutively invited to participate in the study. Participants were recruited according to the date of the
fertility consultation. Inclusion criterions were BMI ≥18
kg/m2 and ≤38 kg/m2, 18 to 40 years of age, having
undergone not more than three unsuccessful cycles and
no significant health risk relevant to the ART procedure
and outcome (metabolic and vascular diseases including
diabetes mellitus, metabolic syndrome, fatty liver dis-
eases and atherosclerosis, severe endometriosis (stage III
or IV) and/or adenomyosis). Participants were not diag-
nosed with any mental disorders and had no significant
physical or mobility impairments. Infertility could specifically affect various life dimensions
of individuals or couples, such as depression, anxiety, social
isolation, sexual dysfunction, psychological and social dis-
tress (PSD), and poorer marital adjustment [12–15]. It has
been hypothesized that depression and anxiety may sub-
stantially have a negative effect on female reproduction or
assisted reproductive treatment (ART) due to hormonal,
neuroendocrine, or immunologic functioning and lead to
poor outcomes [16–20]. The relationship between psycho-
social stress in relation to the success of IVF/ICSI is still
under discussion [21, 22]. It was demonstrated that pre-
treatment levels of perceived anxiety and depression were
significantly related to treatment outcome in in vitro
fertilization (IVF) [23, 24]. For this reason, it is necessary to
explore the fertility related PSD. Data collection was carried out during the routine
examination on the 3rd day of the unstimulated cycles. 62 women participated in the study between December
2018 and June 2019, which means 82.66% response rate. Self-administered questionnaires were given to partici-
pants, who filled them at home in a conventional paper-
pencil form. Questionnaires were returned on the 21st
day of the unstimulated cycles. 2 participants were ex-
cluded due to high rate of missing questionnaire data. The selection of the study population including patient
recruitment, exclusion criteria, and refusals are pre-
sented on the flowchart (Fig. 1.) Benefits of regular physical activity to maintain phys-
ical, mental and social health are not called into question
[25–31]. Positive effects on women’s health are proven
in different contexts [32–35] underlining its importance
in pregnancy as well [36–39]. Background National, regional and global trends in prevalence of infertil-
ity indicate its public health importance [1–7]. Multiple defi-
nitions on infertility are used in parallel from demographic
[1, 8] or epidemiological point of view [9], or it could even
be considered as a disability [10]. The current study is based
on the clinical definition which describes infertility as “a dis-
ease of the reproductive system defined by the failure to
achieve a clinical pregnancy after 12 months or more of
regular unprotected sexual intercourse” following the Inter-
national Classification of Diseases (ICD-11) [11]. (Continued from previous page) (
p
p g )
Regarding the number of embryos (R2 = 0.757, F = 17.692, p < 0.001, B Constant = 1.342) positive relationship was found
with GPAQ-H RPA MET (B = 0.004, p < 0.001) and negative with BMI (B = -0.167, p = 0.038). It was disclosed (R2 = 0.958, F =
408.479, p < 0.001) that higher Very Vigorous Activity (ActiGraph) was accompanied with higher hCG (B = 63.703, p ≤
0.001). However, time spent with moderate PA (GPAQ-H) (B = 0.002, SE = 0.001, Wald = 3.944, p = 0.047, OR = 1.002) was
significantly associated with live births. Conclusions: Amount of PA alone did not have a positive effect on outcome of ART. Type and intensity seemed to be
more significant. Existing differences in response to infertility due to recreational PA suggest the importance of the
development of a specific intervention. The robust overestimation of PA in self-reports highlights the need to improve
physical literacy of women undergoing ART. Keywords: Physical activity, GPAQ, Accelerometer, Assisted reproduction, IVF, Outcomes, Psychosocial factors, Lifestyle,
Pregnancy rate, Live birth Study design and sample
Study design Depending on intensity or
duration, certain studies disagree on health effects of ex-
ercise or even PA in relation to ART [40–43]. These PA
studies primarily focus on outcomes of ART and less on
the PSD aspects during the course of the therapy. © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 14 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 General characteristics of the sample The major socio-demographic and clinical characteristics
of the study population have been presented in Table 1. The data of 60 female patients in reproductive age (34.6 ±
5.2 years), with mostly normal weight (70.0%, BMI 18.5– Therefore, the aim of the current research was to de-
scribe PA and PSD patterns and their combined effects
on the course and outcomes of the treatment in ART
patients using a multi-causal model. Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 3 of 14 Fig. 1 Flow chart of the study population selection including patient recruitment, exclusion criteria, and refusals Fig. 1 Flow chart of the study population selection including patient recruitment, exclusion criteria, and refusals Complete clinical data was available regarding 45 IVF/
ICSI patients. 24.9 kg/m2) were analysed in the study. They were sam-
pled from a larger proportion with higher educational de-
gree
(58.6%)
and
with
satisfactory
economic
status
(96.6%). 95.0% of them worked and 75.0% had urban resi-
dence. Each participant was either married or lived with a
partner, and in average the duration of the partnership
was 8 years (7.6 ± 3.8) with an around 5-years-long (59.0 ±
38.4 month) child-wish. We found various cases of infertil-
ity and types of treatments. However, these primarily nul-
liparous
women
(84.4%)
typically
received
IVF/ICSI
(82.3%)
with
mostly
non-male
indication
(75.6%). 76.7% of the participants rated their physical health par-
ticularly good or excellent. In general, they self-reported a
health-conscious lifestyle regarding diet, tobacco use and
PA, and quality of sleep was satisfactory (86.2%) as well. Lifestyle change was also examined, but we cannot report
relevant changes back to 5 years ago, since the beginning
of ART, or in the current month. 50.0% of the participants
claimed to be physically active, one tenth of them exer-
cised 4–7 times per week (Table 1). Page 4 of 14 Prémusz et al. General characteristics of the sample BMC Public Health 2021, 21(Suppl 1):1480 Assessment of distress The cultural adaptation, efficient translation, and val-
idation of the Hungarian version were composed by our
research group [54–56]. A total of 120 healthy adults
(age 21.53 ± 1.75 years, 46.66% male) were included in
the validity and reliability study, their last 7 days PA by
GPAQ-H was compared with IPAQ-Hungarian Long
version and Actigraph GT3X accelerometer data at the
University of Pécs. Although, the validity GPAQ-H was
fair to moderate (MVPA R = 0.290, p = 0.001) but it was
acceptable, as by similar European studies. Conse-
quently, it could be claimed that the GPAQ-H proved to
be a valid and reliable questionnaire to measure the
healthy Hungarian general population’s physical activity
patterns [57]. Beck Depression Inventory (BDI-13) was applied for
reporting respondents’ mental health status [23, 44–48]. The questionnaire represents how the subjects were feel-
ing the week before. Each question has a set of four pos-
sible responses, ranging in intensity (0–3). A total score
is computed reflecting the outcome index of depression. The validated Hungarian version of the short-form of
the inventory with 13 items was completed by the re-
spondents [49, 50]. To examine infertility-related distress with an infertil-
ity specific scale, the Fertility Problem Inventory (FPI)
was included. FPI is a 46-item questionnaire developed
to measure the level of infertility-related stress [51]. The
scale consists of five subscales identifying the following
domains: social concerns, sexual concerns, relationship
concerns, rejection of childfree lifestyle and need for
parenthood. Higher score indicates that the individual is
experiencing more psychological stress than the average
individual seen for infertility (85–98% reflects moder-
ately high stress, and above 98% reflects very high level
of stress). The former Hungarian version [52] was accur-
ately redefined, results will be published separately. For calculation of energy expenditure (MET) of PA
following the guidelines of both of the questionnaires
MPA by 4 MET, VPA by 8 MET and walking by 3.3
MET should be multiplied [53, 56, 58, 59]. We decided
to apply the values of the updated “Physical Activity
Guidelines for Americans”, which calculates with 3, 6
and 2.5 METs respectively [60]. Assessment of PA Triaxial ActiGraph GT3X+ accelerometers (ActiGraph,
Pensacola, FL) were used to collect data on PA with
standard device initialization (sample rate of 30 Hz, 60 s
epochs and normal filter option). To describe PA and exercise habits, participants self-
reported on the type and frequency of exercise in a PA
diary and reported all kinds of physical activity in
GPAQ-H. These self-reports were compared with ob-
jective measures collected by the Triaxial ActiGraph
GT3X+ accelerometers. Participants were instructed to wear the accelerometer
on the right hip (near the iliac crest) for a week, from
the time they woke up in the morning until they
retreated at the end of the day, except for the duration
of any water-based activities, such as swimming or bath-
ing. The Actigraph GT3X + device measures the strength
of the movement in three spatial directions, as well as
their duration. The device converts acceleration into a
quantifiable and measurable digital signal. It allows us to
accurately assess daily activities and classifies it into
categories. General characteristics of the sample BMC Public Health 2021, 21(Suppl 1):1480 Table 1 General characteristics of women undergoing ART (N = 60)
Socio-demographic Data (N = 60)
Health Status and Lifestyle (N = 60)
Reproduction (N = 60)
Medical records by IVF/ICSI (N = 45)
Age (years)
BMI (kg/m2)
Indication (Self-report)
N (%)
Indication
Mean (SD)
34.6 (±5.2)
Mean (SD)
24.2 (±4.9)
Female
26 (43.3)
Poor semen quality
11 (24.4%)
Education
Underweight (< 18.5)
5 (8.3%)
Male
4 (6.7)
Fallopian tube
11 (24.4%)
2003Low
6 (10.0%)
Normal weight (18.5–24.9)
37 (61.7%)
Dual
14 (23.3)
Endometriosis
7 (15.6%)
Intermediate
18 (30.0%)
Overweight (25–29.9)
7 (11.7%)
Undefined
13 (21.7)
Other female
4 (8.9%)
High
36 (60.0%)
Obesity (> 30)
11 (18.3%)
Diagnosis in progress
3 (5.0)
Unexplained
12 (25.6%)
Marital status (N = 44)
Self-Rated Physical Health
Type of ART Treatment
Procedures
Married
47 (78.3%)
Poor
0
IVF/ICSI
50 (82.3)
Cycle 1
11 (24.4%)
Partner
13 (21.7%)
Fair
1 (1.7%)
IUI
1 (2.1)
Cycle 2
16 (35.6%)
Place of residence
Neither good nor bad
13 (21.7%)
OI
2 (4.2)
Cycle 3
12 (26.7)
County seat
18 (30.0%)
Good
37 (61.7%)
HSG
2 (4.2)
Cycle 4
6 (13.3%)
City
27 (45.0%)
Excellent
9 (15.0%)
Examination in progress
5 (31.1)
Serum oestradiol - pmol/l
1692 ± 2073
Village
15 (25.0%)
Healthy Diet
Child-wish (months)
Mean ± SD
Progesterone - nmol/l
30.42 ± 21.29
Income
Pay attention
56 (93.3%)
Mean (SD)
59.0 ± 38.4
FSHa - IU
2493 ± 2925
Low
2 (3.3%)
Not really / No attention
4 (6.7%)
Relationship (years)
Gonadotropina - IU
0.86 ± 0.19
Medium
34 (56.6%)
Tobacco Use
Mean (SD)
7.6 ± 3.8
No. of oocytes
7.87 ± 4.96
High
24 (40.0%)
Occasional
2 (3.3%)
Gravidity (N = 45)
N (%)
No. of matured oocytesb
5.44 ± 3.93
Non-Smoker
58 (96.7%)
Nulligravid
25 (55.6)
No. of Grade 1 embryos
3.31 ± 2.98
Exercise
Multigravid
20 (44.4)
No of transferred embryos
1.46 ± 0.84
Regularly 4–7 days weekly
6 (10.0%)
Parity(N = 45)
hCG on day 12 - IU
364.9 ± 912.3
Regularly1–3 days weekly
24 (40.0%)
Nulliparous
38 (84.4)
Chemical pregnancies
22 (48.89%)
Not
30 (50.0)
Multiparous
7 (15.6)
Clinical pregnancies
13 (28.9%)
ART Assisted Reproductive Therapy, BMI Body Mass Index, HSG Hysterosalpingogram, ICSI Intracytoplasmic Sperm Injection, IUI Intrauterine Insemination, IVF In-Vitro Fertilization, OI Ovulation Induction, a: Total dose
administrated b Metaphase II) Page 5 of 14 Prémusz et al. Procedure and measurements
Assessment scales activities can be classified and walking activities should
be also distinguished. Our study indicates data in min/week format for easier
comparison with accelerometer data. Total MVPA min/
week (all vigorous + all moderate activities’ mins), mod-
erate and vigorous activities in min/week, and weekly
sitting time in min/week values were calculated [53, 54]. Socio-demographic characteristics were obtained using
questions regarding age, educational level, income, mari-
tal status, duration of partnership, duration of infertility,
BMI and lifestyle habits. Global physical activity questionnaire (GPAQ-H) The GPAQ version 2 was used in our research, which
was developed by the WHO. This self-administered form
comprises 16 items that measure the physical activity
levels of a typical active week (7 days) of adults. The
questionnaire contains three domains of PA: work,
transportation, and recreational activities. The duration
and frequency of physical activity (min/day) were re-
corded in case of all three above-mentioned domains. The evaluation of the intensity of certain activities is well
introduced for the respondents in the questionnaires’
manual, based on the extent of increase in breathing or
heart rate [53]. Results were expressed in time (minutes)
or in energy expenditure (MET: Metabolic Equivalent of
Task). According to intensity, moderate and vigorous Sixty or more motionless minutes were defined as
“non-wear time”. A minimum of 480 min of wear-time
was required daily and a minimum of 5–7 days with
valid wear-time including at least one weekend day was
required for inclusion in the analysis [61]. Finally, all
valid days of recording were averaged and multiplied by
seven
to
provide
the
comparability
with
the
questionnaires. Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 6 of 14 Page 6 of 14 Page 6 of 14 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 before (> 2). Only metaphase II oocytes, identified by the
presence of the first polar body, were chosen for
fertilization. Embryo transfers were done 3–5 days after
the oocyte retrieval. Only Grade 1 staged embryos were
transferred, according to the Consensus embryo scoring
system of ESHRE. To evaluate the success of the treat-
ment, transvaginal ultrasound examination was per-
formed 21 days after the embryo transfer to detect
gestational sac [67]. ActiLife 6 software was used to initialize the acceler-
ometer and to download results, and row data was con-
verted with Freedson cut points [62]. The average of
daily moderate to vigorous physical activity (MVPA)
(min/day) and sedentary behaviour (SB) (min/day) was
calculated [63], with a sensitivity and specificity of more
than 98 and 99%, respectively [64]. Based on the additional physical activity diaries, there
were no contact sports or water-based activities per-
formed, which may restrain the participants to wear the
accelerometer. However, four participants were excluded
due to invalid wear-time. The average number of valid
days was 6.32. One patient was excluded due to high risk of implant-
ation failure, in her 8th cycle. Physical activity categories by guidelines Statistical analyses were performed using IBM SPSS Sta-
tistics 25.0 for Mac (SPSS Inc., Chicago, IL, USA). Nor-
mality of data distribution was tested by Kolmogorov-
Smirnov test. Mann-Whitney U-test was used to com-
pare continuous variables. The association between two
continuous variables was tested by Spearman’s rank cor-
relation. To define predicting factors of primary and sec-
ondary outcomes of IVF from pre-treatment habitual
PA, psycho-socio-demographic and baseline biomedical
variables, we conducted a multivariate linear regression
using the stepwise method. Logistic regression analysis
was conducted to evaluate the effects of all the above pa-
rameters on live births. A post-hoc statistical power ana-
lysis was performed using G*Power software, version
3.1.9.6
for
Mac
(Franz
Faul,
Christian-Albrechts-
Universität
Kiel,
Kiel,
Germany)
[70]. Data
was
expressed as mean ± SD as well as medians with 25th
and 75th percentiles and the significance level of p<0.05
was considered in each case. Following the recommendation of the American Con-
gress of Obstetricians and Gynaecologists (ACOG),
pregnant women should engage in moderate intensity
exercise for 150 min per week [65, 66]. However, there
are no definitive physical activity guidelines for women
attempting conception, or before or during assisted
reproduction treatment. To interpret our results, physical activity was catego-
rized by meeting the key values of Physical Activity
Guidelines for Americans for adults as inactive (any ac-
tivity beyond basic movement from daily life activities),
insufficiently active (less than 150 min of moderate-
intensity physical activity (MPA) or 75 min of vigorous-
intensity physical activity (VPA) or the equivalent com-
bination of them per week), active (equivalent of 150
min to 300 min of MPA a week), or highly active (more
than 300 min of MPA a week) [60]. Equivalent values
were calculated through doubling by vigorous values and
added to moderates. PAQs scoring protocols are focusing on cumulative
values of PA performed on average weeks or the week
prior to the measurement. GPAQ categorises the level of
physical activity as High, Moderate or Low by summing
total PA [53], whereas the PA guidelines of PAGAC and
ACOG focus on aerobe or exercise-type PA in relation to
health enhancing effects. Therefore, we decided to analyse
our data using the recreational type of activities following
the PAGAC and ACOG categories [60, 65, 66]. Global physical activity questionnaire (GPAQ-H) The remaining patients
took part in cycles 1–4. General and infertility-related distress The validated Hungarian short-form of the Beck Depres-
sion Inventory (BDI-13) was applied [44–47, 49, 50] to
reflect on distress in general. 68.96% of the respondents
scored less than 5 points, which indicates normal mood
state; and 20.68% belonged to the category of mild de-
pression (6–11 points). Two patients reported severe
depression. Comparative analysis of physical activity patterns Pre-treatment
physical
activity
patterns
of
women
undergoing ART were summarised in Additional file 1. Intensity, frequency and mode of PA were described
using the GPAQ-H questionnaire and ActiGraph GT3X
accelerometer. Comparing the data of the measurements, we found sig-
nificant differences between the subjective instrument
and the objective measures in all of the marked scores
except for vigorous Accelerometer and GPAQ-H means
(p = 0.255). (Table 3.) Regarding GPAQ-H, respondents performed an aver-
age of 461.50 ± 785.56 min/week moderate and 158.00 ±
467.34 min/week vigorous PA in work and only 35.00 ±
82.70 min/week vigorous activity in recreation/leisure
time domain. However, medians (0.00) revealed that vig-
orous PA during work or leisure time are not common
in the studied group. They preferred moderate intensity
recreational activities for 2 h per week (124.80 ± 339.56). To validate subjective PA results, we examined the
correlation between accelerometer and questionnaires
according to moderate, vigorous, MVPA activities, and
sitting time values. The GPAQ-H vigorous PA showed significant correl-
ation with light accelerometer values (R = 0.310, p =
0.090). Time spent with transportation and the respect-
ive MET values showed moderate correlation with light
activities (R = 0.506, p = 0.004) a tendency-like relation-
ship (R = 0.349, p = 0.055) with objectively measured
weekly steps, yet moderate negative correlation with sed-
entary time (R = -0.511, p = 0.003). Nevertheless, they spent 268.75 ± 521.77 min/week on
average with active transportation, for example with
walking or cycling, which covers 806.25 ± 1565.30 MET
energy expenditure. Means significantly differed in these
relations also, as only 120 min transportation was char-
acteristic. They
spent
6.53 h
per
day
sedentary
(2745.17 ± 1755.39 min/week). If
we
categorise
their
performance,
27
women
(60.00%) reported notable leisure time PA, and only 18
of them (40.00%) reached the 150 min/week RMPA rec-
ommendation. 9 (20.00%) persons spent more than 240
min/week with recreational type PA, just like in the
PAGA Highly active category. Comparison of pre-
treatment physical activity characteristics of women
undergoing IVF by physical activity categories is pre-
sented in Table 4. Analysing the data by intensity, we found that respon-
dents
spent
786.32 ± 998.92 min
(2910.65 ± 3932.02
MET) with moderate to vigorous activities (MVPA). In
total, considering all types and intensities of activities
lasting more than 10 min, women performed around
16.98 h
(1018.95 ± 1225.72 min/week)
or
3716.90 ±
4588.16 MET PA. Fertilization protocol For the purpose of measuring the level of infertility-
related stress, FPI was applied and moderately high Glo-
bal stress (183.33 ± 28.19) was explored. In the five do-
mains of the questionnaire we found similar values as in
the pilot study [71]. Average stress by Rejection of child-
free lifestyle (23.25 ± 6.04), moderately high stress by So-
cial concern (41.40 ± 9.84) and very high stress level by
Sexual-
(38.62 ± 7.77)
and
by
Relationship
concern
(48.53 ± 9.68). Stress related to Need for parenthood was
low again, but markedly higher than in our first pilot Publications of the Human Reproduction Scientific Re-
search Group by Bódis and Várnagy described the de-
tailed protocol of fertility treatments [67–69]. Patient
enrolment into IVF procedure was approved by two in-
dependent physicians. The fertilization was performed
with traditional IVF or intracytoplasmatic sperm injec-
tion (ICSI) depending on the andrological status (sperm
count less than 15 M/ml), the maternal age (> 35) and
the number of the previous IVF cycles the patient had Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 7 of 14 study (31.68 ± 8.35 vs 23.1 ± 5.7). Table 2 shows these
results. study (31.68 ± 8.35 vs 23.1 ± 5.7). Table 2 shows these
results. 7060.28 steps daily (49,422.73 ± 16,351.52 counts/week)
based on objective measures. 7060.28 steps daily (49,422.73 ± 16,351.52 counts/week)
based on objective measures. Comparative analysis of physical activity patterns Relationship between psychosocial distress aspects and PA
Relationship between generic PSD, measured with BDI
and PA patterns was not found. Our results on GPAQ-
H revealed that recreational PA could counteract with
some aspects of infertility related distress, since time
spent with moderate RPA, total time and total MET of
RPA negatively correlated with Social Concern (R =
-0.378 p = 0.013, R = -0.386 p = 0.012 and R = -0.360 p =
0.023 respectively) and Relationship Concern of FPI
(R = -0.365 p = 0.019, R = -0.368 p = 0.018 and R = -0.342
p = 0.033 respectively). However, time spent with vigor-
ous RPA was also significantly correlated to ‘Rejection of Regarding the ActiGraphs, light activity was the most
typical
with
1239.87 ± 329.50 min/week,
moderate
(233.35 ± 132.00 min/week)
and
vigorous
activities
(4.65 ± 13.27 min/week) lag behind the subjective mea-
sures,
very
vigorous
activity
was
almost
negligible
(3.70 ± 15.73 min/week). They performed around 4 h
MVPA (241.70 ± 145.10 min/week) and took in average Table 2 Pre-treatment distress characteristics of women
undergoing ART (N = 45)
BDI
Mean
SD
Median
IQR lower
IQR upper
General Stress
4.92
4.82
4.00
1.00
8.00
FPI
Domains
Mean
SD
Median
IQR lower
IQR upper
Social Concern
41.40
9.84
42.50
33.25
50.75
Sexual Concern
38.62
7.77
41.00
34.25
45.00
Relationship Concern
48.53
9.68
50.00
42.00
57.00
Rejection Concern
23.25
6.04
23.00
18.00
28.00
Need for Parenthood
31.68
8.35
31.00
28.00
37.00
Global Stress
183.33
28.19
179.50
165.00
202.50 Table 2 Pre-treatment distress characteristics of women
undergoing ART (N = 45) Table 3 Comparison of pre-treatment physical activity characteristics
of women undergoing IVF based on accelerometer, self-administered
GPAQ-H questionnaires and ActiGraph GT3X mean values difference Table 3 Comparison of pre-treatment physical activity characteristics
of women undergoing IVF based on accelerometer, self-administered
GPAQ-H questionnaires and ActiGraph GT3X mean values difference
Intensity
p
Mean Difference
(min/week)
Sedentary
0.000
−5980.21
Moderate
0.002
626.75
Vigorous
0.255
184.06
MVPA
0.001
805.23 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 8 of 14 childfree lifestyle’ (R = 0.354 p = 0.021). Relationship between IVF outcomes and physical activity Relationship between IVF outcomes and physical activity
If we divided IVF patients regarding the presence of clin-
ical pregnancy, we can conclude that PA patterns differ. Due to high SD, significant difference was detected only
in case of GPAQ-H recreational PA MET means (p =
0.048). Minutes spent with recreation per week also
showed slight difference, but a level of significance was
not reached (p = 0.067). In both cases, means of the
pregnant group were higher. If we divided IVF patients regarding the presence of clin-
ical pregnancy, we can conclude that PA patterns differ. Due to high SD, significant difference was detected only
in case of GPAQ-H recreational PA MET means (p =
0.048). Minutes spent with recreation per week also
showed slight difference, but a level of significance was
not reached (p = 0.067). In both cases, means of the
pregnant group were higher. In Model 1 (R2 = 0.367) the number of oocytes, as the
dependent variable was influenced positively by the
GPAQ-H recreation MET (F = 10.994, p = 0.004; B =
0.005, p = 0.004, B Constant = 4.604). If the tendencies were analysed by the subjective and
objective measures, it could be seen that pregnant
women spent more time and energy expenditure prior
to the treatment with recreational type- or with vigorous
activities, which refers to exercise. In contrast, in the
non-pregnant group cumulative values of PA were
higher, but in relation to work or in total, we assume
that the amount of PA alone did not have a positive ef-
fect. Type and intensity of PA seems to be significant. (Additional file 2). The number of Grade 1 embryos was also examined as
a dependent variable in Model 2 (R2 = 0.757, F = 17.692,
p < 0.001, B Constant = 1.342). Positive significant rela-
tionship was found with GPAQ-H recreational physical
activity MET (B = 0.004, p < 0.001) and negative relation-
ship with BMI (B = −0.167, p = 0.038). When hCG levels on day 12 were considered as a
dependent variable, multivariate linear regression dis-
closed in Model 3 (R2 = 0. 0.958, F = 408.479, p < 0.001)
that higher Very Vigorous Activity level measured with
ActiGraph was accompanied with higher hCG levels
(B = 63.703, p ≤0.001). Relationship between IVF outcomes and physical activity Consistent with the above results, if the pre-treatment
PA measures undergoing IVF/ICSI were analysed by sec-
ondary outcomes, correlations can only be found with
time spent with recreation. Significant relationship was
found with the number of retrieved oocytes (R = 0.315,
p = 0.045), number of matured oocytes (R = 0.339, p =
0.030) and slight trend with Grade 1 embryos (R = 0.294,
p = 0.062) by women who reached at least 150 min RPA
measured by GPAQ-H. Logistic regression analysis On the basis of biomedical, psycho-socio-demographic
and PA variables, logistic regression analysis was con-
ducted to evaluate the effects of all the above parameters
on live births. Contrary to our previous findings, the re-
sults indicated that total time (min/week) spent with
moderate PA measured with GPAQ (beta coefficient
[B] = 0.002, standard error [SE] = 0.001, Wald = 3.944, Comparative analysis of physical activity patterns A relationship
similar to the above cannot be described by ActiGarph
biomedical variables, we conducted multivariate linear
regression using the stepwise method
Table 4 Comparison of pre-treatment physical activity characteristics of women undergoing IVF (N = 45) by physical activity
categories
Physical Activity Categories
Inactive
Insufficiently active
Active I
Highly active
Total
Groups
0 min/week
≤149 min/week
150–299 min/week
≥300 min/week
Non-pregnant
14
6
4
8
32
43.75%
18.75%
12.50%
25.00%
100.00%
77.78%
66.67%
44.44%
88.89%
71.11%
Pregnant
4
3
5
1
13
30.77
23.08
38.46
7.69
100.00%
22.22%
33.33%
55.56%
11.11%
28.88%
Total
18
9
9
9
45
40.00%
20.00%
20.00%
20.00%
100.00%
100.00%
100.00%
100.00%
100.00%
100.00% Table 4 Comparison of pre-treatment physical activity characteristics of women undergoing IVF (N = 45) by physical activity Table 4 Comparison of pre-treatment physical activity characteristics of women undergoing IVF (N = 45) by physical activity childfree lifestyle’ (R = 0.354 p = 0.021). A relationship
similar to the above cannot be described by ActiGarph. biomedical variables, we conducted multivariate linear
regression using the stepwise method. We applied 3 models, which included women’s age,
education, BMI, child-wish, duration of infertility and
number of cycles, QoL and PSD parameters, and PA
values as covariates. In the first step, we adjusted for
age, education, and BMI. In the second step child-wish,
duration of infertility and number of cycles were add-
itionally adjusted. In the third step, we adjusted sub-
scales of BDI and FPI as well, and finally, in the fourth
step PA parameters as GPAQ-H and ActiGraph data
were also included. Discussion It was assumed that the abundance of pre-treatment PA
would decrease psychosocial distress domains in ART
patients and thereby enhance reproductive performance. To assess the effects of psychosocial and lifestyle factors
with special regard to physical activity on course and
success of ART an observational cohort study was con-
ducted with a follow-up of primary and secondary out-
comes. To the best of our knowledge the current study
was the first one in Hungary which gave a detailed de-
scription on the physical activity patterns of the specific
cohort of patients undergoing assisted reproductive
treatment using ActriGraph GT3X accelerometers and
GPAQ-H questionnaire. Regarding the examination of PA patterns, PAQs rou-
tinely overestimated all types and intensity of PA, but
showed moderate correlation with objective values. Self-
reported time spent sedentary was strongly correlated
with questionnaires and accelerometer measures. Cumu-
lative values of PA in average were analogous to the
Hungarian general population, but medians demon-
strated that most of these women completely avoided
vigorous forms of PA and showed pre-treatment PA pat-
terns like women during pregnancy. In general, 68.96% of the studied sample reported nor-
mal mood state (BDI-13), however they could be charac-
terised by moderately high infertility-related distress
(FPI), with moderately high level by Social- and very
high level by Sexual concern. Relationship between generic PSD, measured with BDI
and PA patterns cannot be described. Our GPAQ-H re-
sults revealed that recreational PA could counteract with
some aspects of infertility related distress, RPA nega-
tively correlated with Social Concern and Relationship
Concern of FPI. Significant difference cannot be de-
scribed using PAGA PA categories or the 240 min cut
off point regarding PSD. g p
g
y
Compared to the general Hungarian population, the
subpopulation of the studied women cannot be de-
scribed as inactive, however only 60.00% of the sample
examined reported notable leisure time PA and only
40.00% reached the recommended level of 150 min/week
recreational
moderate
physical
activity. They
spent
16.98 h per week with all forms of activity and spent
6.53 h per day sedentary. 50.00% of the women in the
sample reported regular exercise, which could be consid-
ered as a relatively active subpopulation in Hungary
compared to previous national studies [79, 80]. However,
Ács et al. Multivariate linear regression analysis To define predicting factors of primary and secondary
outcomes
of
IVF
from
pre-treatment
habitual
PA,
psycho-socio-demographic
variables,
and
baseline Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 9 of 14 Page 9 of 14 p = 0.047, OR = 1.002) significantly associated with live
births. p = 0.047, OR = 1.002) significantly associated with live
births. month period using the Epidemiologic Studies Depres-
sion Scale (CES-D) and the State Anxiety Subscale of
the State-Trait Anxiety Inventory (STAI). In a binary lo-
gistic model including covariates (woman’s age, ethni-
city, income, education, parity, duration of infertility,
and time interval), pre-treatment depression and anxiety
were not significant predictors of the outcome measures. In
linear
regression
models
including
covariates
(woman’s age, income, education, parity, duration of in-
fertility, assessment point, time since last treatment
cycle, and pre-IVF depression or anxiety), experience of
a failed IVF were associated with higher post-IVF de-
pression and anxiety, which draw attention to the sup-
port of patients to prepare for and cope with treatment
and treatment failure [78]. Discussion Similarly to other studies emphasizing the
importance of PA on BMI [34], in a randomised con-
trolled pilot trial authors proved, that the high-intensity
interval training significantly improved insulin sensitiv-
ity, VO2 peak and abdominal fat. However, due to low
number of participants (intervention group N = 8, con-
trol group N = 10) they could not draw a conclusion on
pregnancy rate [82]. Regarding GPAQ, our respondents performed moder-
ate intensity PA during work and preferred that during
recreation. However, mean values showed some vigorous
activity in work (158.00 ± 467.34 min/week) and recre-
ation (35.00 ± 82.70 min/week). Medians demonstrated
that most of these women completely avoided vigorous
forms of movement. Regarding female reproduction,
there is a wide consensus on the beneficial effects of PA
on gestation. Most studies draw attention to the risk of
frequent vigorous PA on fertility [87, 88] and on success
of ART [43, 77]. “Walking the way to better health” general recommen-
dations promote 10,000 steps daily [83, 84]. With
7060.28 steps per day measured with accelerometers,
our respondents cannot be categorised as inactive com-
pared to a Nature letter by Althoff et al. on worldwide
activity inequality, which mentioned the average daily
steps to be around 5300 in Hungary, 5000 Worldwide
and 4800 in the US, measured with smartphones [85]. In
an update published in ACSM’s journal, Medicine & Sci-
ence in Sports & Exercise authors revealed that 7000–
9000 steps per day may trigger in health benefits, which
are associated with current public health guidelines’ em-
phasis on minimal amounts of time spent in MVPA, the
federally recommended amounts of 150 to 300 min per
week [86]. To describe PA levels, both instrumental and self-
reported studies were published. Evenson et al. discussed
that the adjusted odds of intrauterine gestation are
higher among IVF patients who had higher continuous
active living (OR 1.96, 95% CI 1.09–3.50), sports/exercise
(OR 1.48, CI 1.02–2.15), and total activity (OR 1.52, 95%
CI 1.15–2.01) indices in the past year [89]. Regarding
the
benefits
of
pre-treatment
activity,
Moran et al. reported positive effect of lifestyle interven-
tion including exercise and diet in conjunction with
ART in overweight and obese women and described ele-
vated successful pregnancy rate (12 / 18 vs 8 / 20) in the
intervention group compared to controls (Moran, Tsa-
gareli, Norman, & Noakes, 2011). Discussion Women were divided as per the abundance of clinical
pregnancy. Pregnant women spent more time and en-
ergy expenditure with recreational type- or with vigor-
ous activities, which refers to exercise. In contrast, in the
non-pregnant group cumulative values of PA were
higher, but in relation with work or in total, we assume
that this alone did not have a positive effect. Type and
intensity of PA seems to be significant. Significant rela-
tionship could be described with the number of retrieved
oocytes, number of matured oocytes and sligth relation-
ship with Grade 1 embryos by women who reached at
least 150 min RPA measured by GPAQ-H. Palomba et al. in their observational cohort study
assessed the relationship between RPA and reproductive
performance in connection with lifestyle interventions in
obese infertile women who received ART (N = 216). Number of pregnancies (16/41, 39.0% versus 28/175,
16.0%, respectively; p = 0.002) and live births (10/41,
24.4% versus 13/175, 7.4%, respectively; p = 0.004) were
significantly higher in 41 obese patients who did regular
physical activity compared to 175 obese controls who
did not. After adjusting for confounders, the relative
risks for a clinical pregnancy and live birth were 3.22
(95% CI 1.53–6.78; P = 0.002) and 3.71 (95% CI 1.51–
9.11; P = 0.004) in active patients, and RPA significantly
correlated with improved reproductive performance irre-
spective of bodyweight loss [40]. In our study we could
conclude significant difference between pregnant and
non-pregnant groups by GPAQ-H recreational PA MET
means (p = 0.048), which underline the importance of
leisure time activities (inter alia) against PA in general. It could be claimed that the GPAQ-H measurement
tool is a valid and reliable questionnaire to measure the
healthy Hungarian general population’s physical activity
patterns [57]. By the validation study of GPAQ-H, Bland
Altman plots revealed mean differences between the
GPAQ-H and accelerometer data. The plots showed that
GPAQ-H overestimates vigorous activities by 212.75
min/week (331.82–757.42) and MVPA values by 104.93
min/week (−1016.98–807.11). In our current study we
observed similar overestimation of vigorous activities
with 331.10 vs 184.06 min/week, and considerable over-
estimation of MVPA 481.37 vs 805.23 min/week in case
of GPAQ-H and IPAQ-SFH. In the validation study a
high difference, 6336.79 min/week (CI 3638.18–9035.40)
was revealed regarding sitting, as GPAQ-H largely
underestimated the time spent sedentary [57].. Our On the other hand, adverse effects of excessive PA are
also demonstrated. Discussion reported 10% improvement in PA habits based
on representative Eurobarometer data from 2018: Hun-
garians’ regular sport participation and physical activity
is 33%, which is below the EU average (40%). Authors
noted that 42% of Hungarian citizens spent more than
2.5 and less than 5.5 h sedentary. With 6.53 h daily sit-
ting time (2745.17 ± 1755.39 min/day), our results are
slightly elevated but are in line with the above findings
[81]. Comparison of fertility-specific and general question-
naires can be found in literature in relation to ART pa-
tients [72–74]. Cserepes et al. conducted research using
FertiQoL and Beck Depression Inventory on a Hungar-
ian sample (126 couples). Female members of the cou-
ples reported poorer QoL than males. Subscales of the
Core module scored between 69.01 ± 16.33 (Emotional
Scale) and 80.26 ± 13.85 (Social scale), total QoL was de-
scribed as 77.27 ± 12.05. These values were markedly
higher than in our sample [75]. Domar et al. underline the role of improving mental
health with psychological interventions in improved
pregnancy rates among infertile women [76]. Other
studies shift focus to lifestyle behaviours: Domar et al. made
surprising
observations
regarding
interfering
health behaviours as exhausting exercise, smoking, regu-
lar consumption of alcohol and caffeinated beverages
and taking herbal supplements during IVF cycles [77]. In
our sample more health-conscious lifestyle could be
observed. The amount of PA in our research increased unex-
pectedly in line with age and also with BMI, suggesting a
more health-conscious behaviour by women exposed to
higher risk. The current study did not include questions
on health literacy, so we can only assume that these par-
ticipants had some supposed knowledge on the relation-
ship between reduced conception rate and overweight/
obesity, insulin resistance or amount of visceral fat. Lungren, Kiel and co-authors developed a study protocol The Fertility Experiences Project examined 202 first
IVF cycle patients to predict the influence of psycho-
logical distress on IVF treatment outcome and subse-
quent PSD, in a prospective cohort study over an 18- Page 10 of 14 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 study also revealed a difference in the measurement of
sitting time between accelerometer and GPAQ-H −
5980.21 min/week. to provide knowledge on the results of a high-intensity
interval training before ART in subfertile overweight or
obese women and include the program in regular fertil-
ity care [82]. Discussion Gudmundsdottir et al. found that
women who are active on most days, tended to experi-
ence fertility problems 3.2 times more often. In this
study exercising to exhaustion also led to 2.3 times more
fertility impairments than low intensity PA [90]. Based
on the data by Morris et al. on lifetime exercise (level of Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 11 of 14 Page 11 of 14 cannot confirm the emphasized importance of PSD and
age on reproductive performance. evidence: II-2), exercising 4 h or more per week indicate
40% less likelihood of having a live birth (OR 0.6, CI
0.4–0.8), it is 3 times more likely to lead to cycle
cancellation and 2 times more likely to lead to implant-
ation failure or pregnancy loss (OR 2.8, CI 1.5–5.3; OR
2.0, CI 1.4–3.1; OR 2.0, CI 1.2–3.4 respectively) com-
pared to non-exercise [43]. In the current research dur-
ing the follow-up of IVF outcomes, particular attention
was given to the women in our sample who reported at
least 4 h exercise weekly (18.2%). In our study neither
negative nor positive effects can be concluded by ex-
ceeding 240 or even 300 min of activity per week. Sig-
nificant relationship could be described in relation to
reproductive performance (number of retrieved oocytes
and number of matured oocytes) by women who
reached at least 150 min pre-treatment RPA measured
by GPAQ-H. To define predicting factors of primary and secondary
outcomes of IVF from the point of view of PA and PSD,
we conducted a multivariate linear regression using the
stepwise method. We applied 3 models, which included
women’s age, education, BMI, child-wish, duration of in-
fertility and number of cycles and PSD parameters, and
PA values as covariates. In Model 1 the number of oo-
cytes was influenced positively by the GPAQ-H recre-
ation MET, in Model 2 the number of Grade 1 embryos
was positively correlated with GPAQ-H recreational
physical activity MET and negatively with BMI. It was
disclosed in Model 3 that higher Very Vigorous Activity
level measured with ActiGraph was accompanied by
higher hCG levels. Gaskins and co-authors reported similar findings on
maternal PA and sedentary behaviour in relation to
ART’s reproductive outcomes. They found no associ-
ation between MVPA time or total MET and outcomes
as probability of implantation, clinical pregnancy or live
birth. Limitations The limitations of the study include the sample’s non-
representative nature. To avoid potential confounders,
patients were carefully selected, but made the study
population modest. Objective measurement of PA pat-
terns cannot be conducted by all patients and complete
medical record was also missing in a portion of patients. Regarding PA immediately after IVF, Evenson et al. could not find any association between accelerometer-
measured activity or sedentary behaviour with IVF out-
comes. They
described
that
after
embryo
transfer
women engaged only in light activity (ME 3.0 h/day) and
sedentary
behaviours
(ME
9.0 h/day). Although
the
current research focused on pre-treatment habitual PA,
measurement of post-treatment PA in relation to QoL,
PSD,
and
success
rates
could
also
offer
research
potential. A post-hoc sample size estimation (using G*Power
for Mac version 3.1.9.6) for the multivariate linear re-
gression analysis (significance set at 5%, power set at
0.8, effects size at 0.15, and number of predictors at
2) showed that a total of 55 subjects would have been
required to ensure adequate statistical power for ana-
lyses. The final sample of 45 subjects did not meet
the sample requirements. Whereas the sample size
was relatively suboptimal, given the limited study
power, i.e. 71.84%, to detect the difference in primary
and secondary outcomes of ART. Espinós et al. reported in their meta-analyses based
on 8 RCTs, that although lifestyle programmes im-
proved pregnancy rates (RR: 1.43, CI: 95% 1.02 to
2.01; I2 = 60%; 8 RCTs; N = 1098), they had no impact
on live births (RR: 1.39, CI: 95% 0.90 to 2.14; I2 =
64%; 7RCTs; N = 1034) and increased risk of miscar-
riage in obese infertile women [91]. In our sample,
positive association was found between moderate PA
and live births and no relationship with the ratio of
miscarriage. Attention was drawn by Gaskins et al. to the use of
intermediate outcomes of IVF as surrogates of women’s
reproductive performance [93]. Ongoing pregnancy has
been considered as acceptable surrogate for live birth, as
well as clinical pregnancy in or study. However, the
major potential limitation of using ongoing pregnancies
as the primary outcome of ART is the significant odds
of pregnancy loss between the pregnancy confirmation
and live birth. Discussion However, specific leisure time activities (aerobics,
rowing, exercising with ski or stair machine) were posi-
tively associated with live birth (p-trend = 0.02). Positive effects were also concluded in the meta-
analysis of eight published studies (N = 3683 infertile
couples) of Rao et al., which reported an increasing but
not statistically significant trend in the implantation rate
for physically active women when compared with phys-
ically inactive women (OR = 1.95, 95% CI 0.99–3.83,
I2 = 77%). Rates of clinical pregnancy and live births in
physically active women were significantly higher than
those in physically inactive women (OR = 1.96, 95% CI
1.40, 2.73, I2 = 42% and OR = 1.95, 95% CI 1.06–3.59,
I2 = 82%, respectively) [41]. Conclusion Infertility-specific scales could provide a more appropri-
ate information on PDS of ART patients compared to
general scales. GPAQ-H could be used as a valid meas-
urement tool for mapping PA habits of ART patients,
noting that the robust difference between objective and
subjective measures (self-reports) of PA highlight the
need to improve physical literacy of women undergoing
ART. Abbreviations ACOG: American congress of obstetricians and gynaecologists; ART: Assisted
reproductive therapy; BDI: Beck depression inventory; BMI: Body mass index;
ESHRE: European society of human reproduction and embryology;
FertiQol: Fertility quality of life tool; FPI: Fertility problem inventory; GPAQ-
H: Global physical activity questionnaire-hungarian version; HADS: Hospital
anxiety and depression; ICD-11: International classification of diseases 11th
revision; ICSI: Intracytoplasmic sperm injection; IQR: Interquartile range;
IUI: Intrauterine insemination; IVF: In-vitro fertilization; MVPA: Moderate to
vigorous physical activity; MET: Metabolic equivalent of task; OI: Ovulation
induction; PAQs: Physical activity questionnaires; RCT: Randomized controlled
trial 2. Calhaz-Jorge C, De Geyter C, Kupka MS, de Mouzon J, Erb K, Mocanu E,
Motrenko T, Scaravelli G, Wyns C, Goossens V. Assisted reproductive
technology in Europe, 2013: results generated from European registers by
ESHRE. Hum Reprod. 2017;32(10):1957–73. 3. De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T,
Scaravelli G, Smeenk J, Vidakovic S, Goossens V, et al. ART in Europe, 2014:
results generated from European registries by ESHRE†: the European IVF-
monitoring consortium (EIM)‡ for the European Society of Human
Reproduction and Embryology (ESHRE). Hum Reprod. 2018;33(9):1586–601. 4. De Geyter C, Calhaz-Jorge C, Kupka MS, Wyns C, Mocanu E, Motrenko T,
Scaravelli G, Smeenk J, Vidakovic S, Goossens V, et al. ART in Europe, 2015:
results generated from European registries by ESHRE†. Hum Reprod Open. 2020;2020(1):hoz038. https://doi.org/10.1093/hropen/hoz038. Acknowledgements Authors wish to thank the women who underwent ART for participating in
the study at such a challenging time of their life. 5. ESHRE. Annual Report 2017. Grimbergen: ESHRE; 2018. 6. McLaren JF. Infertility evaluation. Obstet Gynecol Clin N Am. 2012;39(4):
453–63. 6. McLaren JF. Infertility evaluation. Obstet Gynecol Clin N Am. 2012;39(4):
453–63. Received: 31 January 2021 Accepted: 1 February 2021
Published: 23 April 2021 Additional file 2 Mean differences of pre-treatment physical activity
measures undergoing IVF/ICSI (N = 45) by primary outcome. Funding g
The publication costs were funded by the Economic Development and
Innovation Operational Programme GINOP 2.3.2-15-2016-00047 grant. The
authors declare that the design of the study and collection, analysis, and
interpretation of data and writing of the manuscript are independent of
GINOP. References 1. Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a
systematic analysis of 277 health surveys. PLoS Med. 2012;9(12):e1001356. 1. Mascarenhas MN, Flaxman SR, Boerma T, Vanderpoel S, Stevens GA. National, regional, and global trends in infertility prevalence since 1990: a
systematic analysis of 277 health surveys. PLoS Med. 2012;9(12):e1001356. Limitations An analysis of 121,744 women with failed first treat-
ment revealed that age is a key predictor of failure to
have a live birth following IVF as well as the risk of hin-
dered performance, while increased duration of infertil-
ity is also associated with poorer outcomes at every
stage [92]. Comparing our results to our models, we For more impressive results on the effects of physical
activity on the effectiveness of fertility programmes, a Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 12 of 14 Page 12 of 14 detailed objective assessment of physical activity, in-
creased number of participants, and further examina-
tions on outcome measures, with live birth’s rate as end
point are needed in a well-powered randomized con-
trolled prospective study. the sample, BP and MH contributed in collection and analysis of participants
anthropometric and accelerometer data, VP, PÁ and AM contributed to the
data collection and analysis, and to the drafting and final editing of the
manuscript. About this supplement This article has been published as part of BMC Public Health Volume 21
Supplement 1, 2021: Level and Determinants of Physical Activity in the V4
Countries – Part 2. The full contents of the supplement are available online
at URL. https://bmcpublichealth.biomedcentral.com/articles/supplements/
volume-21-supplement-1. 7. Bernard A, Krizsa F: Generally about infertility. Modern diagnostic and
therapy in infertility [A meddőségről általában In: Kaáli, S: A meddőség
korszerű diagnosztikája és kezelése] Medicina Könyvkiadó, Budapest 2006. 8. WHO: Infecundity, infertility, and childlessness in developing countries. DHS
Comparative Reports 2004, 9. 9. World Health Organization. Reproductive health indicators: guidelines for
their generation, interpretation and analysis for global monitoring. Geneva:
WHO Press; 2006. Supplementary information 1Faculty of Health Sciences, University of Pécs, Vorosmarty u. 4, Pécs 7621,
Hungary. 2MTA-PTE Human Reproduction Scientific Research Group,
University of Pécs, Édesanyák u. 17, Pécs H-7624, Hungary. 3Department of
Obstetrics and Gynaecology, Medical School, University of Pécs, Édesanyák u. 17, Pécs H-7624, Hungary. pp
y
Supplementary information accompan y
Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09522-7. Supplementary information accompanies this paper at https://doi.org/10. 1186/s12889-020-09522-7. Additional file 1 Pre-treatment physical activity characteristics of
women undergoing ART (N = 45) based on accelerometer, self-
administered GPAQ-H questionnaire and ActiGraph GT3X data. Received: 31 January 2021 Accepted: 1 February 2021
Published: 23 April 2021 Availability of data and materials The dataset supporting the conclusions of this article is available from the
corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Consent for publication Consent for publication
Not applicable. Ethics approval and consent to participate The ethical approval was granted for the study by Ethics Committee of
University of Pécs (Nr. 6533). Participants were informed about the research
aim and methods before signing the informed consent form. The
investigation conforms to the principles outlined in the Declaration of
Helsinki. Based on our results, recreation type of pre-treatment
PA could positively influence domains of infertility re-
lated QoL and PSD during ART and improve reproduct-
ive performance in relation to primary and secondary
outcomes. Existing differences in response to infertility
due to PA suggest the need for the development of a
specific intervention. Authors’ contributions Beck AT, Beck RW. Screening depressed patients in family practice. A rapid
technic. Postgrad Med J. 1972;52(6):81–5. 24. Smeenk JM, Verhaak CM, Vingerhoets AJ, Sweep CG, Merkus JM, Willemsen
SJ, van Minnen A, Straatman H, Braat DD. Stress and outcome success in
IVF: the role of self-reports and endocrine variables. Hum Reprod. 2005;
20(4):991–6. 48. Khademi A, Alleyassin A, Aghahosseini M, Ramezanzadeh F, Abhari AA. Pretreatment Beck depression inventory score is an important predictor for
post-treatment score in infertile patients: a before-after study. BMC
Psychiatry. 2005;5:25. 25. WHO. Global action plan on physical activity 2018–2030: more active
people for a healthier world. Switzerland: World Health Organization; 2018. 49. Kopp M, Fóris N. A szorongás kognitív viselkedésterápiája. Budapest:
Végeken Sorozat; 1993. 26. Merom D, Sinnreich R, Aboudi V, Kark JD, Nassar H. Lifestyle physical
activity among urban Palestinians and Israelis: a cross-sectional
comparison in the Palestinian-Israeli Jerusalem risk factor study. BMC
Public Health. 2012;12(1):90. 50. Reynolds WM, Gould JW. A psychometric investigation of the standard and
short form Beck depression inventory. J Consult Clin Psychol. 1981;49(2):
306–7. 51. Newton CR, Sherrard W, Glavac I. The fertility problem inventory: measuring
perceived infertility-related stress. Fertil Steril. 1999;72(1):54–62. 27. Acs P, Stocker M, Fuge K, Paar D, Olah A, Kovacs A. Economic and public
health benefits: the result of increased regular physical activity. Eur J Integr
Med. 2016;8:8–12. 52. Cserepes RE, Kollar J, Sapy T, Wischmann T, Bugan A. Effects of gender roles,
child wish motives, subjective well-being, and marital adjustment on
infertility-related stress: a preliminary study with a Hungarian sample of
involuntary childless men and women. Arch Gynecol Obstet. 2013;288(4):
925–32. 28. Ács P, Stocker M, Oláh A. The determination of economic and public health
benefits achievable by increasing regular physical exercise. Apstract Appl
Stud Agribusiness Commerce. 2013;8(1):5–14. 53. Department of Chronic Diseases and Health Promotion Surveillance and
Population-Based Prevention World Health Organization. Global Physical
Activity Questionnaire (GPAQ) Analysis Guide V2. Geneva; 2012. [https://
www.who.int/ncds/surveillance/steps/GPAQ%20Instrument%20and%2
0Analysis%20Guide%20v2.pdf]. Accessed 02 Mar 2018. 29. Ács P, Prémusz V, Morvay-Sey K, Kovács A, Makai A, Elbert G. A sporttal,
testmozgással összefüggésben lévő mutatók változása Magyarországon és
az Európai Unióban az elmúlt évek eredményeinek nyomán. Sport és
Egészségtudományi Füzetek. 2018;2(1):61–76. 30. Börjesson M, Hellénius M, Jansson E. FYSS - physical activity in the
prevention and treatment of disease. Stockholm: Elanders; 2010. 54. Herrmann SD, Heumann KJ, Der Ananian CA, Ainsworth BE. Authors’ contributions All authors read and approved the final manuscript. VP conceived, designed
and managed the study, MK, JB, ÁV and KL contributed to the study
conceptualization and provided critical editorial input to the interpretation of
the data, OM contributed to interpretation of the psychometric properties of 11. WHO. International statistical classification of diseases and related health
problems: 11th revision (ICD-11). Geneva: World Health Organization; 2018. Page 13 of 14 Page 13 of 14 Page 13 of 14 Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 12. Rashidi B, Montazeri A, Ramezanzadeh F, Shariat M, Abedinia N, Ashrafi M. Health-related quality of life in infertile couples receiving IVF or ICSI
treatment. BMC Health Serv Res. 2008;8:186. 34. Ruiz-Montero PJ, Castillo-Rodriguez A, Mikalački M, Nebojsa C, Korovljev D. 24-weeks Pilates-aerobic and educative training to improve body fat mass
in elderly Serbian women. Clin Interv Aging. 2014;9:243–8. 35. Fusz K, Pakai A, Kívés Z, Szunomár S, Regős A, Oláh A. Work schedules in
the Hungarian health care system and the sleep quality of nurses. Orv Hetil. 2016;157(10):379–84. 13. Zurlo MC, Della Volta MFC, Vallone F. The association between stressful life
events and perceived quality of life among women attending infertility
treatments: the moderating role of coping strategies and perceived
couple’s dyadic adjustment. BMC Public Health. 2019;19(1):1548. 36. Davies GA, Wolfe LA, Mottola MF, MacKinnon C. Joint SOGC/CSEP clinical
practice guideline: exercise in pregnancy and the postpartum period. Can J
Appl Physiol. 2003;28(3):330–41. 14. Sumera A, Raafay S, Ayesha MI, Faisal IK, Syed FA, Annum S, Syed F-U-H. Knowledge, perceptions and myths regarding infertility among selected
adult population in Pakistan: a cross-sectional study. BMC Public Health. 2011;11:760. 37. Russo LM, Nobles C, Ertel KA, Chasan-Taber L, Whitcomb BW. Physical
activity interventions in pregnancy and risk of gestational diabetes mellitus
a systematic review and meta-analysis. Obstet Gynecol. 2015;125(3):576–82. 15. Reading AE, Chang LC, Kerin JF. Attitudes and anxiety levels in women
conceiving through invitro fertilization and gamete intrafallopian transfer. Fertil Steril. 1989;52(1):95–9. 38. Prémusz V, Makai A, Melczer C, Perjés B, Ács P, Bódis J, Lampek K, Várnagy
Á. Habituális fizikai aktivitás és életminőség összefüggése várandósság alatt
a WHO Global Physical Activity Questionnaire alapján. Magy Noorv Lapja. 2018;81(6):343–50. 16. Greil AL. Infertility and psychological distress: a critical review of the
literature. Soc Sci Med. 1997;45(11):1679–704. 39. Tendais I, Figueiredo B, Mota J, Conde A. Physical activity, health-related
quality of life and depression during pregnancy. Authors’ contributions Cad Saude Publica. 2011;
27(2):219–28. 17. Magiakou MA, Mastorakos G, Webster E, Chrousos GP: The hypothalamic-
pituitary-adrenal axis and the female reproductive system. In: Adolescent
Gynecology and Endocrinology: Basic and Clinical Aspects. Volume 816,
edn. Edited by Creatsas G, Mastorakos G, Chrousos GP; 1997: 42–56. 40. Palomba S, Falbo A, Valli B, Morini D, Villani MT, Nicoli A, La Sala GB. Physical
activity before IVF and ICSI cycles in infertile obese women: an
observational cohort study. Reprod BioMed Online. 2014;29(1):72–9. edn. Edited by Creatsas G, Mastorakos G, Chrousos GP; 1997: 42–56 18. Ferin M. Stress and the reproductive cycle. J Clin Endocrinol Metab. 1999;
84(6):1768–74. 19. Dobson H, Ghuman S, Prabhakar S, Smith R. A conceptual model of the
influence of stress on female reproduction. Reproduction. 2003;125(2):
151–63. 41. Rao M, Zeng Z, Tang L. Maternal physical activity before IVF/ICSI cycles
improves clinical pregnancy rate and live birth rate: a systematic review and
meta-analysis. Reprod Biol Endocrinol. 2018;16(1):11. 42. Rich-Edwards JW, Spiegelman D, Garland M, Hertzmark E, Hunter DJ, Colditz
GA, Willett WC, Wand H, Manson JE. Physical activity, body mass index, and
ovulatory disorder infertility. Epidemiology. 2002;13(2):184–90. 20. Lynch CD, Sundaram R, Maisog JM, Sweeney AM, Louis GMB. Preconception
stress increases the risk of infertility: results from a couple-based prospective
cohort studyuthe LIFE study. Hum Reprod. 2014;29(5):1067–75. 43. Morris SN, Missmer SA, Cramer DW, Powers RD, McShane PM, Hornstein
MD. Effects of lifetime exercise on the outcome of in vitro fertilization. Obstet Gynecol. 2006;108(4):938–45. 21. Boivin J, Takefman JE. Stress level across stages of in-vitro fertilization in
subsequently pregnant and nonpregnant women. Fertil Steril. 1995;64(4):
802–10. 44. Beck AT, Ward CH, Mendelson M, Mock J, Erbaugh J. An inventory for
measuring depression. Arch Gen Psychiatry. 1961;4:561–71. 22. Luppa P, Muller B, Jacob K, Kimmig R, Strowitzki T, Hoss C, Weber MM,
Engelhardt D, Lobo RA. Variations of steroid-hormone metabolites in serum
and urine in polycystic-ovary-syndrome after nafarelin stimulation -
evidence for an altered corticoid excretion. J Clin Endocrinol Metab. 1995;
80(1):280–8. 45. Beck AT, Steer RA, Carbin MG. Psychometric properties of the Beck
depression inventory: twenty-five years of evaluation. Clin Psychol Rev. 1988;
8(1):77–100. 46. Beck AT. Cognitive therapy of depression. New York: Guilford press; 1979. 23. Smeenk JM, Verhaak CM, Eugster A, van Minnen A, Zielhuis GA, Braat DD. The effect of anxiety and depression on the outcome of in-vitro fertilization. Hum Reprod. 2001;16(7):1420–3. 47. Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Authors’ contributions Validity and
reliability of the global physical activity questionnaire (GPAQ). Meas Phys
Educ Exerc Sci. 2013;17(3):221–35. 31. Fusz K, Tóth Á, Varga B, Rozmann N, Oláh A. Different work schedules of
nurses in Hungary and their effects on health. Ideggyogy Sz. 2017;70(3–4):
136–9. 55. Armstrong T, Bull F. Development of the World Health Organization global
physical activity questionnaire (GPAQ). J Public Health. 2006;14(2):66–70. 32. Szalai M, Szirmai A, Fuge K, Makai A, Erdelyi G, Premusz V, Bodis J. Special
aspects of social support: Qualitative analysis of oncologic rehabilitation
through a belly dancing peer support group. Eur J Cancer Care. 2017;26(6):
10.1111/ecc.12656. https://doi.org/10.1111/ecc.12656. 56. Cleland CL, Hunter RF, Kee F, Cupples ME, Sallis JF, Tully MA. Validity of the
global physical activity questionnaire (GPAQ) in assessing levels and change
in moderate-vigorous physical activity and sedentary behaviour. BMC Public
Health. 2014;14. 33. Szalai M, Lévay B, Szirmai A, Papp I, Prémusz V, Bódis J. A clinical study to
assess the efficacy of belly dancing as a tool for rehabilitation in female
patients with malignancies. Eur J Oncol Nurs. 2015;19(1):60–5. 57. Ács P, Betlehem J, Oláh A, Bergier B, Morvay-Sey K, Makai A, Prémusz V. Cross-cultural adaptation and validation of the global physical activity Prémusz et al. BMC Public Health 2021, 21(Suppl 1):1480 Page 14 of 14 Page 14 of 14 questionnaire among healthy Hungarian adults. BMC Public Health. 2020;
20(1):1056. https://doi.org/10.1186/s12889-020-08477-z. 80. Gabnai Z, Müller A, Bács Z, Bácsné Bába É. The economic burden of physical
inactivity at national level [a fizikai inaktivitás nemzetgazdasági terhei]. Egészségfejlesztés. 2019;60(1):20–30. questionnaire among healthy Hungarian adults. BMC Public Health. 2020;
20(1):1056. https://doi.org/10.1186/s12889-020-08477-z. 58. Bull FC, Maslin TS, Armstrong T. Global physical activity questionnaire
(GPAQ): nine country reliability and validity study. J Phys Act Health. 2009;
6(6):790–804. 81. Ács P, Prémusz V, Morvay-Sey K, Kovács A, Makai A, Elbert G. Changes of
sport and physical activity indicators in Hungary and in the European Union
according to the results from recent years (A sporttal, testmozgással
összefüggésben lévő mutatók változása Magyarországon és az Európai
Unióban az elmúlt évek eredményeinek nyomán). Sport és
Egészségtudományi Füzetek. 2018;2(1):61–76. 59. The IPAQ group. Guidelines for data processing and analysis of the
International Physical Activity Questionnaire (IPAQ)–short and long forms. 2005. [https://docs.google.com/viewer?a=v&pid=sites&srcid=
ZGVmYXVsdGRvbWFpbnx0aGVpcGFxfGd4OjE0NDgxMDk3NDU1YWRlZTM]. Accessed 20 Jan 2018. 82. Lundgren KM, Romundstad LB, von During V, Morkved S, Kjotrod S,
Moholdt T. Authors’ contributions Exercise prior to assisted fertilization in overweight and obese
women (FertilEX): study protocol for a randomized controlled trial. Trials. 2016;17. 60. U.S. Department of Health and Human Services. Physical Activity Guidelines
for Americans, 2nd ed. Washington, DC: Department of Health and Human
Services; 2018. 83. Duncan MJ, Brown WJ, Mummery WK, Vandelanotte C. 10,000 steps
Australia: a community-wide eHealth physical activity promotion
programme. Br J Sports Med. 2018;52(14):885–6. 61. Trost SG, Owen N, Bauman AE, Sallis JF, Brown W. Correlates of adults'
participation in physical activity: review and update. Med Sci Sports Exerc. 2002;34(12):1996–2001. 84. HHS USDoHaHS: The Physical Activity Guidelines for Americans, 2nd edition,
Date Accessed 08/06, 2019, from In: Physical Activity Guidelines Advisory
Committee; 2018. 62. Freedson PS, Melanson E, Sirard J. Calibration of the computer science and
applications, Inc. accelerometer. Med Sci Sports Exerc. 1998;30(5):777–81. 63. Matthews CE, Ainsworth BE, Thompson RW, Bassett DR Jr. Sources of
variance in daily physical activity levels as measured by an accelerometer. Med Sci Sports Exerc. 2002;34(8):1376–81. 85. Althoff T, Sosič R, Hicks JL, King AC, Delp SL, Leskovec J. Large-scale physical
activity data reveal worldwide activity inequality. Nature. 2017;547(7663):
336–9. 64. Skotte J, Korshoj M, Kristiansen J, Hanisch C, Holtermann A. Detection of
physical activity types using triaxial accelerometers. J Phys Act Health. 2014;
11(1):76–84. 86. Tudor-Locke C, Craig CL, Brown WJ, Clemes SA, De Cocker K, Giles-Corti B,
Hatano Y, Inoue S, Matsudo SM, Mutrie N, et al. How many steps/day are
enough? For adults. Int J Behav Nutr Phys Act. 2011;8(1):79. 65. ACOG. ACOG Committee Opinion No. 650: physical activity and exercise
During pregnancy and the postpartum period. Obstet Gynecol. 2015;126(6):
e135–42. 87. Green BB, Daling JR, Weiss NS, Liff JM, Koepsell T. Exercise as a risk factor for
infertility with ovulatory dysfunction. Am J Public Health. 1986;76(12):1432–6. 88. Wise LA, Rothman KJ, Mikkelsen EM, Sorensen HT, Riis AH, Hatch EE. A
prospective cohort study of physical activity and time to pregnancy. Fertil
Steril. 2012;97(5):1136–42. 66. ACOG. ACOG Committee opinion. Number 267, January 2002: Exercise during
pregnancy and the postpartum period. Obstet Gynecol. 2002;99(1):171–3. 67. Várnagy Á, Kőszegi T, Györgyi E, Sulyok E, Prémusz V, Bódis J. Levels of total
antioxidant capacity and 8-hydroxy-2′-deoxyguanosine of serum and
follicular fluid in women undergoing in vitro fertilization: focusing on
endometriosis. Hum Fertil. 2020;23(3):200–8. 89. Evenson KR, Calhoun KC, Herring AH, Pritchard D, Wen F, Steiner AZ. Authors’ contributions Association of physical activity in the past year and immediately after
in vitro fertilization on pregnancy. Fertil Steril. 2014;101(4):1047–U1482. 90. Gudmundsdottir SL, Flanders WD, Augestad LB. Physical activity and fertility
in women: the north-Trondelag health study. Hum Reprod. 2009;24(12):
3196–204. 68. Bódis J, Várnagy Á. Non-invazív vizsgálati lehetőségek az in vitro fertilizációs
kezelés hatásfokának növelésére. Magy Noorv Lapja. 2015;78(5):226–31. 69. Gödöny K, Laki K, Várnagy Á, Kovács K, Sulyok E, Berenténé Bene J, Melegh
B, Bódis J. Az in vitro fertilizáció sikerességét befolyásoló biomarkerek
vizsgálata: a karnitin jelentősége. Egészség-Akadémia. 2014;5(4):247–53. 91. Espinós JJ, Solà I, Valli C, Polo A, Ziolkowska L, Martínez-Zapata MJ. The
effect of lifestyle intervention on pregnancy and birth outcomes on obese
infertile women: a systematic review and meta-analysis. Int J Fertil Steril. 2020;14(1):1. 70. Faul F, Erdfelder E, Lang AG, Buchner A. G*power 3: a flexible statistical
power analysis program for the social, behavioral, and biomedical sciences. Behav Res Methods. 2007;39(2):175–91. 92. Bhattacharya S, Maheshwari A, Mollison J. Factors associated with failed
treatment: an analysis of 121,744 women embarking on their first IVF cycles. PLoS One. 2013;8(12):e82249. 71. Prémusz V, Makai A, Gács B, Nagy Á, Perjés B, Ács P, Lampek K, Várnagy Á. Preliminary study on pre-treatment physical activity and quality of life in
infertility. Exerc Qual Life J. 2019;11(2):5–17. 93. Messerlian C, Gaskins AJ: Epidemiologic approaches for studying assisted
reproductive technologies: design, methods, analysis and interpretation. Curr Epidemiol Rep 2017, 4(2):124–132. 93. Messerlian C, Gaskins AJ: Epidemiologic approaches for studying assisted
reproductive technologies: design, methods, analysis and interpretation. Curr Epidemiol Rep 2017, 4(2):124–132. 72. Aarts JW, van Empel IW, Boivin J, Nelen WL, Kremer JA, Verhaak CM. Relationship between quality of life and distress in infertility: a validation
study of the Dutch FertiQoL. Hum Reprod. 2011;26(5):1112–8. Publisher’s Note 73. Dural O, Yasa C, Keyif B, Celiksoy H, Demiral I, Yuksel Ozgor B, Gungor
Ugurlucan F, Bastu E. Effect of infertility on quality of life of women: a
validation study of the Turkish FertiQoL. Hum Fertil. 2016;19(3):186–91. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. 74. Boivin J, Takefman J, Braverman A. The fertility quality of life (FertiQoL) tool:
development and general psychometric properties. Hum Reprod. 2011;
26(8):2084–91. 75. Cserepes RE, Korosi T, Bugan A. A meddőséggel összefüggő életminőség
jellemzői magyar pároknál [Characteristics of infertility-specific quality of life
in Hungarian couples]. Orv Hetil. 2014;155(20):783–8. 76. Domar AD, Gross J, Rooney K, Boivin J. Exploratory randomized trial on the
effect of a brief psychological intervention on emotions, quality of life,
discontinuation, and pregnancy rates in in vitro fertilization patients. Fertil
Steril. 2015;104(2):440–451.e447. 77. Domar AD, Conboy L, Denardo-Roney J, Rooney KL. Lifestyle behaviors in
women undergoing in vitro fertilization: a prospective study. Fertil Steril. 2012;97(3):697–701.e691. 78. Pasch LA, Gregorich SE, Katz PK, Millstein SG, Nachtigall RD, Bleil ME, Adler
NE. Psychological distress and in vitro fertilization outcome. Fertil Steril. 2012;98(2):459–64. 79. Bácsné Bába É, Fenyves V, Szabados G, Pető K, Bács Z, Dajnoki K. Sport
involvement analysis in Hungary, in the north great plain region. Sustainability. 2018;10(5):1629. 79. Bácsné Bába É, Fenyves V, Szabados G, Pető K, Bács Z, Dajnoki K. Sport
involvement analysis in Hungary, in the north great plain region. Sustainability. 2018;10(5):1629.
|
https://openalex.org/W2749530997
|
https://zenodo.org/record/1161816/files/DCROUTE_HIPC_2016.pdf
|
English
| null |
DCRoute: Speeding up Inter-Datacenter Traffic Allocation while Guaranteeing Deadlines
| null | 2,017
|
cc-by
| 8,007
|
DCRoute: Speeding up Inter-Datacenter Traffic
Allocation while Guaranteeing Deadlines Sriram Rao
Microsoft
sriramra@microsoft.com Cauligi S. Raghavendra
University of Southern California
raghu@usc.edu Mohammad Noormohammadpour
University of Southern California
noormoha@usc.edu improve fault-tolerance and user quality of service by making
multiple copies of data and getting data closer to end users. Most of these transfers have to be completed before a deadline
in order to meet customer service level agreements (SLAs)
and they can take hours to complete [7]. For example, search
engines may have to exchange data among datacenters in order
to synchronize their search databases and storage applications
may need to back up user data over certain periods. Abstract—Datacenters provide the infrastructure for cloud
computing services used by millions of users everyday. Many such
services are distributed over multiple datacenters at geograph-
ically distant locations possibly in different continents. These
datacenters are then connected through high speed WAN links
over private or public networks. To perform data backups or data
synchronization operations, many transfers take place over these
networks that have to be completed before a deadline in order to
provide necessary service guarantees to end users. Upon arrival of
a transfer request, we would like the system to be able to decide
whether such a request can be guaranteed successful delivery. If yes, it should provide us with transmission schedule in the
shortest time possible. In addition, we would like to avoid packet
reordering at the destination as it affects TCP performance. Previous work in this area either cannot guarantee that admitted
transfers actually finish before the specified deadlines or use
techniques that can result in packet reordering. In this paper,
we propose DCRoute, a fast and efficient routing and traffic
allocation technique that guarantees transfer completion before
deadlines for admitted requests. It assigns each transfer a single
path to avoid packet reordering. Through simulations, we show
that DCRoute is at least 200 times faster than other traffic
allocation techniques based on linear programming (LP) while
admitting almost the same amount of traffic to the system. As mentioned in [8], inter-datacenter traffic can be cate-
gorized into three groups: interactive traffic that has to be
transmitted as soon as possible since it is in the critical
path of user experience, elastic traffic that requires timely
delivery which can be modeled in the form of a deadline, and
background traffic which is bandwidth hungry and has less
priority than the other two categories of traffic. DCRoute: Speeding up Inter-Datacenter Traffic
Allocation while Guaranteeing Deadlines In this paper,
we focus on elastic traffic with user specified deadlines. Previous work in the context of inter-datacenter traffic
scheduling either fails to consider the negative effects of packet
reordering caused by multiplexing packets over different paths
(AMOEBA [6]) or cannot guarantee that admitted requests will
actually complete transmission before the deadlines specified
by customers (B4 [9], SWAN [8], TEMPUS [7]). In addi-
tion, AMOEBA and TEMPUS which are the state-of-the-art
techniques in this area, model the allocation problem as large
linear programs (LP), with possibly hundreds of thousands
of variables, solving which incurs large memory and CPU
overhead and can take a long time. Index Terms—Datacenter; Routing; Traffic Allocation; Traffic
Scheduling; Deadlines; Wide Area Networks; I. INTRODUCTION Cloud Computing allows customers to build online applica-
tions that can cost effectively scale as necessary [1]. It provides
a massive pool of resources for online applications that can be
flexibly obtained when needed and then returned back to the
pool at a later time. Such resources can be provided to different
applications on top of the infrastructure built and maintained
by a cloud company. Examples of hosted online applications
include video streaming, data storage and sharing, and big
data processing. Most companies that provide cloud computing
services own multiple datacenters placed in different cities and
countries in order to improve availability, reduce end-to-end
delays for end users, and provide customized regional services. At the time of writing this paper, Amazon has more than
two dozen availability zones each consisting of one or more
discrete datacenters [2], Microsoft has 22 regions with plans
to build 8 more [3], and Google relies on more than a dozen
datacenters [4]. Avoiding packet reordering allows data to be instantly
delivered to applications upon arrival of packets. In addition,
inter-datacenter networks have characteristics similar to WAN
networks (including asymmetric link delays and large delays
for links that connect distant locations) for which multiplexing
packets over different paths has been shown to considerably
degrade TCP performance [10]. Putting out of order packets
and segments back in order can be expensive in terms of
memory and CPU usage, especially when transmitting at high
rates. As explained in [11], TCP needs to buffer as much as the
bandwidth-delay product (BDP) of the network in lossless
and 2 × BDP in lossy networks to put out of order packets
back in order. For high speed inter-datacenter networks with
tens of gigabits of speed and tens of milliseconds of latency
considerable amount of buffering may be needed. In [12],
authors show how Vanilla kernel uses 50% more CPU in Many applications hosted on these datacenters need to
transfer data to their peers in other datacenters for the purpose
of data replication and synchronization [5], [6]. The aim is to Central
Controller
DC1
DC2
DC3
Fig. 1. Problem Setup Central
Controller
DC1
DC2
DC3
Fig. 1. Problem Setup presence of severe packet reordering and present Juggler, a
reordering resilient network stack designed for low latency
datacenter networks which can reduce the extra CPU utiliza-
tion to 10%: still a considerable amount. • Works much faster than other techniques while ad-
mitting almost equal traffic to the system. The input to DCRoute is the network topology as well
as a list of transfers including their volumes and deadlines
which are submitted to DCRoute in the order of arrival. DCRoute assigns a single path to every transfer and generates
a transmission schedule which specifies the rate at which every
transfer should be sent over their assigned path during current
timeslot. In this paper, we verify the performance of DCRoute
through long running simulations. In practice, label switching
(such as VLAN tagging) can be used to enforce paths and rate-
limiting at the end hosts can be used to enforce transmission
rates as in SWAN [8]. Upon arrival of a request, a central controller decides
whether it is possible to allocate it considering some criteria
that includes the total available bandwidth over future times-
lots. If there is not enough room to allocate a request, the
request is rejected and can be submitted to the system again
later with a new deadline. A request is considered active if it is accepted into the
system and its deadline has not passed yet. Some active
requests may take many timeslots to complete transmission. The total unsatisfied demand of an active request is called the
residual demand of that request. • Guarantees that admitted transfers complete prior to
specified deadlines. • Schedules all packets of each transfer over the same
path to avoid packet reordering. • Works much faster than other techniques while ad-
mitting almost equal traffic to the system. I. INTRODUCTION For higher latency
networks, due to large variation of RTT over multiple paths,
reordering may further increase CPU utilization. Central
Controller In [13], we proposed RCD, a technique that speeds up
the traffic allocation problem by scheduling transfers close to
their deadlines. Through simulations, we showed that RCD
speeds up the allocation process by allowing new transfers to
be scheduled only considering the residual bandwidth which
would result in creation of much smaller LP models. However,
we did not discuss the reordering problem and only evaluated
our technique for a single link scenario. Fig. 1. Problem Setup inter-datacenter request R is represented with four parameters
src(R), dst(R), vol(R) and dl(R) which stand for source,
destination, volume of data to be transferred and the deadline
prior to which the transfer has to be completed. In this paper, we propose DCRoute, a fast and efficient
routing algorithm which eliminates the need for LP modeling
and: Similar to previous work [6], we assume arriving requests
are put into a queue and processed in the order of arrival and
that there is no preemption: once a request is allocated, it
cannot be unallocated from the system. At each moment, we
have two parameters tnow and tend which represent current
timeslot and the latest deadline among all active requests,
respectively. A request arriving sometime in timeslot t can
be allocated starting timeslot t + 1 since the schedule and
transmission rate for current timeslot is already decided upon
and broadcast into all datacenters. Also, at any moment t, tnow
is the timeslot that includes t (current timeslot), and tnow + 1
is the next available timeslot for allocation (next timeslot). • Guarantees that admitted transfers complete prior to
specified deadlines. II. PROBLEM DESCRIPTION Figure 1 shows our problem setup which is comprised
of multiple datacenters in different locations managed by a
central controller. The datacenters are connected using high
speed WAN links. Applications hosted on these datacenters
will make transfers that may take hours to complete and
have to be finished before specified deadlines. Due to large
transfer time, the time it takes for the source datacenter to
request a path from the central controller and the time it takes
to setup such a path is considered negligible. However, the
time it takes for the scheduling algorithm to prepare a traffic
schedule depends on the scheduling algorithm which we aim
at minimizing. In addition, in order to avoid packet reordering,
we would prefer to send all packets of a transfer on a single
path. Allocation Problem: Given active requests R1 through Rn
with residual demands D1 to Dn (0 ≤Di, 1 ≤i ≤n), is
it possible to allocate a new request Rn+1? If yes, what is a
possible schedule? An important characteristic of network traffic is that the
size of smallest traffic unit (which is a packet) is significantly
smaller than link capacities (which are in the range of gigabits
nowadays). This allows us to solve the allocation problem by
forming a linear program (LP) considering capacity constraints
of the network edges as well as demand constraints of requests. The answer will give us a possible allocation if the constructed
LP is feasible. Although this solution maybe straightforward,
considering the number of active requests, number of links
in network graph, and how far we are planning ahead into
the future (tend), the resulting LP could be large and may
take a long time to solve. One of the ways to speed up this
process is to limit the number of possible paths between every As in [6], [8], we assume the timeline is divided into
properly sized timeslots over which the transmission rate
is constant. Using a slotted timeline allows for schedules
with variable transmission rates over time. In our model, an t
t
t
Transfer 1:
Deadline = d1 & Volume = V1
Transfer 2:
Deadline = d2 & Volume = V2
d1
d2
d1
V1
V1
V2
t
Transfer 3:
Deadline = d3 & Volume = V3
d2
d1
V1
V2
d3
V3
now
now
now
now
Fig. 2. II. PROBLEM DESCRIPTION An example of “As Late As Possible” allocation Figure 2 provides an example of the ALAP allocation
technique. As can be seen, when the first transfer is received
the timeline is empty and therefore it is allocated adjacent
to its deadline. The second transfer is allocated as close as
possible to its deadline. The benefit of this type of scheduling
is that requests do not use resources until it is absolutely
necessary. This means resources will be available to other
requests that currently demand them. Now when the third
transfer is submitted, the resources are free and it just grabs
as much bandwidth as needed. If we had allocated the first
two requests closer to current time we may have had to either
reject the third transfer or move the first two transfers ahead
freeing resources for the third transfer. Transfer 1:
Deadline = d1 & Volume = V1 t
Deadline d1 & Volume V1
V1 g
Assume requests R1 through Rn are current active requests
and we would like to allocate Rn+1. For every request
Ri, 1 ≤i ≤n, we either have dl(Ri) ≤dl(Rn+1) or
dl(Ri) > dl(Rn+1). In the former case, since there is no
preemption, there is no way to increase the chance of new
request being accepted by shifting the traffic allocation of
request i away as it has to be completed before dl(Rn+1). For the latter case, since we allocated all requests ALAP, the
traffic is already shifted out of Rn+1’s window as much as
possible. As a result, it is possible to decide on admission
of new request by just looking at the residual bandwidth on
the links. For a single link, since all requests use a single
shared resource (link capacity) this technique allows us to
optimally decide whether a new request can be allocated. For
a network, each request is routed on multiple links and there
are many ways to schedule requests ALAP. If some link is
used by multiple requests that are routed on different edges,
how traffic is allocated on the common link can affect multiple
other links which will affect the requests that use those links
later on. Despite this uncertainty, we will show that using this
feature, we can greatly speed up the allocation process. II. PROBLEM DESCRIPTION t
Transfer 2:
Deadline = d2 & Volume = V2
d1
d2
d1
V1
V2
now
now Using ALAP alone can result in poor utilization as we
always push traffic towards future timeslots and leave the
current timeslot underutilized. In order to maximize utilization,
upon beginning of a new timeslot, the scheduler looks at
future timeslots and pulls as much traffic as possible from
the closest timeslots in the future to the upcoming timeslot. Pulling from closest timeslot allows the ALAP characteristic
of allocation to hold true afterwards: all residual demands will
still be allocated as close to their deadlines as possible. Fig. 2. An example of “As Late As Possible” allocation pair of nodes [7], for example, using k-shortest paths [6]. While solving the LP, another speedup method is to limit the
number of considered active requests based on some criterion
[6] such as having a common link with the new request. It is
also possible to use customized iterative methods to solve the
resulting LP models faster based on the solutions of previous
LP models in a way similar to the water filling process [7]. While pulling traffic from future timeslots, a request can
span over multiple links and if there is some traffic fully
occupying the next timeslot on one of those links, it is
impossible to pull traffic back for that request from the future
timeslots to the next timeslot. In such cases, we may need
to pull traffic from requests that may not be the closest to
current timeslot. This can result in an allocation in which some
requests are not scheduled ALAP because they can be pushed
further into the future. To address this problem, after pulling
traffic from future timeslots, we sweep requests allocated on
future timeslots and push them forward as much as possible. Our proposition is to avoid building an LP model in the
first place by trying to allocate new requests only knowing the
residual bandwidth on the edges for different timeslots. III. DCROUTE DCRoute relies on the following three techniques: • Requests are initially allocated as late as possible
(ALAP) [14] • Utilization is maximized by pulling traffic from closest
future timeslots into the upcoming timeslot Figure 3 shows an example of this process. There are three
different requests all of which having the same deadline. It • A variant of BFS search is used for path selection riant of BFS search is used for path selection is not possible to pull back the green request as the link E1
is already occupied. Therefore, we have to pull the orange
request (PullBack phase). Afterwards, the allocation is not
ALAP anymore, so we push the green request towards its
deadline (PushForward phase). The final allocation is ALAP
and the utilization of upcoming timeslot is maximum. III. DCROUTE Algorithm 1 1: procedure ALLOCATE(R)
2:
for t = tend + 1 to R.dl do
3:
for e ∈E do
4:
St,e ←St−1,e
5:
tend ←max(R.dl, tend)
6:
for e ∈E do
7:
e.use ←T rue
8:
for v ∈V do
9:
{v.v, v.r, v.rv, v.rs} ←{F alse, 0, 0, 0}
10:
SE ←array of edges sorted by SR.dl,e descending
11:
i ←0, j ←1, flag ←T rue, rb ←BFS test(R)
12:
while rb.r is not −1 do
13:
if (rb.rv > 0) and flag then
14:
flag ←F alse
15:
while SR.dl,SE[i] > rb.rv do
16:
SE[i].use ←F alse
17:
i ←i + 1
18:
SE[i].use ←F alse, i ←i + 1
19:
rn ←BFS test(R)
20:
if rn.r == −1 then break
21:
α ←rb.r × R.vol + rb.rs
22:
β ←rn.r × R.vol + rn.rs
23:
if (α > β) or ((α == β) and (rb.rv > rn.rv)) then
24:
j ←i + 1, rb ←rn, flag ←T rue
25:
while i ≥j do
26:
i ←i −1, SE[i].use ←T rue
27:
path ←edges of the path ending at R.dst on the BFS
28:
tree starting at R.src
29:
if PathAllocate(path, R, F alse) then
30:
return PathAllocate(path, R, T rue)
31:
else
32:
return F alse
33: procedure BFS TEST(R)
34:
for v ∈V do
35:
{v.v, v.r, v.rv, v.rs} ←{F alse, 0, 0, 0}
36:
Q ←a queue having R.src as first element
37:
R.src.v ←T rue
38:
while Q is not empty do
39:
node ←head of the Q removed
40:
if node == R.dst then
41:
return {node.r, node.rv, node.rs}
42:
for next ∈all neighbors of node do
43:
e ←edge connecting node to next
44:
if e.use == T rue and next.v == F alse then
45:
add next to Q
46:
next.v ←T rue
47:
next.r ←next.r + 1
48:
next.rv ←max(node.rv, SR.dl,e)
49:
next.rs ←node.rv + SR.dl,e
50:
return {−1, −1, −1}
51: procedure PATHALLOCATE(path, R, apply)
52:
vol ←R.vol
53:
for t = R.dl to tnow + 2 step −1 do
54:
space ←vol
55:
for e ∈path do
56:
space ←min(space, 1 −Stnow+1,e)
57:
if space > 0 then
58:
vol ←vol −space
59:
if apply then
60:
for e ∈path do
61:
add space of R to edge e at time t
62:
for t′ = t to tend do
63:
St′,e ←St′,e + space
64:
return vol == 0 A. DCRoute Algorithms We assume a graph G(V, E) connecting datacenters with
M bidirectional links. For simplicity, we also assume all
links/edges have equal capacity of 1.0. Every edge e has a
boolean use property which identifies whether that edge can
be used in the course of routing. Also each node v has 4
parameters v, r, rv and rs. If p is the path on BFS tree from
src(R) ending at v, these parameters represent whether v has
been visited before, the number of hops to v from source, the
load of bottleneck link, and the sum of loads on all edges
from source to v, respectively. Moreover, we have variables
St,m that represent the total sum of traffic over link m from
time tnow + 1 to t. Every time a new request is submitted to
the system, tend is updated so that it covers all active requests. Finally, we define the active window as the set of all timeslots
over all edges from time tnow +1 to tend. Our algorithms only
operate on the active window. Allocate(R): Algorithm 1 is executed upon arrival of a
new request R and performs multiple BFS searches. In every
search, we calculate multiple costs, remove edges with highest
costs, and compare the result with previous steps. While
examining different paths, our heuristic finds the path with
most preferred characteristics: the total sum of traffic before
dl(R) over the chosen path will be minimal compared to other
paths when R gets allocated on that path. In each round, the path assignment algorithm starts by
choosing the path with the least number of hops and calculates
the total cost of sending the request on that path. Also, the
bottleneck link on that path is identified. If the total cost
is less than the best path found in previous steps, this path
replaces the best path. Next, all edges with an equal or higher
cost than the bottleneck edge are removed from the network. This process continues until there is no path from source to
destination. Now, if it is possible to allocate the request on the
selected path, we apply the allocation, otherwise, the request
is rejected since admitting it might result in many more future
requests to be rejected. PullBack(): Algorithm 2 looks at the timeslots starting
tnow + 2 to tend and pulls back traffic to tnow + 1 (next
timeslot to be scheduled). A. DCRoute Algorithms An example of improving utilization while keeping the final allocation ALAP Algorithm 2
1: procedure PULLBACK()
2:
for t = tnow + 1 to t = tend do
3:
for e ∈E do
4:
reqs ←all requests allocated on e at t
5:
for R ∈reqs do
6:
vol ←how much of R allocated on e at t
7:
path ←path assigned to R upon allocation
8:
for e′ ∈path do
9:
vol ←min(vol, 1 −Stnow+1,e′)
10:
if vol > 0 then
11:
for e′ ∈path do
12:
move vol of R from edge e′ at t
13:
to edge e′ at tnow + 1
14:
for t′ = tnow + 1 to t′ = t do
15:
St′,e′ ←St′,e′ + vol
Algorithm 3
1: procedure PUSHFORWARD()
2:
for t = tnow + 2 to t = tend do
3:
for e ∈E do
4:
reqs ←all requests allocated on e at t
5:
for R ∈reqs do
6:
vol ←how much of R allocated on e at t
7:
path ←path assigned to R upon allocation
8:
for t2 = R.dl to t2 = t + 1 step −1 do
9:
free ←min(vol, free space on e at t2)
10:
for e′ ∈path do
11:
free ←min(free, free space on e′ at t2)
12:
if free > 0 then
13:
for e′ ∈path do
14:
remove free of R from edge e′ at t
15:
add free of R to edge e′ at t2
16:
for t′ = t to t′ = t2 do
17:
St′,e′ ←St′,e′ −free Algorithm 4
1: procedure WALK()
2:
Broadcast the schedule for tnow + 1 to all datacenters
3:
for e ∈E do
4:
D ←Stnow+1
5:
for t = tnow + 1 to t = tend do
6:
St,e ←St,e −D
7:
tnow ←tnow + 1
8:
tend ←max(tend, tnow + 1) Algorithm 4
1: procedure WALK()
2:
Broadcast the schedule for tnow + 1 to all datacenters
3:
for e ∈E do
4:
D ←Stnow+1
5:
for t = tnow + 1 to t = tend do
6:
St,e ←St,e −D
7:
tnow ←tnow + 1
8:
tend ←max(tend, tnow + 1) limiting every transfer to a single path results in 2% less
utilization in the worst case. A. DCRoute Algorithms To use the aggregate bandwidth on multiple paths, one
can use Multi-Path TCP (MPTCP) [15] which allows sending
traffic from multiple interfaces of a single host. MPTCP is
based on multiple sub-flows that behave similar to single TCP
flows. While using MPTCP, the overall CPU and memory
footprint can increase significantly compared to TCP which is
the cost paid for increased bandwidth. However, by carefully
choosing which parts of the data goes over what path, it is
possible to improve the CPU footprint [11]. Algorithm 3
1: procedure PUSHFORWARD()
2:
for t = tnow + 2 to t = tend do
3:
for e ∈E do
4:
reqs ←all requests allocated on e at t
5:
for R ∈reqs do
6:
vol ←how much of R allocated on e at t
7:
path ←path assigned to R upon allocation
8:
for t2 = R.dl to t2 = t + 1 step −1 do
9:
free ←min(vol, free space on e at t2)
10:
for e′ ∈path do
11:
free ←min(free, free space on e′ at t2)
12:
if free > 0 then
13:
for e′ ∈path do
14:
remove free of R from edge e′ at t
15:
add free of R to edge e′ at t2
16:
for t′ = t to t′ = t2 do
17:
St′,e′ ←St′,e′ −free To increase network utilization, it is possible to use MPTCP
and apply DCRoute to all sub-flows created. To do that, one
can divide the total demand of a request over multiple sub-
flows and assign the same deadline as the deadline of the
original request to all of them. If all sub-flows can be allocated
with DCRoute, then the request is accepted. Deciding on the number of sub-flows and the portion of
traffic that goes over each one of them is not a trivial problem. It may be possible to extend DCRoute by ranking the paths
found in Allocate procedure and choosing a subset of the best
paths. Further discussion of this subject is out of the scope of
this paper. Walk(): Algorithm 4 is executed when the allocation for
next timeslot is final. This algorithm tells each datacenter of
the decided allocation and adjusts request demands accord-
ingly by deducting what is scheduled to be sent from the total
demand. A. DCRoute Algorithms An example of improving utilization while keeping the final allocation ALAP
thm 2
dure PULLBACK()
r t = tnow + 1 to t = tend do
for e ∈E do
reqs ←all requests allocated on e at t
for R ∈reqs do
vol ←how much of R allocated on e at t
path ←path assigned to R upon allocation
for e′ ∈path do
vol ←min(vol, 1 −Stnow+1,e′)
if vol > 0 then
Algorithm 4
1: procedure WALK()
2:
Broadcast the schedule for tnow + 1 to all datacenters
3:
for e ∈E do
4:
D ←Stnow+1
5:
for t = tnow + 1 to t = tend do
6:
St,e ←St,e −D
7:
tnow ←tnow + 1
8:
tend ←max(tend, tnow + 1) B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
t
t
t B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
t
t
t Fig. 3. A. DCRoute Algorithms When pulling back traffic, all edges
on a request’s path have to be checked for unused capacity
and updated together as we pull traffic back. PullBack(): Algorithm 2 looks at the timeslots starting
tnow + 2 to tend and pulls back traffic to tnow + 1 (next
timeslot to be scheduled). When pulling back traffic, all edges
on a request’s path have to be checked for unused capacity
and updated together as we pull traffic back. PushForward(): After pulling some traffic back, it may be
possible for some other traffic to be pushed ahead even further. Algorithm 3 scans all future timeslots starting tnow + 2 and
makes sure that all demands are allocated ALAP. If not, it
moves as much traffic as possible to the future timeslots until
all residual demands are ALAP. A
B
C
D
E3
E1
E2
The red dashed line is the
deadline for all 3 requests A
B
C
D
E3
E1
E2
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
PullBack()
PushForward()
The red dashed line is the
deadline for all 3 requests
t
t
t
t
t
t
t
t
t
Fig. 3. An example of improving utilization while keeping the final allocation ALAP C
D
E3
E1
E2
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
B→C
B→D
B→D
A→D
A→D
E1
E2
E3
Tnow+1
PullBack()
PushForward()
red dashed line is the
dline for all 3 requests
t
t
t
t
t
t
t
t
t
Fig. 3. A. Google’s GScale Network GScale network [9] comprised of 12 nodes and 19 links (at
2013, they have 15 datacenters as of 2016 [4]) is a private
network that connects Google data centers. We used the same
topology to evaluate DCRoute as well as other allocation
schemes. Figure 4 shows the rejection rate of different tech-
niques for different arrival rates from low load (λ = 1) to high
load (λ = 15). We have included the schemes that potentially
multiplex traffic over multiple paths just to provide a lower
bound. Comparing with PMC and SPMC schemes over all
arrival rates, DCRoute performs < 2% worse than the one
with minimum rejection rate. Also, compared to all schemes,
DCRoute rejects at most 4% more traffic. All simulations were performed on a machine with an
Intel Core i7-6700T CPU and 24 GBs of memory and the
algorithms are coded in Java. To solve linear programs faster,
we used Gurobi Optimizer [16] with a free academic license. Gurobi Optimizer can speedup the LP solving process using
several techniques including parallel processing. All simula-
tions presented here are performed 3 times and the average is
reported. We compare DCRoute with the following allocation
schemes for all of which we used the same objective function
as [6]: Figure 4 shows the relative time to process a request using
different schemes. This time is calculated dividing the total
time to allocate/adjust all requests over all timeslots by the
total number of requests. DCRoute is about 3 orders of
magnitude faster than either PMC or SPMC. It should be
noted that the rate at which time complexity grows drops
as we move towards higher arrival rates since there is less
capacity available for new requests and many arriving requests
get rejected by failing simple capacity constraint checks. Global LP: This technique is the most general and flexible
way of allocation which routes traffic over all possible edges. All active requests are considered for all timeslots on all edges
creating a potentially large linear program. The solution here
gives us a lower bound on traffic rejection rate. K-Shortest Paths: Same as Global LP, however, only the
K-Shortest Paths between each pair of nodes are considered
in routing. The traffic is allocated using a linear program over
such paths. We simulated four cases of K ∈{1, 3, 5, 7}. B. DCRoute and Multi-Path Routing In this section, we perform simulations to evaluate the per-
formance of DCRoute. We generate synthetic traffic requests
with Poisson arrival and input the traffic to both DCRoute and
a few other techniques that can be used for traffic allocation. Two metrics are being measured and compared: allocation As mentioned earlier, to avoid packet reordering, DCRoute
maps every transfer to exactly one path and if there is no single
path that can allocate a transfer, it will be rejected. Although
this sounds too restrictive, we show in the next section that time and portion of rejected traffic both of which are desired
to be small. • Shortest Path, Minimum Cost (SPMC): Amongst all
shortest paths that result in a feasible solution and have
the least number of hops, we choose the one with smallest
objective value. Simulation Parameters: We used the same traffic dis-
tributions as described in [6]. Requests arrive with Poisson
distribution of rate λ. Also, total demand of each request
R is distributed exponentially with mean
1
8 proportional to
the maximum transmission volume possible prior to dl(R). In
addition, the length of requests is exponentially distributed for
which we assumed a mean of 10 timeslots. We performed the
simulations over 500 timeslots. A. Google’s GScale Network It
is obvious that as K increases, the overall rejection rate will
decrease as we have higher flexibility for choosing paths and
multiplexing traffic. B. Variable Network Size B. Variable Network Size We simulated different methods against four networks from
5 to 20 nodes: (N, M) ∈{(5, 7), (10, 17), (15, 27), (20, 37)} (N, M) ∈{(5, 7), (10, 17), (15, 27), (20, 37)} Pseudo-Integer Programming (PIP): In terms of traffic
rejection rate, comparing DCRoute with the previous two
techniques is not fair as they allow multiplexing packets on
multiple paths. The aim of this technique is to find a
lower bound on traffic rejection rate when all packets
of each request are sent over a single path. To do so,
the general way is to create an integer program involving a
list of possible paths (maybe all paths) for the new request
and fixed paths for requests already allocated. The resulting
model would be a non-linear integer program which cannot
be solved using standard optimization libraries available. We
instead created a number of linear programs each assigning
one of the possible K-Shortest Paths for the newly arriving
request. We then compare the objective values manually and
choose the best possible path. In our implementation, we chose
K = 20. This K seems to be more than necessary as we
saw negligible improvement in traffic rejection rate even when
increasing K from 5 to 7. Using PIP, the path over which
a request is transferred is decided upon admission and does
not change afterwards. We implemented two versions of this
scheme: Pseudo-Integer Programming (PIP): In terms of traffic
rejection rate, comparing DCRoute with the previous two
techniques is not fair as they allow multiplexing packets on
multiple paths. The aim of this technique is to find a
lower bound on traffic rejection rate when all packets
of each request are sent over a single path. To do so,
the general way is to create an integer program involving a
list of possible paths (maybe all paths) for the new request
and fixed paths for requests already allocated. The resulting
model would be a non-linear integer program which cannot
be solved using standard optimization libraries available. We
instead created a number of linear programs each assigning
one of the possible K-Shortest Paths for the newly arriving
request. We then compare the objective values manually and
choose the best possible path. In our implementation, we chose
K = 20. B. Variable Network Size This K seems to be more than necessary as we
saw negligible improvement in traffic rejection rate even when
increasing K from 5 to 7. Using PIP, the path over which
a request is transferred is decided upon admission and does
not change afterwards. We implemented two versions of this
scheme: In our topology, each node was connected to 3 or 4 other
nodes at most 2 hops away. The arrival rate was kept constant
at λ = 6.0 for all cases. Figure 5 shows the rejection rate of different schemes for
different network sizes. As network size increases, since λ is
kept constant, the total cpacity of network increases compared
to the total demand of requests. As a result, for a scheme that
multiplexes request traffic over different paths, we expect to
see decrease in rejection rate. For the K-Shortest Paths case
with K ∈{1, 3} we see increase in rejection rate which we
think is because these schemes cannot multiplex packets that
much. Increasing the network size for these cases can cause
more requests to have common links as the network is sparsely
connected and create more bottlenecks resulting in a higher
rejection rate. PMC has a high rejection rate for small networks since
choosing the minimum cost path might result in selecting
longer (more hops) paths that create larger number of bottle-
necks due to collision with other requests. Increasing network
size, there are more paths to choose from and that results in
less bottlenecks and therefore less rejection rate. In contrast,
SPMC enforces the selection of paths with smaller number of
hops resulting in lower rejection rates for small networks (due • Pure Minimum Cost (PMC): We choose the path that
results in smallest objective value. • Pure Minimum Cost (PMC): We choose the path that
results in smallest objective value. Arrival Rate ()
2
4
6
8
10
12
14
% of Rejected Traffic
0
5
10
15
20
25
30
35
40
45
DCRoute
Global LP
K-Shortest Paths (K=1)
K-Shortest Paths (K=3)
K-Shortest Paths (K=5)
K-Shortest Paths (K=7)
SPMC
PMC
Arrival Rate ()
2
4
6
8
10
12
14
Relative Time to Process a Request
100
101
102
103
104
105
Fig. 4. B. Variable Network Size Total % of rejected traffic and relative request processing time for GScale network with 12 nodes and 19 links Arrival Rate ()
2
4
6
8
10
12
14
Relative Time to Process a Request
100
101
102
103
104
105 Arrival Rate ()
2
4
6
8
10
12
14
% of Rejected Traffic
0
5
10
15
20
25
30
35
40
45
DCRoute
Global LP
K-Shortest Paths (K=1)
K-Shortest Paths (K=3)
K-Shortest Paths (K=5)
K-Shortest Paths (K=7)
SPMC
PMC ig. 4. Total % of rejected traffic and relative request processing time for GScale network with 12 nodes and 19 links (λ = 6). We only considered the K-Shortest Paths techniques
with K ∈{3, 7} since during previous tests they provided the
best utilization at the least processing cost. It appears that if
the timeslots are short enough, making them shorter does not
improve the load accepted into the system: DCRoute rejects
2% more traffic compared to K-Shortest Paths schemes for all
timeslot resolutions. Although DCRoute is always more than
2 orders of magnitude faster than K-Shortest Paths schemes,
it does have a slightly higher growth rate in processing time
as the timeslot resolution increases. to request paths colliding less) and more rejections as network
grows due to less diversity of chosen paths. Compared to these two approaches, DCRoute balances the
choice between smaller and longer paths. The assigned path
has the least sum of load on the entire path and the least
bottleneck load among all such paths. Paths with heavily
loaded links and unnecessarily larger number of hops are
avoided. As a result, rejection rate compared to min(PMC,
SPMC) is relatively small (< 3%) for all network sizes. Also,
as Figure 5 shows, similar to previous simulation, DCRoute
is almost three orders of magnitude faster than PIP schemes
and more than 200 times faster than all considered schemes. D. Discussion We do not directly compare our scheme with any of the
previous schemes, such as AMOEBA or TEMPUS, since we
pursue the following two objectives together which is not the
case for previous work: C. Effect of Timeslot Length As mentioned earlier, the timeline is divided into timeslots. In this paper, we do not discuss the exact duration of timeslots,
however we would like to see how smaller timeslots affect
the amount of rejected traffic as well as the speed of different
methods. The transmission rate of transfers only changes when
moving from one timeslot to the next. Having timeslots that
last longer than most requests would essentially result in a
fixed transmission rate for such transfers. This causes less
critical transfers (with a later deadline) to have to share the
bandwidth with more critical transfers (with a closer deadline),
reducing the flexibility of the system and increasing the
number of rejected transfers. Having very short timeslots on
the other hand can result in unnecessary scheduling overhead
and practical complications. For example, rate-limiters at the
end hosts may not be able to converge to specified rate if they
have to change rate too often. Therefore, a proper timeslot
length could be a few times smaller than most requests but
large enough to avoid unnecessary processing overhead. • Avoiding any packet reordering • Guaranteeing deadlines for admitted transfers • Guaranteeing deadlines for admitted transfers However, since AMOEBA is the most similar to DCRoute,
we would like to provide an approximate comparison. We
argue that AMOEBA is essentially an extension to the K-
Shortest Paths LP technique (K = 10) introduced here. By
intelligently avoiding LP modeling for some special cases,
AMOEBA provides a speedup of 30 times compared to 10-
Shortest Paths LP for (λ = 8) [6]. For the same arrival rate and
request volume/deadline distribution, DCRoute performs more
than 1000 times faster than 10-Shortest Paths LP technique. Also in a previous work called RCD [13], we showed how
the close to deadline scheduling technique can speed up the
traffic allocation by up to 15 times compared to AMOEBA
while resulting in same utilization over a single link. DCRoute
is based on RCD coupled with a path selection heuristic that
eliminates the need for LP modeling. As shown in Figure 6, we divide every timeslot into 2 to 5
smaller timeslots. We again use GScale topology and consider Network Size (# Nodes)
5
10
15
20
% of Rejected Traffic
0
2
4
6
8
10
12
14
16
18
20
= 6.0
DCRoute
Global LP
K-Shortest Paths (K=1)
K-Shortest Paths (K=3)
K-Shortest Paths (K=5)
K-Shortest Paths (K=7)
SPMC
PMC
Network Size (# Nodes)
5
10
15
20
Relative Time to Process a Request
100
101
102
103
104
105
= 6.0
Fig. 5. Total % of rejected traffic and relative request processing time for different network sizes
Timeslot Resolution (# timeslots per original timeslot)
1
2
3
4
5
Relative Request Processing Time
100
101
102
103
104
= 6.0
Timeslot Resolution (# timeslots per original timeslot)
1
2
3
4
5
% of Rejected Traffic
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
= 6.0
DCRoute
K-Shortest Paths (K=3)
K-Shortest Paths (K=7)
Fig. 6. Total % of rejected traffic and relative request processing time as timeslot resolution increases
V. CONCLUSIONS AND FUTURE WORK
In this paper, we proposed DCRoute, a routing algorithm
and 2% less traffic compared to schemes that schedule all
packets of each transfer on the same path. • Guaranteeing deadlines for admitted transfers Finally, we studied the effect of timeslot resolution and Network Size (# Nodes)
5
10
15
20
% of Rejected Traffic
0
2
4
6
8
10
12
14
16
18
20
= 6.0
DCRoute
Global LP
K-Shortest Paths (K=1)
K-Shortest Paths (K=3)
K-Shortest Paths (K=5)
K-Shortest Paths (K=7)
SPMC
PMC
Network Size (# Nodes)
5
10
15
20
Relative Time to Process a Request
100
101
102
103
104
105
= 6.0
Fig. 5. Total % of rejected traffic and relative request processing time for different network sizes Network Size (# Nodes)
5
10
15
20
% of Rejected Traffic
0
2
4
6
8
10
12
14
16
18
20
= 6.0
DCRoute
Global LP
K-Shortest Paths (K=1)
K-Shortest Paths (K=3)
K-Shortest Paths (K=5)
K-Shortest Paths (K=7)
SPMC
PMC Network Size (# Nodes)
5
10
15
20
Relative Time to Process a Request
100
101
102
103
104
105
= 6.0 Fig. 5. Total % of rejected traffic and relative request processing time for different network sizes Timeslot Resolution (# timeslots per original timeslot)
1
2
3
4
5
Relative Request Processing Time
100
101
102
103
104
= 6.0 1
2
3
4
5
% of Rejected Traffic
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
= 6.0
DCRoute
K-Shortest Paths (K=3)
K-Shortest Paths (K=7) Timeslot Resolution (# timeslots per original timeslot)
1
2
3
4
5
Relative Request Processing Time
100
101
102
103
104
= 6.0
Timeslot Resolution (# timeslots per original timeslot)
1
2
3
4
5
% of Rejected Traffic
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
= 6.0
DCRoute
K-Shortest Paths (K=3)
K-Shortest Paths (K=7)
Fig. 6. Total % of rejected traffic and relative request processing time as timeslot resolution increases Fig. 6. Total % of rejected traffic and relative request processing time as timeslot resolution increases V. CONCLUSIONS AND FUTURE WORK and 2% less traffic compared to schemes that schedule all
packets of each transfer on the same path. In this paper, we proposed DCRoute, a routing algorithm
for Inter-Datacenter networks which guarantees that trans-
fers complete before their deadlines and to avoid reordering,
schedules all packets of a request on the same path. Inspired
by the fact that allocating ALAP allows for new requests to
be scheduled considering only residual bandwidth, DCRoute
performs much faster than schemes based on LP modeling. It
is more than 2 orders of magnitude faster than all simulated
schemes and 3 orders of magnitude faster than schemes that
do not multiplex transfer packets on multiple paths. Finally, we studied the effect of timeslot resolution and
found out that making timeslots smaller than necessary does
not increase the admitted load and only incurs extra processing
costs. In this paper, we evaluated DCRoute with synthetic
traffic. Evaluation of DCRoute using real inter-datacenter
traffic is suggested as a future work. In addition, studying the
effects of link failures and developing methods to properly
handle them can be a subject for future work. ACKNOWLEDGEMENT We showed that DCRoute admits at most 4% less traffic
compared to schemes that multiplex packets on multiple paths
(which provide an estimate of lower bound on rejected traffic) We would like to thank the anonymous reviewers of HiPC
whose suggestions and comments helped us improve the
quality of this paper. REFERENCES [1] Michael Armbrust, Armando Fox, Rean Griffith, Anthony D. Joseph,
Randy Katz, Andy Konwinski, Gunho Lee, David Patterson, Ariel
Rabkin, Ion Stoica, and Matei Zaharia. A view of cloud computing. Commun. ACM, 53(4):50–58, April 2010. p
[2] Amazon web services (aws) - cloud computing services. https://aws. amazon.com/. [3] Microsoft azure: Cloud computing platform & services. https://azure. microsoft.com/. [4] Compute engine - iaas - google cloud platform. https://cloud.google. com/compute/. p
[5] Ravi Kiran R Poluri, Samir V Shah, Rui Chen, and Lin Huang. Datacenter synchronization, October 16 2012. US Patent 8,291,036. [6] Hong Zhang, Kai Chen, Wei Bai, Dongsu Han, Chen Tian, Hao Wang,
Haibing Guan, and Ming Zhang. Guaranteeing deadlines for inter-
datacenter transfers. In Proceedings of the Tenth European Conference
on Computer Systems, page 20. ACM, 2015. p g
[7] Srikanth Kandula, Ishai Menache, Roy Schwartz, and Spandana Raj
Babbula. Calendaring for wide area networks. ACM SIGCOMM
Computer Communication Review, 44(4):515–526, 2015. [8] Chi-Yao Hong, Srikanth Kandula, Ratul Mahajan, Ming Zhang, Vijay
Gill, Mohan Nanduri, and Roger Wattenhofer. Achieving high utilization
with software-driven wan. In Proceedings of the ACM SIGCOMM 2013
Conference on SIGCOMM, SIGCOMM ’13, pages 15–26, New York,
NY, USA, 2013. ACM. [9] Sushant Jain, Alok Kumar, Subhasree Mandal, Joon Ong, Leon
Poutievski, Arjun Singh, Subbaiah Venkata, Jim Wanderer, Junlan Zhou,
Min Zhu, et al. B4: Experience with a globally-deployed software
defined wan. ACM SIGCOMM Computer Communication Review,
43(4):3–14, 2013. [10] M. Laor and L. Gendel. The effect of packet reordering in a backbone
link on application throughput. IEEE Network, 16(5):28–36, Sep 2002. [11] Costin Raiciu, Christoph Paasch, Sebastien Barre, Alan Ford, Michio
Honda, Fabien Duchene, Olivier Bonaventure, and Mark Handley. How
hard can it be? designing and implementing a deployable multipath tcp. In Proceedings of the 9th USENIX conference on Networked Systems
Design and Implementation, pages 29–29. USENIX Association, 2012. [12] Yilong Geng, Vimalkumar Jeyakumar, Abdul Kabbani, and Mohammad
Alizadeh. Juggler: a practical reordering resilient network stack for
datacenters. In Proceedings of the Eleventh European Conference on
Computer Systems, page 20. ACM, 2016. [13] M. Noormohammadpour, C. S. Raghavendra, S. Rao, and A. M. ACKNOWLEDGEMENT Madni. Rcd: Rapid close to deadline scheduling for datacenter networks. In
World Automation Congress (WAC), pages 1–6. IEEE, 2016. [14] K.Y. Li and R.J. Willis. An iterative scheduling technique for resource-
constrained project scheduling. European Journal of Operational
Research, 56(3):370 – 379, 1992. [15] Alan Ford, Costin Raiciu, Mark Handley, and Olivier Bonaventure. Tcp
extensions for multipath operation with multiple addresses, 2013. [16] Inc. Gurobi Optimization. Gurobi optimizer reference manual, 2016. . Gurobi Optimization. Gurobi optimizer reference manual, 2
|
https://openalex.org/W3045717117
|
http://telkomnika.uad.ac.id/index.php/TELKOMNIKA/article/download/15199/9152
|
English
| null |
An energy-efficient cluster head selection in wireless sensor network using grey wolf optimization algorithm
|
Telkomnika
| 2,020
|
cc-by-sa
| 8,704
|
TELKOMNIKA Telecommunication, Computing, Electronics and Control
Vol. 18, No. 6, December 2020, pp. 2822~2833
ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018
DOI: 10.12928/TELKOMNIKA.v18i6.15199 TELKOMNIKA Telecommunication, Computing, Electronics and Control
Vol. 18, No. 6, December 2020, pp. 2822~2833
ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/KPT/2018
DOI: 10.12928/TELKOMNIKA.v18i6.15199 TELKOMNIKA Telecommunication, Computing, Electronics and Control
Vol. 18, No. 6, December 2020, pp. 2822~2833
ISSN: 1693-6930, accredited First Grade by Kemenristekdikti, Decree No: 21/E/K
DOI: 10.12928/TELKOMNIKA.v18i6.15199 Corresponding Author: Corresponding Author: Sangsoon Lim,
Department of Computer Engineering,
Sungkyul University,
Anyang, South Korea. Email: lssgood80@gmail.com An energy-efficient cluster head selection in wireless sensor
network using grey wolf optimization algorithm Kaushik Sekaran1, R. Rajakumar2, K. Dinesh3, Y. Rajkumar4, T. P. Latchoumi5,
Seifedine Kadry6, Sangsoon Lim7 Kaushik Sekaran1, R. Rajakumar2, K. Dinesh3, Y. Rajkumar4, T. P. Latchoumi5,
Seifedine Kadry6, Sangsoon Lim7
1Department of Computer Science and Engineering, Vignan Institute of Technology & Science, India
2,4,5Department of Computer Science and Engineering, Vignan's Foundation for Science, Technology & Research, India
3School of Computing and Science and Engineering, Vellore Institute of Technology, India
6Department of mathematics and computer science, Faculty of Science, Beirut Arab University, Lebanon
7Department of Computer Engineering, Sungkyul University, South Korea Keywords: Cluster head
Grey wolf optimization
Network lifetime
Residual energy
Wireless sensor network This is an open access article under the CC BY-SA license. This is an open access article under the CC BY-SA license. Corresponding Author:
Sangsoon Lim,
Department of Computer Engineering,
Sungkyul University,
Anyang, South Korea. Email: lssgood80@gmail.com ABSTRACT Clustering is considered as one of the most prominent solutions to preserve the
energy in the wireless sensor networks. However, for optimal clustering, an
energy efficient cluster head selection is quite important. Improper selection
of cluster heads (CHs) consumes high energy compared to other sensor nodes
due to the transmission of data packets between the cluster members and the
sink node. Thereby, it reduces the network lifetime and performance of the
network. In order to overcome the issues, we propose a novel cluster head
selection approach using grey wolf optimization algorithm (GWO) namely
GWO-CH which considers the residual energy, intra-cluster and sink distance. In addition to that, we formulated an objective function and weight parameters
for an efficient cluster head selection and cluster formation. The proposed
algorithm is tested in different wireless sensor network scenarios by varying
the number of sensor nodes and cluster heads. The observed results convey
that the proposed algorithm outperforms in terms of achieving better network
performance compare to other algorithms. Received Jan 7, 2020
Revised Mar 22, 2020
Accepted Jun 25, 2020 1.
INTRODUCTION Wireless sensor networks can be expounded as a collation of crammed dissipation of ad-hoc sensor
nodes that acts as a watchdog which provides contiguous information about its surroundings which are coagulated
in a central processing node called sink. Due to its compactness and low value, it has been predominantly used
across different kinds of monopoly such as military, health, education, design and engineering sectors. It has
grabbed its attention because of the applications that cater to diverse variants have been discovered. In a packed
environment of nodes, routing poses a hefty concern. It is obvious since nodes are optimal in size, energy is also
an argument where lots of research has been concerted. Since the nodes are battery-powered devices which are
deployed in a downtrodden area, it is not possible to reconstitute back which poses a limitation in case of a vast
set up of wireless sensor networks [1-3]. One of the most poignant stipulations in the deployment of nodes in
wireless sensor network (WSN) is to exercise the energy that is stored in the nodes. Countering to this need, many Journal homepage: http://journal.uad.ac.id/index.php/TELKOMNIKA TELKOMNIKA Telecommun Comput El Control 2823 protocols and schemes have been evolved. Since routing mainly confides on battery power, clustering captures
its attention among the researches due to its efficiency during information exchange. Clustering can be defined as
a grouping of nodes based on parameters such as proximity, range, power, and location, [4-6]. Cluster-based
sensors aids to utilize the resources efficiently in wireless sensor networks. Clustering facilitates the cluster
members to transmit data only to cluster heads (CHs) and then the CHs transmits the collected data to the base
station and thereby reducing the energy consumption and minimizing the routing overhead as shown in Figure 1. However, the communication cost of data is higher than the processing; therefore, clustering the sensors will be
beneficial. The central processing unit is mainly responsible for the intimating the common mob about the
happenings that have been captured from the down-trodden environment. Clustering provides many leverages
which include; a) ease of deployment; b) wide area coverage; c) fault tolerance; and d) energy conservation. During the dissipation of information from one node to the other, several nodes may contain the same redundant
information resulting in huge energy consumption. However, the selection of cluster heads poses a problem
against the lifetime of the network [7, 8]. Figure 1. 2.1. Heuristic-based clustering algorithm Since diverse algorithms catering to different needs are there, low-energy adaptive clustering
(LEACH) [20] is of the predominant clustering algorithm which elects the cluster head with some feasibility. It provides aggregation of the crammed data thus reducing the unwanted traffic and energy consumption of
the network [21], thereby increasing the longevity of the network. However, it does not provide any adequate
information about the number of cluster heads in a network. Sometimes it may opt a node with low energy as
a cluster head thereby shortening the lifetime of the network. Other most popular algorithms include
power-efficient gathering in sensor information systems (PEGASIS) and hybrid energy-efficient distributed
(HEED). PEGASIS [22] is an addendum to that of LEACH protocol. It is more advantageous in the sense
because it aggregates all the data and sends it to the central processing unit. However, it introduces an additional
lag if nodes are distant. It is unsuitable for large scale WSNs which involves multi-hop communication. HEED
[23] is also an extension of the LEACH; it suffers from serious communication overhead between a cluster
head and a base station. In the case of E-LEACH [14], the cluster head communication between different
clusters is highly efficient, but in the case of larger networks, it fails to select the nodes with low energy. TL-LEACH [24] increases the lifetime of the network, but it wastes the energy while performing
communication between cluster heads and the other nodes. M-LEACH carries an advantage by considering
mobility in a routing protocol. It assumes that all the nodes are congruent, and it does not care about the
formation of the cluster while clustering. B-LEACH [25] is another extension where the communication is
entirely depending upon the position of the cluster heads which needs no information about all the other nodes
inside the cluster. Therefore, the residual energy of the CHs gets drained which further reduces the lifetime of
the network. LEACH-C [26, 27] outperforms LEACH-A, LEACH-B, and MTE because the central processing
unit takes care of the location and the energy of all the nodes in the network, hence cluster formation and cluster
maintenance will not get affected. The only disadvantage is that it is not vigorous. E-LEACH is much energy
efficient in case of multi-hop communication. It enhances the cluster head selection process by considering the
higher residual energy available at a particular time within a cluster. 1.
INTRODUCTION Our contributions in this paper
are described as follows: −
Proposed cluster head selection using grey wolf optimization with energy efficient parameters. −
Proposed cluster head selection using grey wolf optimization with energy efficient parameters. −
Proposed an objective function and weight parameters to select the optimal CHs and efficient cluster form −
Proposed an objective function and weight parameters to select the optimal CHs and efficient cluster formation
T
d
d
k
i h
i
WSN
i
d ffi
d
i h
h
l
i h Proposed an objective function and weight parameters to select the optimal CHs and efficient cluster formatio
Tested proposed work with various WSN scenarios and efficacy compared with other algorithms. posed work with various WSN scenarios and efficacy compared with other algorithms. The rest of the paper formulated as follows. Section 2 deals with the literature study of the existing
mechanism to select the cluster heads. Section 3 discussed the preliminaries of GWO algorithm and energy
consumption models. The proposed methodologies with its formulated objective function and weight parameter
for cluster formation presented in section 4. The experimentation results are discussed in section 5 and finally,
conclusion and future works are provided in section 6. 2.1. Heuristic-based clustering algorithm Though V-LEACH [28] has been proposed
as an alternative to LEACH, it elects additional CHs to that of main CHs in order to mitigate the failure of the
main CHs. Hence whenever the main CHs, fails the additional CHs selected takes care of its position and
perform the flooding. The algorithm suffers from deprivation that it does not bother about the cluster formation
process. 2.
LITTERATURE REVIEW Vast research has been plunged in the area of wireless sensor networks in order to perpetuate the lifetime
of the network. Since the selection of cluster heads is an NP-hard problem each algorithm has its own flaws as well. Algorithms devised for increasing the longevity of the network can be broadly categorized into 1) heuristic and
2) meta-heuristic approaches. Elaborations of these approaches are as follows: 1.
INTRODUCTION Working flow of cluster head and base station (BS) in wireless sensor network (WSN) Figure 1. Working flow of cluster head and base station (BS) in wireless sensor network (WSN) Grey wolf optimization algorithm is family of the swarm intelligence techniques which is inspired by
the behaviour of grey wolf (i.e. leadership and hunting strategies). This algorithm has been utilized by different
domains researchers to solve their domain related problems due to its simplicity and ease of implementations. Grey wolf optimization (GWO) algorithm has few parameters to solve the non-deterministic polynomial
(NP)-hard problems within the course of iterations. This algorithm is used to solve different domain problems
such as Localization in WSN [9], economic load dispatch problem [10], feature selection [11], engineering
problems [12], unit commitment problems [13] and so on. Clustering in WSN is considered an NP-hard problem
which can be solved using an efficient optimization algorithm. In this paper, we proposed an optimal cluster head
selection mechanism based on grey wolf optimization algorithm namely GWO-CH. This algorithm considers
the residual energy, intra-cluster and Sink distance to select the optimal cluster heads. In addition to that, we
introduced an objective function which includes the essential parameters to select the optimum. In GWO
algorithm, we incorporated the efficient search agent representation scheme to represent the energy efficient
cluster head selection. On the other hand, we proposed a weight parameter for cluster formation. This parameter
guides the sensor nodes to join their respective cluster head groups. The sensor node with high weight will be
moved to the corresponding clusters. Thereby, that sensor will act as cluster members under the CHs and transmits
their information to the base station through the CHs. The experimentation of the proposed algorithm is tested in
the different scenarios of sensor nodes by varying the number of sensor nodes and the CHs. To analyze the efficacy
of the proposed work is compared with the other algorithms namely end-to-end secure low energy adaptive
clustering hierarchy (E-LEACH) [14], genetic algorithms (GA) [15, 16], cuckoo search (CS) [17], particle swarm An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
2824
2824 ISSN: 1693-6930 optimization-C (PSO-C) [18], and fruit fly optimization algorithm (FFOA) [19]. Our contributions in this paper
are described as follows: optimization-C (PSO-C) [18], and fruit fly optimization algorithm (FFOA) [19]. TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833 3.1. Grey wolf optimization Grey wolf optimization [28] is a recently proposed swarm intelligence algorithm which mimics
the intelligent behavior of grey wolves which includes leadership and hunting characteristics of the grey wolf. Grey wolf works in a pack of 5-12 members which follows a very strict social hierarchy. Grey wolf pack
consists of four level hierarchy namely alpha, beta, delta, and omega. Alpha is the first level in the hierarchy
which is considered as the first leader of the pack. It is responsible for all the decision making a process like
hunting the prey, approaching the prey and instructing the wolves in the entire pack. The second level in
the hierarchy is beta, which guides the alpha in decision making and also acts as alpha whenever the alpha is
passed away. In most cases, beta is also called as subordinate wolves. Delta is the third level in the hierarchy
which also known as caretaker and finally. Omega is the last level in the hierarchy which obeys the decision
of the three above leaders and also maintains the safety and integrity in the wolf pack. GWO working process
is mathematically modelled as follows: 2.2. Meta-heuristic approaches
Meta-Heuristic algorithm The method offers a wide range of
coverage leveraging a better lifetime for the nodes as well as the network and it proves its efficiency even after
increasing the number of clusters compared with LEACH and PSO approach [8]. Sariga et al. [33] proposed a meta-heuristic ACO based unequal clustering (MHACO-UC) algorithm that
concentrates mainly on preserving the lifetime of the CHs, by using a distance estimation function. It also keeps
knowledge about the nearness of the nodes present in the clusters and in the entire network thereby propagating
the information to the central processing unit and this increases the longevity of the lifetime of the network. Tauseef
Ahmad et al. [34] proposed an algorithm that concentrates mainly on selecting the cluster head that has the optimum
energy using bee colony optimization algorithm. The author provides a significant contribution in identifying
the proximities of the nodes inside the cluster and between the cluster heads using an optimized fitness function. Amit Sarkar et al. [35] utilized the firefly algorithm for increasing the lifetime of the network and the liveliness of
the nodes by electing optimal cluster heads. Cyclic randomization is employed which outperforms the traditional
cluster head selection algorithms respectively. Srinivasa Rao et al. [8] came up with a solution based on particle
swarm optimization approach to address the issues such as energy and clustering. It employs a geometric method to
elect a cluster head and as flooding occurs, the higher energy nodes are only used and the nodes with lower energy
are preserved from propagating the information to the central processing unit thereby preserving the lifetime of
the network. Kia et al. [26] a new hybrid protocol based on cuckoo search optimization have been proposed in order
to conserve energy while flooding the information inside the clusters by selecting the optimal cluster heads. It employs an energy conservator in order to increase the longevity of the network. Dattatraya et al. [19] proposed
a hybrid algorithm by combining glom worm swarm optimization (GSO) and fruit fly optimization (FFOA). GSO
suffers from low computational speed and low searching capacity. Fruit fly optimization algorithm (FFOA) has its
own merging rate. Hence hybridizing both yields perfect results thereby outperforming the traditional cluster head
selection algorithms. 2.2. Meta-heuristic approaches
Meta-Heuristic algorithm Meta-Heuristic algorithms act as the most promising approach for NP-hard combinatorial problems. Since they mimic from nature, it concentrates mainly on the aspirant which has a high survival rate. Meta-heuristic algorithms are broadly categorized into two types namely evolutionary and swarm intelligence
approaches [29]. Genetic algorithm [15, 30] and simulated annealing are the most popular evolutionary
algorithms. Some of the swarm intelligence approaches are ant colony optimization (ACO), fish colony
optimization (FCO), bird flocking behaviour, particle swarm optimization (PSO), firefly algorithm (FA) [31],
bat algorithm (BA), cuckoo search (CS), artificial bee colony optimization (ABC), fish swarm optimization
(FSO), glow-worm swarm optimization (GSO), grey wolf optimizer (GWO), fruit fly optimization algorithm
(FFOA). Sweta Potthuri et al. [32] proposed a hybrid differential evolution and simulated annealing (DESA)
algorithm which aims to increase the liveliness of the network by selecting the cluster heads which has optimal TELKOMNIKA Telecommun Comput El Control 2825 energy, thereby preventing the energy loss. The author in [15] proposed an energy efficient clustering algorithm
in order to extend the lifetime of the network. It uses a genetic algorithm (GA), where the cluster heads are
elected by using appropriate fitness function until the information is propagated through to the central
processing unit i.e. base station. Osama Helmy et al. proposed an algorithm that provides energy consumption
thereby increasing the longevity of the network. The different approaches such as preying, and swarming are
employed in order to achieve the selection of the optimal cluster head. The method offers a wide range of
coverage leveraging a better lifetime for the nodes as well as the network and it proves its efficiency even after
increasing the number of clusters compared with LEACH and PSO approach [8]. energy, thereby preventing the energy loss. The author in [15] proposed an energy efficient clustering algorithm
in order to extend the lifetime of the network. It uses a genetic algorithm (GA), where the cluster heads are
elected by using appropriate fitness function until the information is propagated through to the central
processing unit i.e. base station. Osama Helmy et al. proposed an algorithm that provides energy consumption
thereby increasing the longevity of the network. The different approaches such as preying, and swarming are
employed in order to achieve the selection of the optimal cluster head. 3.1.3. Seeking and attacking the prey The parameter 𝐴 is a random vector which is used to explore and exploit the search position of
the grey wolves. Every course of iterations, this parameter has been adjusted in the range of[−𝑎 , 𝑎 ], where
the value 𝑎 linearly decreases from 2 to 0. GWO algorithm exploits the prey if |𝐴 | < 1 otherwise it seeks for
new prey if |𝐴 | > 1. In addition to that, parameter 𝐶 lies in the range of [0, 2] which aids the algorithm to avoid
the local optima stagnation by providing some random weight to the position update. However, tuning
the parameter 𝑎 provides better results compare to the generic GWO algorithm. In this proposed work, we
tuned the parameter 𝑎 for better results. The working flow of the GWO algorithm is mathematically modelled
and it is shown in algorithm 1. 3.1.1. Encircling process The position update using alpha, beta and delta are graphically represented in Figure 2. Figure 2. Position update in GWO Figure 2. Position update in GWO TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833 3.1.1. Encircling process All the grey wolves in the pack start encircling the prey before it starts the hunting process. The encircling process is mathematically formulated and it is given in the (1) and (2). 𝐷⃗⃗ = |𝐶 . 𝑋𝑝
⃗⃗⃗⃗ (𝑘) −𝑋 (𝑘)|
(1)
𝑋 (𝑘+ 1) = |𝑋𝑝
⃗⃗⃗⃗ (𝑘) −𝐴 . 𝐷⃗⃗ |
(2) 𝐷⃗⃗ = |𝐶 . 𝑋𝑝
⃗⃗⃗⃗ (𝑘) −𝑋 (𝑘)| (2) where, 𝐷⃗⃗ represents the distance between the prey and wolf, 𝑋 determines the current position of the wolf in 𝑘
iterations and 𝑋𝑝
⃗⃗⃗⃗ is the position of the prey. The constant parameters 𝐴 and 𝐶 are measured using the (3) and (4). 𝐴 = 2𝑎 . 𝑟𝑎𝑛𝑑1
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ −𝑎
(3)
𝐶 = 2. 𝑟𝑎𝑛𝑑2
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗
(4) 𝐴 = 2𝑎 . 𝑟𝑎𝑛𝑑1
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ −𝑎
𝐶 = 2. 𝑟𝑎𝑛𝑑2
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ 𝐴 = 2𝑎 . 𝑟𝑎𝑛𝑑1
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ −𝑎 An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
2826 2826 ISSN: 1693-6930 where, 𝑟𝑎𝑛𝑑1
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ and 𝑟𝑎𝑛𝑑1
⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗⃗ determines the arbitrary vectors generated between the range of [0,1]. These values
aids to adjust the position of the grey wolf randomly at any position towards the prey. Parameter 𝑎 is aids to
control the movement of the algorithm which linearly reduces from 2 to 0 over certain generations. 3.1.2. Hunting process g p
In the hunting process, all the dominant wolves 𝜔 adjust their positions using non-dominant wolves
𝛼, 𝛽, and 𝛿. The position update using these non-dominant wolves have mathematically modelled and it is
given in (5-7). 𝐷𝛼
⃗⃗⃗⃗⃗ = |𝐶1
⃗⃗⃗⃗ . 𝑋𝛼
⃗⃗⃗⃗ −𝑋 |, 𝐷𝛽
⃗⃗⃗⃗ = |𝐶2
⃗⃗⃗⃗ . 𝑋𝛽
⃗⃗⃗⃗ −𝑋 |, 𝐷𝛿
⃗⃗⃗⃗ = |𝐶3
⃗⃗⃗⃗ . 𝑋𝛿
⃗⃗⃗⃗ −𝑋 |
(5)
𝑋1
⃗⃗⃗⃗ = |𝑋𝛼
⃗⃗⃗⃗ −𝐴1
⃗⃗⃗⃗ . 𝐷𝛼
⃗⃗⃗⃗⃗ |, 𝑋2
⃗⃗⃗⃗ = |𝑋𝛽
⃗⃗⃗⃗ −𝐴2
⃗⃗⃗⃗ . 𝐷𝛽
⃗⃗⃗⃗ |, 𝑋3
⃗⃗⃗⃗ = |𝑋𝛽
⃗⃗⃗⃗ −𝐴3
⃗⃗⃗⃗ . 𝐷𝛽
⃗⃗⃗⃗ |
(6) (5) (6) Using in (5) and (6) are used to update the position of the grey wolf and it is shown in (7). Using in (5) and (6) are used to update the position of the grey wolf and it is shown in (7). 𝑋 (𝑘+ 1) = 0.33 ∗∑
𝑋𝑖
⃗⃗⃗
3
𝑖=1
(7 𝑋 (𝑘+ 1) = 0.33 ∗∑
𝑋𝑖
⃗⃗⃗
3
𝑖=1 (7) The position update using alpha, beta and delta are graphically represented in Figure 2. TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833
Algorithm 1: Grey wolf optimization algorithm
Input – Initialize the population size of the wolves 𝑋𝑖= (1,2,3, … 𝑛) and parameters A, C
Step 1: Randomly generate solutions 𝑋𝑖 within the boundary regions
Step 2: Evaluate the fitness of the wolves 𝑓𝑖
Step 3: Select the first best solution as Alpha, second best as beta, third best as Delta
and rest as Omega.
Step 4: Update the position of the grey wolves and its parameters 3.2. Energy consumption model In this paper, the energy consumption model is used based on the suggestions of the author in [36]. In this model, the energy has been utilized by the transmitter and receiver for transmitting and receiving their
signals and to operate the radio amplifiers. The energy consumption of a sensor for transmitting (𝐸𝑇𝑋) n-bit of
information is mathematically represented in (8). 𝐸𝑇𝑋(𝑛, 𝜃) = { 𝑛× 𝐸𝑒𝑙𝑒𝑐 + 𝑛× 𝜀𝑓𝑠× 𝜃2
𝑛× 𝐸𝑒𝑙𝑒𝑐 + 𝑛× 𝜀𝑚𝑝× 𝜃4
𝑖𝑓 𝜃< 𝜑
𝑖𝑓 𝜃≥𝜑
(8) (8) where, 𝐸𝑒𝑙𝑒𝑐 represented as energy utilized per bit to operate the transmitter or receiver. 𝜀𝑓𝑠 and 𝜀𝑚𝑝determined
as the free space model and multipath of amplification power. 𝜑 and 𝜃 denoted as the threshold and distance
for transmitting the information from one sensor location to other sensors. where, 𝐸𝑒𝑙𝑒𝑐 represented as energy utilized per bit to operate the transmitter or receiver. 𝜀𝑓𝑠 and 𝜀𝑚𝑝determined
as the free space model and multipath of amplification power. 𝜑 and 𝜃 denoted as the threshold and distance
for transmitting the information from one sensor location to other sensors. g
At the same time, energy consumed by the receiver for receiving n-bit of information (𝐸𝑅𝑋(𝑛))
mputed as follows; 𝐸𝑅𝑋(𝑛) = 𝑛× 𝐸𝑒𝑙𝑒𝑐
(9) 𝐸𝑅𝑋(𝑛) = 𝑛× 𝐸𝑒𝑙𝑒𝑐 (9) he total energy consumption (𝐸𝑡𝑜𝑡𝑎𝑙) of a sensor node for transmitting and receiving the 𝑛-bit information
athematically calculated as follows; 𝐸𝑇𝑜𝑡𝑎𝑙= 𝐸𝑇𝑋(𝑛, 𝜃) + 𝐸𝑅𝑋(𝑛)
(10) 𝐸𝑇𝑜𝑡𝑎𝑙= 𝐸𝑇𝑋(𝑛, 𝜃) + 𝐸𝑅𝑋(𝑛) (10) A sensor node lifetime is computed based on the initial energy of the node and the remaining energy of th
node after transmitting and receiving the 𝑛-bit information. It is expressed as follows; 𝐿 =
𝐸𝑖𝑛𝑡𝑖𝑎𝑙
𝐸𝑡𝑜𝑡𝑎𝑙
(11) 𝐿 =
𝐸𝑖𝑛𝑡𝑖𝑎𝑙
𝐸𝑡𝑜𝑡𝑎𝑙 (11) where, 𝐸𝑖𝑛𝑡𝑖𝑎𝑙 represents initial energy of the sensor node (i.e. 2J in our work) and 𝐸𝑡𝑜𝑡𝑎𝑙 represented as
the total consumed the energy of the sensor node. In our work, the network lifetime considered based on
the number of iterations until the last node of death. where, 𝐸𝑖𝑛𝑡𝑖𝑎𝑙 represents initial energy of the sensor node (i.e. 2J in our work) and 𝐸𝑡𝑜𝑡𝑎𝑙 represented as
the total consumed the energy of the sensor node. In our work, the network lifetime considered based on
the number of iterations until the last node of death. 4.
PROPOSED ALGORITHM The proposed algorithm mainly contributes to selecting the cluster heads by considering the residual
energy and distance measurement of the sensor nodes. Initially, all the sensor nodes send their information
(node_id, residual energy, location) to the base station. Our proposed GWO algorithm executed at the base
station to select the optimal CH (i.e. by sensor node information) and to form the optimal clusters. In order to
process the cluster formation, we have formulated the weight function which involves the intra-cluster distance
information, residual energy, and neighborhood ratio of CHs respectively. Algorithm 1: Grey wolf optimization algorithm Algorithm 1: Grey wolf optimization algorithm TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833
g
y
p
g
Input – Initialize the population size of the wolves 𝑋𝑖= (1,2,3, … 𝑛) and parameters A, C
Step 1: Randomly generate solutions 𝑋𝑖 within the boundary regions
Step 2: Evaluate the fitness of the wolves 𝑓𝑖
Step 3: Select the first best solution as Alpha, second best as beta, third best as Delta
and rest as Omega. Step 4: Update the position of the grey wolves and its parameters g
y
p
g
Input – Initialize the population size of the wolves 𝑋𝑖= (1,2,3, … 𝑛) and pa g
y
p
g
Input – Initialize the population size of Input – Initialize the population size of Step 1: Randomly generate solutions 𝑋𝑖 within the boundary regions Step 3: Select the first best solution as Alpha, second best as b
and rest as Omega. TELKOMNIKA Telecommun Comput El Control TELKOMNIKA Telecommun Comput El Control tep 5: Evaluate the new fitness of all wolve p
Step 6: Update the alpha, beta, and delta p
p
p
Step 7: Repeat step 5 to 7 until condition Step 7: Repeat step 5 to 7 until condition satisfies Output – visualize the Alpha wolf Output – visualize the Alpha wolf 4.2. Cluster formation In WSN, selecting the optimal CHs will lead to a proper cluster formation and it aids to prolong
the network lifetime. In this work, we select the CHs using the residual energy, neighborhood ratio and distance
from BS. To create an optimal cluster formation, we formulate weight function which guides the sensor node
to join in their respective CHs. The derived weight function is mathematically represented in the (15). 𝐶𝐻𝑤(𝑇𝑖, 𝐶𝐻𝑗) = 𝐾∗
𝐸𝑅(𝐶𝐻𝑗)
𝜃(𝑇𝑖,𝐶𝐻𝑗)×𝜃(𝐶𝐻𝑗,𝐵𝑆)×𝐷𝑁(𝐶𝐻𝑗)
(15) (15) where 𝐾 is the constant parameter value (i.e. 𝐾= 1). 𝐸𝑅(𝐶𝐻𝑗) represented as the residual energy of the 𝑗𝑡ℎ
cluster head. 𝜃(𝑇𝑖, 𝐶𝐻𝑗) denoted as the distance between the ith target sensor node (i.e. normal sensor node)
and jth cluster head. 𝜃(𝐶𝐻𝑗, 𝐵𝑆) represented as the distance between 𝑗𝑡ℎ cluster head and the base station. 𝐷𝑁(𝐶𝐻𝑗) denoted as the neighborhood ratio of the 𝑗𝑡ℎ CH. The 𝑖𝑡ℎ sensor node with high weight value can
able to join in a 𝑗𝑡ℎ cluster head. where 𝐾 is the constant parameter value (i.e. 𝐾= 1). 𝐸𝑅(𝐶𝐻𝑗) represented as the residual energy of the 𝑗𝑡ℎ
cluster head. 𝜃(𝑇𝑖, 𝐶𝐻𝑗) denoted as the distance between the ith target sensor node (i.e. normal sensor node)
and jth cluster head. 𝜃(𝐶𝐻𝑗, 𝐵𝑆) represented as the distance between 𝑗𝑡ℎ cluster head and the base station. 𝐷𝑁(𝐶𝐻𝑗) denoted as the neighborhood ratio of the 𝑗𝑡ℎ CH. The 𝑖𝑡ℎ sensor node with high weight value can
able to join in a 𝑗𝑡ℎ cluster head. 4.1. The objective function for CH selection In this work, we derived the objective function which utilizes the intra-cluster distance among
the sensors and the distance from the target node. Let us assume 𝑓1 be a function of mean intra-cluster and
the target distance of the CHs. In order to select the optimal CHs, the 𝑓1 to be minimized. Let us assume 𝑓2 be
the function which is inverse of total current energy of all the selected CHs. In order to provide better results
both the objective function is to be minimized and it to be within (𝑓1, 𝑓2) ∈[0,1]. The objective function 𝑓1 is represented as; The objective function 𝑓1 is represented as; min 𝑓1 = ∑
1
𝑛𝑖
𝑚
𝑖=1
(∑
𝜃(𝑇𝑗, 𝐶𝐻𝑖) + 𝜃(𝐶𝐻𝑖, 𝐵𝑆)
𝑛𝑖
𝑖=1
)
(12) (12) where, 𝜃(𝑇𝑗, 𝐶𝐻𝑖) represented as the distance between the target node 𝑗 to the cluster head 𝑖. 𝜃(𝐶𝐻𝑖, 𝐵𝑆)
denoted as the distance between the cluster head 𝑖 to the base station. 𝑛 and 𝑚 denoted as the number of target
sensor nodes and cluster heads. where, 𝜃(𝑇𝑗, 𝐶𝐻𝑖) represented as the distance between the target node 𝑗 to the cluster head 𝑖. 𝜃(𝐶𝐻𝑖, 𝐵𝑆)
denoted as the distance between the cluster head 𝑖 to the base station. 𝑛 and 𝑚 denoted as the number of target
sensor nodes and cluster heads. The objective function 𝑓2 is mathematically represented as; The objective function 𝑓2 is mathematically represented as; An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
2828 2828 ISSN: 1693-6930 min 𝑓2 =
1
∑
𝐸𝐶𝐻𝑖
𝑚
𝑖=1 n 𝑓2 =
1
∑
𝐸𝐶𝐻𝑖
𝑚
𝑖=1
(13) min 𝑓2 =
1
∑
𝐸𝐶𝐻𝑖
𝑚
𝑖=1 (13) where, 𝐸𝐶𝐻𝑖 denoted as the residual energy of the cluster head 𝑖. In order to minimize both the objective
function, we use GWO algorithm to select the optimal CH to linearly decrease the function. The combined
objective function is mathematically represented in (14). where, 𝐸𝐶𝐻𝑖 denoted as the residual energy of the cluster head 𝑖. In order to minimize both the objective
function, we use GWO algorithm to select the optimal CH to linearly decrease the function. The combined
objective function is mathematically represented in (14). 𝐹= 𝜇× 𝑓1 + (1 −𝜇)𝑓2,
0 < 𝜇< 1
(14) 𝐹= 𝜇× 𝑓1 + (1 −𝜇)𝑓2,
0 < 𝜇< 1 (14) where 𝜇 is the weight parameter in the range of [0,1]. 4.1. The objective function for CH selection The search agent with minimal objective value is
considered as the CH. where 𝜇 is the weight parameter in the range of [0,1]. The search agent with minimal objective value is
considered as the CH. 5.1. Simulation setup p
In this paper, the algorithms were implemented in MATLAB (version 8.5) with configurations of Intel
Core i5 Processor with 8GB RAM in a Windows 10 platform. The parameter settings of the proposed system
are given in Table 1. To analyze the performance of the proposed system, the state-of-art other algorithms such
as E-LEACH, GA, CS, PSO-C, and FFOA algorithms are used respectively. In our work, we considered
the network region as 300x300 m2, with a varying number of sensors from 400 to 700 and the number of
clusters from 20 to 40. The detailed information about the network considerations is given in Table 1. Table 1. Network configurations
Parameter
Value
Network Field
(300, 300) m2
Base Station Position
(150-400, 150-400)
Sensor Nodes
400-700
Initial Energy
2J
Number of Cluster Heads
20-40
𝐸𝑒𝑙𝑒𝑐, 𝜀𝑓𝑠, 𝜀𝑚𝑝
50 nJ/bit, 10 pJ/bit/m2, 0.0013 pJ/bit/m4
𝑑𝑚𝑎𝑥, 𝜑
100 m, 30 m
Packet Size, Message Size
4000 bits, 500 bits Table 1. Network configurations
Parameter
Value
Network Field
(300, 300) m2
Base Station Position
(150-400, 150-400)
Sensor Nodes
400-700
Initial Energy
2J
Number of Cluster Heads
20-40
𝐸𝑒𝑙𝑒𝑐, 𝜀𝑓𝑠, 𝜀𝑚𝑝
50 nJ/bit, 10 pJ/bit/m2, 0.0013 pJ/bit/m4
𝑑𝑚𝑎𝑥, 𝜑
100 m, 30 m
Packet Size, Message Size
4000 bits, 500 bits To measure the performance of the algorithms, we considered three different cases in WSN with
the varying number of sensors and CHs. Firstly, case#1 deals with the 400 sensor nodes with 20 CHs. Next, case#2
deals with 500 sensor nodes with 30 CHs, case#3 consists of 600 sensor nodes with 30 CHs and finally, case#4 holds
700 sensor nodes with 40 CHs. In addition to that, we have placed the Base station in three different locations namely
mid of the network region (150, 150), corner of the network region (300, 300) and outside of the network region
(400, 400). Owing to the Placement of BS in different locations are used to analyze the performance of packet
delivery information and the network lifetime. Every algorithm has been executed repeatedly for 30 times
and average values of that execution are measured and plotted in the figures. The proposed algorithm has been
tested with different population size and based on the experimentation analysis we fixed the population size as 50. At the same time, the weighted sum of 𝜇 value is fixed as 0.27 based on the experimentation analysis. 5.1. Simulation setup This value
provides better performance compared to values from 0 to 1. The detailed parameters information of the GWO
algorithm is given in Table 2. Table 2. GWO parameters
Parameters
Value
No. of Search agents
50
C
(2 – 0)
a
(0 – 1.5)
𝜇
0.27
Dimension of search agents
20-40 (CHs)
Number of Iterations
100
5 2 Performance analysis 4.3. GWO algorithm for CH selection In the proposed GWO algorithm, the search agent represented as m dimensional cluster heads with its
position (x-axis, y-axis) and sensor id as shown in Figure 3. Initially, the algorithm selects the random cluster
head with their appropriate locations and it computes the objective value for those cluster heads. Next, it selects
the first best search agent 𝛼, second best search agent 𝛽 and third best search agent 𝛿 and rest of the search
agent as 𝜔. With the aid of three best solutions, the remaining search agents update its position and the new
position represented as the new cluster heads which satisfies the objective function. Later, identify the weight
function to determine the appropriate cluster members to join in their respective CHs. The working flow of
the proposed GWO is presented in Algorithm 2. TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833
Figure 3. Representation of search agent in GWO
Algorithm 2: GWO algorithm for CH selection
Input: Number of sensors 𝑆= {𝑆1,𝑆2, … , 𝑆𝑛}, Population size = 𝑁𝑃
Step 1: Randomly initialize the search agent 𝑋𝑖 ∀𝑖,𝑗,
1 ≤𝑖≤𝑁𝑃,
1 ≤𝑗≤𝐷
𝑃𝑖𝑗(0) = (𝑃𝑋𝑖𝑗(0), 𝑃𝑌𝑖𝑗(0))
Step 2: Calculate the fitness 𝑓(𝑋𝑖)
Step 3: Select 𝛼= min𝑓(𝑋𝑖), 𝛽= min𝑓(𝑋𝑖−1), 𝛿= min𝑓(𝑋𝑖−2)
/* 𝛼- first best solution, 𝛽 second best search agent, 𝛿 – third best search agent */
Step 4: while (𝑡< 𝑡𝑚𝑎𝑥) /* 𝑡𝑚𝑎𝑥 – the maximum number of iterations */ Figure 3. Representation of search agent in GWO Figure 3. Representation of search agent in GWO Figure 3. Representation of search agent in GWO Figure 3. Representation of search agent in GWO TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833
Algorithm 2: GWO algorithm for CH selection
Input: Number of sensors 𝑆= {𝑆1,𝑆2, … , 𝑆𝑛}, Population size = 𝑁𝑃
Step 1: Randomly initialize the search agent 𝑋𝑖 ∀𝑖,𝑗,
1 ≤𝑖≤𝑁𝑃,
1 ≤𝑗≤𝐷
𝑃𝑖𝑗(0) = (𝑃𝑋𝑖𝑗(0), 𝑃𝑌𝑖𝑗(0))
Step 2: Calculate the fitness 𝑓(𝑋𝑖)
Step 3: Select 𝛼= min𝑓(𝑋𝑖), 𝛽= min𝑓(𝑋𝑖−1), 𝛿= min𝑓(𝑋𝑖−2)
/* 𝛼- first best solution, 𝛽 second best search agent, 𝛿 – third best search agent *
Step 4: while (𝑡< 𝑡𝑚𝑎𝑥) /* 𝑡𝑚𝑎𝑥 – the maximum number of iterations */ Algorithm 2: GWO algorithm for CH selection
2829 𝑃𝑖𝑗(𝑡+ 1) →{𝑆𝑘| min(𝜃(𝑃𝑖𝑗(𝑡+ 1), 𝑆𝑘)), ∀𝑖, 1 ≤𝑗≤𝑁𝑃 end for d
o
end while end while end while
Step 5: Repeat Step 4 until it reaches the maximum number of iterations
Output: Visualize the best cluster heads 𝐶𝐻= {𝐶𝐻1, 𝐶𝐻2,… , 𝐶𝐻𝑚} TELKOMNIKA Telecommun Comput El Control TELKOMNIKA Telecommun Comput El Control for 𝑖 = 1: 𝑁𝑝
Update the position of search agent 𝑋𝑖
𝑡
Calculate the fitness 𝑓(𝑋𝑖
𝑡)
Update 𝛼, 𝛽 and 𝛿 end for
for 𝑖 = 1: 𝑛
calculate 𝜃(𝑃𝑖𝑗(𝑡+ 1), 𝑆𝑘) end for
for 𝑖 = 1: 𝑛
calculate 𝜃(𝑃𝑖𝑗(𝑡+ 1), 𝑆𝑘) 𝑃𝑖𝑗(𝑡+ 1) →{𝑆𝑘| min(𝜃(𝑃𝑖𝑗(𝑡+ 1), 𝑆𝑘)), ∀𝑖, 1 ≤𝑗≤𝑁𝑃 5.2.1. Performance analysis of TEC In order to measure the performance of energy consumption, firstly we executed the algorithms by
varying the number of sensor nodes from 400 to 700 and the number of cluster heads from 20 to 50. The performance measures of E-LEACH, GA, CS, PSO-C, FFOA, and GWO-CH are shown in Tables 3 and
4 and Figures 4-8 in terms of total energy utilization in all the different cases. In the first case, the BS location
was considered as mid of the network region (150, 150). The observed results notify that the proposed
GWO-CH algorithm outperforms better than E-LEACH, GA, CS, PSO-C, FFOA in terms of total energy
consumption respectively. In addition to that, we have noticed that if the sensors are nearest to the CHs,
the energy consumption for transferring packets from one sensor to other is decreased. Because of the proposed
fitness function which concentrates on the energy consumption of the normal nodes by minimizing the distance
between the sensor and CHs. Table 3. Total energy consumption for 20CHs in case#1 (5000 iterations)
Sensors Nodes = 400
BS (150,150)
BS (300,300)
BS (400,400)
E-LEACH
800.00
800.00
800.00
GA
786.54
794.74
800.00
CS
782.92
784.96
788.82
PSO-C
764.64
774.82
784.68
FFOA
710.28
724.65
746.87
GWO
646.54
680.38
702.49 Table 3. Total energy consumption for 20CHs in case#1 (5000 iterations)
Sensors Nodes = 400
BS (150,150)
BS (300,300)
BS (400,400) Table 4. Total energy consumption for 30CHs in case#2 (5000 iterations)
Sensors Nodes = 500
BS (150,150)
BS (300,300)
BS (400,400)
E-LEACH
1000.00
1000.00
1000.00
GA
954.87
968.75
986.78
CS
914.15
932.87
954.54
PSO-C
880.54
917.54
942.87
FFOA
864.54
886.40
902.14
GWO
804.51
835.21
856.12 On the other hand, we have noticed that when the network size increases then the performance of
the existing algorithm decreases, which was in Figures 4-8. Initially, the performance of the proposed algorithm is
not that much satisfactory compared to PSO-C and FFOA. As the number of iterations increases, the residual energy
of the sensors is decreasing due to the improper cluster head selections. In this case, our proposed algorithms provide
a better solution in case of selecting the proper cluster heads by our derived fitness function. In order to measure
the energy consumption performance, we executed our algorithm by varying the number of sensors from 400 to 700
and the cluster heads from 20 to 50. 5.2. Performance analysis y
The performance of the proposed algorithm has been measured using three metrics namely total
energy consumption (TEC), network lifetime (NL) and packet received by BS (PR-BS). These three-
performance metrics are used to analyze the performance of the proposed algorithm with other algorithms. An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
2830
2830 ISSN: 1693-6930 6.
CONCLUSION In this paper, we introduced a novel cluster head selection algorithm based on GWO using efficient
search agent representation and novel objective function. For the energy efficacy, we have considered
intra-cluster distance, sink distance and the residual energy of sensors respectively. In addition to that, we have
formulated the weighted function for the efficient cluster formation. The experimental results with its
comparison of existing algorithms E-LEACH, GA, CS, PSO-C, and FFOA has been presented. The algorithm
has been executed in the different test cases with a varying number of sensors and CHs. The observed results
convey that the proposed algorithm outperforms better compared to other algorithms in terms of energy
consumption, network lifetime and packet received by the BS. Further, this work can be extended by
formulating novel routing algorithm in the proposed algorithm. Still, we can consider the various issues viz.,
load balancing and fault tolerance in WSN. In this work, we have tested the proposed algorithm in
the homogeneous network. In the future, the same can be tested on heterogeneous networks.
2832 ISSN: 1693-6930 Finally, we specify that the proposed algorithm utilizes the minimum energy consumption and
maximizes the network lifetime achieves better performance in delivering the maximum number of packets. It is also observed that the proposed algorithm achieves the maximum number of packets received when
compared to other algorithms E-LEACH, GA, CS, PSO-C, and FFOA. In the existing algorithm, when the BS
location is at out of the network region then the number of packets received is less but the proposed algorithm
maximizes the number of packets received in terms of selecting the efficient cluster head using the derived
fitness function. ACKNOWLEDGEMENTS This work was supported by the National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIT) (No. NRF-2018R1C1B5038818). This work was supported by the National Research Foundation of Korea (NRF) grant funded by
the Korea government (MSIT) (No. NRF-2018R1C1B5038818). REFERENCES ]
X. Liu, “A survey on clustering routing protocols in wireless sensor networks,” sensors, vol. 12, no. pp. 11113-11153. 2012. ]
M. M. Afsar and M. H. Tayarani-N, “Clustering in sensor networks: A literature survey,” Journal of Network a
Computer Applications, vol. 46, pp. 198-226, 2014. [3]
I. F. Akyildiz, W. Su, Y. Sankarasubramaniam, and E. Cayirci, “Wireless sensor networks: a survey,” Computer
networks, vol. 38, no. 4, pp. 393-422, 2002. pp
[4]
M. C. M. Thein, and T. Thein, “An energy efficient cluster-head selection for wireless sensor networks,”
International Conference on Intelligent Systems Modelling and Simulation IEEE pp 287 291 2010 [4]
M. C. M. Thein, and T. Thein, “An energy efficient cluster-head selection for wireless sensor networks,” 2010
International Conference on Intelligent Systems, Modelling, and Simulation, IEEE, pp. 287-291, 2010. f
g
y
g
, pp
,
]
N. K. Sharma, and M. Kumar, “An energy constrained multi-hop clustering algorithm for wireless sensor network
International Conference on Networking, Springer, Berlin, Heidelberg, pp. 706-713, 2005. [6]
A. Taherkordi, R. Mohammadi, and F. Eliassen, “A communication-efficient distributed clustering algorithm for
sensor networks,” 22nd International Conference on Advanced Information Networking and Applications-
Workshops, pp. 634-638, 2008. ]
P. S. Rao, and H. Banka, “Novel chemical reaction optimization based unequal clustering and routing algorithms f
wireless sensor networks,” Wireless Networks, vol. 23, no. 3, pp. 759-778, 2017. ,
,
,
, pp
,
[8]
P. S. Rao, P. K. Jana, and H. Banka, “A particle swarm optimization based energy efficient cluster head selection
algorithm for wireless sensor networks ” Wireless networks vol 23 no 7 pp 2005 2020 2017 [8]
P. S. Rao, P. K. Jana, and H. Banka, “A particle swarm optimization based energy efficient cluster head
algorithm for wireless sensor networks,” Wireless networks, vol. 23, no. 7, pp. 2005-2020, 2017. [8]
P. S. Rao, P. K. Jana, and H. Banka, A particle swarm optimization based energy efficient clust
algorithm for wireless sensor networks,” Wireless networks, vol. 23, no. 7, pp. 2005-2020, 2017. g
pp
[9]
R. Rajakumar, J. Amudhavel, P. Dhavachelvan, and T. Vengattaraman, “GWO-LPWSN: Grey wolf optimization
algorithm for node localization problem in wireless sensor networks,” Journal of Computer Networks and
Communications, vol. 2, pp. 1-10, 2017. ,
, pp
,
[10] V. K. Kamboj, S. K. Bath, and J. S. Dhillon, “Solution of non-convex economic load dispatch problem using Grey
Wolf Optimizer,” Neural Computing and Applications, vol. TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833 5.2.1. Performance analysis of TEC For efficient performance analysis, the algorithms are executed for 5000
iterations. The overall energy consumption was measured at the final iterations 5000. Figures 4-8 displays that
the proposed algorithm provides better performance compared to other state-of-art algorithms. The efficacy of
the proposed algorithm has been achieved by the novel derived fitness function which handles in selecting
the appropriate cluster heads by minimizing the distance between the sensors and CHs. Finally, the performance of
the algorithm in terms of energy consumption with varying number of sensors from 400 to 700 and cluster heads
from 20 to 50 with 5000 iterations shown in Tables 3 and 4. TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833
Figure 4. Total energy consumption in case 2
with 30 CHs
Figure 5. Total energy consumption in case 3
with 40 CHs Figure 5. Total energy consumption in case 3
with 40 CHs Figure 4. Total energy consumption in case 2
with 30 CHs Figure 4. Total energy consumption in case 2
with 30 CHs Figure 5. Total energy consumption in case 3
with 40 CHs TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833 TELKOMNIKA Telecommun Comput El Control, Vol. 18, No. 6, December 2020: 2822 - 2833 TELKOMNIKA Telecommun Comput El Control
2831 (a)
(b)
(c)
Figure 6. Total energy consumption by placing BS in different locations; (a) case 1 (b) case 2 (c) case 3 (b) (a)
(b) (b) (c) Figure 6. Total energy consumption by placing BS in different locations; (a) case 1 (b) case 2 (c) case 3 (a)
(b)
Figure 7. Comparison of packet received by BS by placing the BS in different locations; (a) case 1 (b) case 2 Figure 7. Comparison of packet received by BS by placing the BS in different locations; (a) case 1 (b) case 2 An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
(a)
(b)
Figure 8. Comparison of packet received by BS by placing the BS in different locations; (a) case 3 (b) case 4 (b) Figure 8. Comparison of packet received by BS by placing the BS in different locations; (a) case 3 (b) case 4 An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
2832 TELKOMNIKA Telecommun Comput El Control 366-379, 2004. 4] V. Loscri, G. Morabito, and S. Marano, “A two-level hierarchy for low-energy adaptive clustering hierarc
(TL-LEACH),” IEEE vehicular technology conference, vol. 62, no. 3, pp. 1809-13, 1999. (
),
gy
f
,
, pp
,
[25] M. Tong, and M. Tang, “LEACH-B: an improved LEACH protocol for wireless sensor network,” 2010 6th
international conference on wireless communications networking and mobile computing (WiCOM), pp. 1-4, 2010. [
]
g,
g,
p
p
,
international conference on wireless communications networking and mobile computing (WiCOM), pp. 1-4, 2010. [26] W. Xinhua, and W. Sheng, “Performance comparison of LEACH and LEACH-C protocols by NS2,” 2010 Ninth
International Symposium on Distributed Computing and Applications to Business, Engineering and Science,
pp. 254-258, 2010. [26] W. Xinhua, and W. Sheng, “Performance comparison of LEACH and LEACH-C protocols by NS2,” 2010 Ninth
International Symposium on Distributed Computing and Applications to Business, Engineering and Science,
pp. 254-258, 2010. pp
7] P. Dabas, and N. Gupta, “LEACH-and-its-Improved-Versions-A-Survey,” International Journal of Scientific
Engineering Research, vol. 6, no. 6, pp. 184-188. 2015. [28] M. B. Yassein, Y. Khamayseh, and W. Mardini, “Improvement on LEACH protocol of wireless sensor network,”
International Journal of Digital Content Technology and its Applications, vol. 33, no. 2, pp. 132-36, 2009. 9] C. P. Lim, and L. C. Jain, “Advances in swarm intelligence,” ICSI: International Conference on Swarm Intelligenc
pp. 1-7, 2009. pp
[30] S. K Jha, and E. M. Eyong, “An energy optimization in wireless sensor networks by using genetic algorithm.,”
Telecommunication Systems, vol. 67, no. 1, pp. 113-121, 2018. 1] K. Sekaran, et al., "Improving the Response Time of M-Learning and Cloud Computing Environments Usi
a Dominant Firefly Approach," IEEE Access, vol. 7, pp. 30203 - 30212, 2019. [31] K. Sekaran, et al., "Improving the Response Time of M-Learning and C
a Dominant Firefly Approach," IEEE Access, vol. 7, pp. 30203 - 30212, 201 1] K. Sekaran, et al., Improving the Response Time of M-Learning and Cloud Computing En
a Dominant Firefly Approach," IEEE Access, vol. 7, pp. 30203 - 30212, 2019. [31] K. Sekaran, et al., Improving the Response Time of M Learning and Cloud C
a Dominant Firefly Approach," IEEE Access, vol. 7, pp. 30203 - 30212, 2019. [32] S. Potthuri, T. Shankar, and A. Rajesh, “Lifetime Improvement in Wireless Sensor Networks using Hybrid
Differential Evolution and Simulated Annealing (DESA),” Ain Shams Engineering Journal, vol. 9, no. 4,
pp. 655-663, 2018. TELKOMNIKA Telecommun Comput El Control 2833 [16] Sekaran, Kaushik, and P. Venkata Krishna, "Big Cloud: a hybrid cloud model for secure data storage through cloud
space," International Journal of Advanced Intelligence Paradigms, vol. 8, no. 2, pp. 229-241, 2016. p
,
f
g
g
,
, pp
,
[17] G. Kia, and A. Hassanzadeh, “HYREP : A Hybrid Low-Power Protocol for Wireless Sensor Networks,” Int
Journal of Engineering, vol. 32, no. 4, pp. 519-527, 2019. [18] N. A. Latiff, C. C. Tsimenidis, and B. S. Sharif, “Energy-aware clustering for wireless sensor networks using particle
swarm optimization,” 2007 IEEE 18th International Symposium on Personal, Indoor and Mobile Radio
Communications, pp. 1-5, 2007. pp
[19] K. N. Dattatraya, and K. R. Rao, “Hybrid based cluster head selection for maximizing network lifetime and energy
efficiency in WSN,” Journal of King Saud University-Computer and Information Sciences, pp. 1-11, 2019. y
f
g
y
p
f
pp
[20] W. R. Heinzelman, A. Chandrakasan, and H. Balakrishnan, “Energy-efficient communication protocol for wireless
microsensor networks,” Proceedings of the 33rd annual Hawaii international conference on system sciences, 2000. [20] W. R. Heinzelman, A. Chandrakasan, and H. Balakrishnan, “Energy-efficient communication protocol for wireless
microsensor networks,” Proceedings of the 33rd annual Hawaii international conference on system sciences, 2000. [21] Sekaran, Kaushik, and P. Venkata Krishna, “Cross region load balancing of tasks using region-based rerouting of
l
d i
l
d
ti
i
t ” I t
ti
l J
l f Ad
d I t lli
P
di
l 9
5 6 [20] W. R. Heinzelman, A. Chandrakasan, and H. Balakrishnan, “Energy-efficient communication protocol for wireless
microsensor networks,” Proceedings of the 33rd annual Hawaii international conference on system sciences, 2000. [21] Sekaran, Kaushik, and P. Venkata Krishna, “Cross region load balancing of tasks using region-based rerouting of
loads in cloud computing environment,” International Journal of Advanced Intelligence Paradigms, vol. 9, no. 5-6,
pp. 589-603, 2017. ,
g
f
f
y
[21] Sekaran, Kaushik, and P. Venkata Krishna, “Cross region load balancing of tasks using region-based rerouting of
loads in cloud computing environment,” International Journal of Advanced Intelligence Paradigms, vol. 9, no. 5-6,
pp. 589-603, 2017. 2] S. Lindsey, and C. S. Raghavendra, “PEGASIS: Power-efficient gathering in sensor information systems
Proceedings, IEEE aerospace conference, vol. 3, pp. 3-3, 2002. [23] O. Younis, S. Fahmy, “HEED a hybrid, energy-efficient, distributed clustering approach for ad hoc sensor networks,”
IEEE Transactions on mobile computing, vol. 4, pp. An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran) REFERENCES 27, no. 5, pp. 1301-1316, 2016. [11] J. K. Seth, and S. Chandra, “Intrusion detection based on key feature selection using binary GWO,” 3rd International
Conference on Computing for Sustainable Global Development, pp. 3735-3740, 2016. 2] M. Kohli, and S. Arora, “Chaotic grey wolf optimization algorithm for constrained optimization problems,” Journ
of computational design and engineering, vol. 5, no. 4, pp. 458-472, 2018. f
p
g
g
g
pp
[13] V. K. Kamboj, “A novel hybrid PSO–GWO approach for unit commitment problem,” Neural Computing and
Applications, vol. 27, no. 6, pp. 1643-1655, 2016. pp
pp
[14] F. Xiangning, and S. Yulin, “Improvement on LEACH protocol of wireless sensor network,” 2007 International
Conference on Sensor Technologies and Applications, pp. 260-264, 2007. [15] M. Sangeetha, and A. Sabari, “Genetic optimization of hybrid clustering algorithm in mobile wireless sensor
networks,” Sensor Review, vol. 38, no. 4, pp. 526-533, 2018. TELKOMNIKA Telecommun Comput El Control TELKOMNIKA Telecommun Comput El Control [33] S. Arjunan, and P. Sujatha, “Lifetime maximization of wireless sensor network using fuzzy based unequal clustering
and ACO based routing hybrid protocol,” Applied Intelligence, vol. 48, no. 8, pp. 1-18, 2017. 4] T. Ahmad, M. Haque, and A. M. Khan, “An Energy-Efficient Cluster Head Selection Using Artificial Bees Colo
Optimization for Wireless Sensor,” Advances in Nature-Inspired Computing and Applications, pp. 189-203, 2018 [34] T. Ahmad, M. Haque, and A. M. Khan, “An Energy-Efficient Cluster Head Selection Using Artificial Bees Colony
Optimization for Wireless Sensor,” Advances in Nature-Inspired Computing and Applications, pp. 189-203, 2018. [35] A. Sarkar, and T. S. Murugan, “Cluster head selection for energy efficient and delay-less routing in wireless sensor
network,” Wireless Networks, vol. 25, no. 1, pp. 303-320, 2019. Optimization for Wireless Sensor, Advances in Nature Inspired Computing and Applications, pp. 189 203, 2018. [35] A. Sarkar, and T. S. Murugan, “Cluster head selection for energy efficient and delay-less routing in wireless sensor
network,” Wireless Networks, vol. 25, no. 1, pp. 303-320, 2019. ,
,
,
, pp
,
[36] S. Mirjalili, S. M. Mirjalili, and A. Lewis, “Grey wolf optimizer,” Advances in engineering software, vol. 69,
pp. 46-61, 2014. An energy-efficient cluster head selection in wireless sensor network using… (Kaushik Sekaran)
|
https://openalex.org/W2899305944
|
https://journals.vsu.ru/sorpchrom/article/download/419/391
|
Russian
| null |
Анализ морфологии поверхности микрофильтрационных мембран МФФК, МПС методом атомной силовой микроскопии
|
Sorbcionnye i hromatografičeskie processy
| 2,018
|
cc-by
| 3,389
|
608 608 УДК 543 Поступила в редакцию 15.02.2017 г. Методом атомной силовой микроскопии исследована морфология поверхности полимерных
микрофильтрационных мембран МФФК, МПС. Анализ полученных результатов позволил установить
различия морфологии поверхности исходных коммерческих пористых микрофильтрационных мате-
риалов и образцов после проведения процесса микрофильтрационного разделения растворов зрелой
мелассной бражки, подвергшихся эксплуатации при избыточном трансмембранном давлении 0,05
МПа. В работе выделены и рассчитаны площади эллиптических глобул полимера, показаны межпо-
ровые участки на выбранных областях сканирования и поровые участки (поры) щелевой формы, яв-
ляющиеся проводящими участками для проницания растворителя. р
у
р
р
р
Ключевые слова: атомно-силовая микроскопия, поверхность, морфология, микрофил
ционная мембрана, поровое пространство. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 Kovaleva О.А., Lazarev S.I., Osipova I.A., Kovalev S.V., Polyansky К.К.
1Tambov State Technical University, Tambov
2Voronezhsky branch Russian University of Economics G.V. Plekhanov, Voronezh 1Tambov State Technical University, Tambov
2Voronezhsky branch Russian University of Economics G.V. Plekhanov, Voronezh The surface morphology of the polymeric microfiltration membranes was investigated by atomic-
force microscopy. Manufactured new and used (which were got after technological solution separation by
microfiltration process) microfiltration membranes MFFK 0.45 µm (fine-pored white film on a nonwoven
substrate based on the hydrophobic PTFE composite membrane) and MPS 0,45 µm (white film based on
polyethersulfone) are produced by LLC «Scientific-production enterprise «Technofilter». The mature mo-
lasses mash from OJSC «BIOHIM» (Rasskazovo) was used in the microfiltration process as process liquid
for used membranes MFFK and MPS. The study of the surface morphology of the microfiltration membranes
was carried out in the flat and topographic mode (for the study of surface topography) and phase contrast (for
recognition of areas differing in porous composition (relative proportion of voids), the adhesion and elastic
properties). Analysis of the results obtained by atomic-force microscopy allowed to establish morphology
differences of the original porous microfiltration materials and samples after solutions separation of the ma-
ture molasses mash by microfiltration with excessive transmembrane pressure 0.05 MPa. The surface con-
trast of the flat 2D and the visual 3D images of the new and used MFFK and MPS membranes were used to
identify porous and interporous sections of membranes before and after the microfiltration process. It is ob-
served in comparison of obtained contrast images of the membranes surfaces that the pores plots of new
MFFK and MPS membranes with the selected scan area 100 µm2 have wider mixed form pores (length and
width are 0.4-0.6 µm), and used membrane have mixed form pores with domination of elongated slits (length Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 609 is 0.3-0.5 µm and width is 0.2-0.3 µm). Reducing of the pores size of the membranes, which were used,
shows that membrane is clogged with particles of yeast and polysaccharides that are present in the treated
vinasse solution. is 0.3-0.5 µm and width is 0.2-0.3 µm). Reducing of the pores size of the membranes, which were used,
shows that membrane is clogged with particles of yeast and polysaccharides that are present in the treated
vinasse solution. Keywords: atomic force microscopy, surface morphology, membrane. Введение Применение методов сканирующей зондовой микроскопии является активно
развивающимся направлением для исследования морфологии поверхности органи-
ческих и неорганических мембранных материалов [1-3]. В работе [4] представлено
исследование морфологии поверхности мембран МФ-4СК и модифицированной по-
лианилином МФ-4СК. В источнике указывается, что АСМ (атомно-силовая микро-
скопия) позволяет характеризовать качественные и количественные изменения рель-
ефа исследуемой поверхности, что является важной информацией для моделирова-
ния течения растворов через эти поверхности. Исследование состояния поверхности
анионообменных мембран МА-40 и МА-41 сорбировавших пектин методом скани-
рующей силовой микроскопии представлено в источнике [5]. В работе отмечается,
что сорбция пектина на мембранах МА-40 и МА-41 повышает и понижает, соответ-
ственно, однородность поверхности, что связано с различной химической природой
ионообменного материала мембран. В работе [6], рассмотрено применения АСМ
для изучения пористой структуры катионообменных мембран, на основании экспе-
риментальных данных авторами рассчитываются пористость и распределение пор по
радиусам. р
у
Механические свойства новых и отработанных засоренных мембран для об-
ратного осмоса из композитного полиамида, используемых для процессов очистки и
опреснения морской воды были исследованы с применением методов атомно-
силовой микроскопии (AСM) в источнике [7]. В работе отмечается, что на величину
отложения на мембранах бактерий влияет гладкость поверхности, которую можно
исследовать методом атомно-силовой микроскопии для оценки биологического об-
растания мембран. Данный метод использовался для определения механических
свойств (сил сцепления, работы когеззии) исходной и использованной в промыш-
ленности мембран. В источнике отмечается, что использование метода (AСM) для
исследования изображения поверхности мембран исходных и бывших в употребле-
нии перегородок облегчает анализ системы применения промышленных мембран. В
работе [8] представлен обзор по современному состоянию исследований нанофильт-
рационных мембран методом атомно-силовой микроскопии, по материалам работы
приводится заключение, что широкий диапазон использования данного метода явля-
ется универсальным инструментом для определения и изучения характеристик по-
верхности. Имеются и другие работы зарубежных ученых исследующих проницае-
мые, структурные и морфологические характеристики мембран методом силовой
микроскопии [9-15], основное внимание которых уделено обратноосмотическим и
нанофильтрационным пористым перегородкам. Проведенный обзор по использованию методов сканирующей силовой мик-
роскопии для исследования структуры поверхности органических и неорганических
мембран позволил сформулировать утверждение, что адекватное использование
процесса мембранного разделения растворов различных производств связано не
только со структурой мембран, но и морфологией поверхности, для расширения
представлений о механизме переноса веществ через поровое пространство. Поэтому
целью данной работы явилось исследование методом атомной силовой микроскопии
морфологии поверхности микрофильтрационных мембран МФФК, МПС. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 610 Эксперимент Применение для биохимических производств высокопроизводительных по-
ристых полимерных материалов, используемых при высоких температурах эксплуа-
тации до 100оС, обладающих хорошей механической прочностью и задерживающей
способностью по белкам и полисахаридам, является перспективной задачей, которая
может решаться при использовании микрофильтрационных материалов типа МФФК
и МПС. Объектами исследования являлись промышленно выпускаемые исходные и
отработанные (полученные после проведения процесса микрофильтрационного раз-
деления технологического раствора) микрофильтрационные мембраны МФФК – 0.45
(мелкопористая пленка белого цвета на нетканой подложке на основе гидрофобной
фторопластовой композиционной мембраны) и МПС – 0.45 (пленка белого цвета на
основе полиэфирсульфона) (ООО НПП «Технофильтр», Владимир) [16, 17]. В каче-
стве технологической жидкости используемой для образцов МФФК, МПС при про-
ведении процесса микрофильтрования, выступала зрелая мелассная бражка
ОАО«Биохим» г. Рассказово, основные характеристики которой представлены в
таблице 1. Таблица
1. Основные
характеристики
зрелой
мелассной
бражки
(ОАО «Биохим», Рассказово)
Основные показатели
и характеристики
Единица
измерения
Значение
Бражной чан № 3
Цвет
-
Темно-коричневый мутный раствор
рН среды
-
5.25± 0.15
Содержание спирта
%об. 9.20
Содержание биомассы дрожжей
кг/м3
11.50
Несброженные сахара
г/см3
0.525 Поверхности исходных и отработанных образцов мембран МФФК и МПС
подвергались сканированию при помощи зондового микроскопа NanoEducator кор-
порации NT-MDT (Россия, Зеленоград) в автоматическом режиме на воздухе при
температуре 22±1°C на основе рекомендаций представленных в руководстве [18]. Размер скана (масштаб сканирования) при исследовании морфологии поверхности
исследуемых образцов исходной и отработанной мембран МФФК, МПА выбирался
одинаковым для двух типов пористых перегородок 10.0х10.0 мкм. Компьютерное
моделирование результатов проводилось средствами программы Scan Viewer. Для визуализации поверхности микрофильтрационных мембран был исполь-
зован градиентный фильтр Превита, позволяющий подчеркивать и усиливать неод-
нородности изображения посредством выделения границ объектов. Для выравнива-
ния поверхности и устранения наклона и искажений изображения образцов поли-
мерных перегородок применено вычитание поверхности, заключающееся в очистке
изображения от проявления различных шумов (от полученного первоначального
скана изображения поверхности мембран вычитался скан изображения поверхности
обработанной с применением фильтра). р
р
ф
р
Исследование морфологии поверхности микрофильтрационных мембран
осуществлялось в плоском и топографическом режиме (для изучения рельефа по-
верхности) и фазового контраста (для распознавания областей отличающихся по по-
розностному составу (относительной доле пустот), адгезионным и упругим свойст-
вам). Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 611 Обсуждение результатов В ходе исследования были получены плоские - 2D (рис. 1, 2 а, б) и наглядные
- 3D (рис. 1, 2 в, г) изображения поверхности исходной (а, в) и отработанной (б, г)
мембран МФФК и МПС до (слева) и после (справа) проведения процесса микро-
фильтрационного разделения технологического раствора. По плоским и наглядным изображениям мембран МФФК и МПС (рис. 1, 2)
выполненным методом атомно-силовой микроскопии можно заметить, что поверх-
ности этих перегородок имеют различную морфологическую структуру, из которой
выделяются участки глобул полимера (активно проявляющихся на отработанных
образцах мембран при заданном разрешении сканирования), пор и межпоровые об-
ласти [19]. [
]
а
б
в
г
Рис. 1. АСМ-изображения поверхности микрофильтрационной мембраны
МФФК: плоские - 2D (а, б) и наглядные - 3D (в, г) изображения исходной (а, в)
и отработанной (б, г) мембран б а б а в г г в Рис. 1. АСМ-изображения поверхности микрофильтрационной мембраны
МФФК: плоские - 2D (а, б) и наглядные - 3D (в, г) изображения исходной (а, в)
и отработанной (б, г) мембран Анализируя АСМ-изображения поверхности микрофильтрационной мембра-
ны МФФК и МПС представленных на рис. 1 и 2 можно отметить, что глобулы поли-
мера на плоских - 2D (а) и наглядных - 3D (в) изображениях исходных мембран ме-
нее выражены, причем при указанном разрешении сканирования эти глобулы, на
наш взгляд, имели эллиптическую форму и только частично попадали во фрейм ска-
нированного участка, а глобулы полимера для плоских - 2D (б) и наглядных - 3D (г)
изображений отработанных мембран, только подтверждали наше предположение об
их эллиптической форме. Площадь эллиптических глобул полимера рассчитывалась по (1): (1) у
b
a
Sгл
⋅
⋅
= π
, где Sгл - площадь эллиптической глобулы полимера, мкм2; π - число пи; a - длина
большой полуоси, мкм; b - длина малой полуоси, мкм. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 612 а
б
в
г
Рис. 2. АСМ-изображения поверхности микрофильтрационной мембраны
МПС: плоские - 2D (а, б) и наглядные - 3D (в, г) изображения исходной (а, в)
и отработанной (б, г) мембран б а б а г в г в в
г
Рис. 2. АСМ-изображения поверхности микрофильтрационной мембраны
МПС: плоские - 2D (а, б) и наглядные - 3D (в, г) изображения исходной (а, в)
и отработанной (б, г) мембран Отработанные образцы мембран (рис. Обсуждение результатов 1, 2 б, г), полученные после микро-
фильтрационного разделения мелассной бражки, лишь подчеркивали рельефность
глобул полимера, пор и межпоровых участков, что является предсказуемым состоя-
нием в результате действия на полимерную мембрану рабочего давления (обжатое
состояние мембраны). Расчетная площадь глобул при измеренных большой а, малой
b полуоси эллипса соответственно составляли
2 SглМФФК=π .а.b=π 4 мкм 7.9 мкм=90.43 мкм2 SглМФФК π а b π 4 мкм 7.9 мкм 90.43 мкм
и SглМПС=π . 2
1
a
a +
. 2
1
b
b +
=π .3.94 мкм 7.54 мкм=93.28 мкм2 (в случае на-
клонного расположения глобулы). и SглМПС=π . 2
1
a
a +
. 2
1
b
b +
=π .3.94 мкм 7.54 мкм=93.28 мкм2 (в случае на-
нного расположения глобулы). и SглМПС=π . 2
1
a
a +
. 2
1
b
b +
=π .3.94 мкм 7.54 мкм=93.28 мкм2 (в случае на-
клонного расположения глобулы). В литературе [20] отмечается, что глобулы полимеров – аморфны, имеют не-
одинаковые размеры вследствие различной длины макромолекул, что обуславливает
их рыхлую упаковку с неравномерной плотностью, что на наш взгляд, также говорит
о способности этих участков мембраны пропускать растворитель наряду с поровыми
областями (порами). Для дальнейшей идентификации межпоровых и поровых участков мембран
принималось решение использовать контрастирование поверхности плоских - 2D
(рис. 3, 4 а, б) и наглядных - 3D (рис. 3, 4 в, г) изображений исходной (а, в) и отрабо-
танной (б, г) мембран МФФК и МПС до (слева) и после (справа) проведения процес-
са микрофильтрационного разделения технологического раствора. Сравнивая полученные контрастные изображения поверхностей мембран
(рис. 3, 4) можно отметить, что участки пор исходных мембран МФФК и МПС при
выбранной площади сканирования (100 мкм2) имеют более широкие поры смешан-
ной формы (длиной и шириной 0.4-0.6 мкм, а отработанные мембраны - поры сме-
шанной формы, где преобладают вытянутые щели (длиной 0.3-0.5 мкм, шириной 0.2-
0.3 мкм). Уменьшение размеров пор обработанных мембран показывает, что мем- Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 613 браны засоряются частицами дрожжей и полисахаридов, которые присутствуют в
растворе обрабатываемой бражки. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4
браны засоряются частицами дрожжей и полисахаридов, которые присутствуют в
растворе обрабатываемой бражки. а
б
в
г
Рис. 3. АСМ-изображения поверхности микрофильтрационной мембраны МФФК:
плоские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран
а
б
в
г
Рис. 4. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 Заключение Проведенный анализ морфологии поверхности микрофильтрационных мем-
бран МФФК и МПС методом атомно-силовой микроскопии позволил получить но-
вые знания о поверхностной структуре мембран с выделением участков расположе-
ния глобул полимера, пор и межпорового связующего участка, что углубляет науч-
ное описание механизма переноса веществ через полимерные области пористого ма-
териала. Полученные экспериментальные и расчетные данные по морфологии поверх-
ности микрофильтрационных исходных и отработанных мембран МФФК и МПС ме-
тодом атомно-силовой микроскопии позволят адекватно моделировать работу полу-
промышленных установок с применением пористых перегородок, так как исследо-
вание структуры поверхностного слоя мембраны необходимо для выяснения меха-
низма обрастания мембран при разделении биохимических и биологических раство-
ров. Обсуждение результатов АСМ-изображения поверхности микрофильтрационной мембраны МПС: пло-
ские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран б р
а р
р
р
р
а
б
в
г
Рис. 3. АСМ-изображения поверхности микрофильтрационной мембраны МФФК:
плоские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран б а в г г в Рис. 3. АСМ-изображения поверхности микрофильтрационной мембраны МФФК:
плоские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран Рис. 3. АСМ-изображения поверхности микрофильтрационной мембраны МФФК:
плоские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран
а
б
в
г
Рис. 4. АСМ-изображения поверхности микрофильтрационной мембраны МПС: пло-
ские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран б а
б а б а б
г в г в Рис. 4. АСМ-изображения поверхности микрофильтрационной мембраны МПС: пло-
ские - 2D (а, б) и наглядные - 3D (в, г) контрастные изображения исходной (а, в)
и отработанной (б, г) мембран 614 Межпоровые участки являются связующими областями между глобулами по-
лимера мембран и порами. При контрастировании поверхности исходных (а, в) и от-
работанных мембран (б, г) (рис. 3, 4) отмечается, что окраска поровых (пор) и меж-
поровых участков мембраны МФФК более светлая, что говорит о хорошей рыхлости
(пористости) данной мембраны по сравнению с менее осветленными образцами по-
верхности полимерной перегородки МПС. References 10. Vrijenhoek E.M., Hong S., Elimelech M., J. of Membrane Science, 2001, Vol. 188, рр. 115-
128. 1. Vasil'eva V.I., Bitjuckaja L.A., Zajchenko
N.A., Grechkina M.V. et al., Sorbtsionnye i khro-
matograficheskie protsessy 2008, Vol. 8, No. 2,
рр. 260-271. 11. Boussu K., Van der Bruggen B., Volodin A.,
J. of Colloid and Interface Science, 2005, Vol. 286, рр. 632-638. 2. D'jakonova O.V., Sokolova S.A., Zjablov
A.N., Zhibrova Ju.A, Sorbtsionnye i khromatogra-
ficheskie protsessy 2008, Vol. 8, No. 5, рр. 863-
868. 12. Lee H.S., Im S.J., Kim J.H., Desalination,
2008, Vol. 219, рр. 48-56. 3. Wyart Y., Georges G., Deumie C., Amra C. et
al., J. of Membrane Science, 2008, Vol. 315, рр
82-92. 13. Xindong L., Lei W., Wanfu H., Juan L. et
al., J. of Engineering Science and Technology Re-
view, 2016, Vol. 9 (3), рр. 74-79. 14. Quanfu A., Feng L., Yanli J., Huanlin C., J. of Membrane Science, 2011, Vol. 367, No 1-2, рр. 158-165. 4. Kalinin V.V., Filippov A.N., Hanukaeva
D.Ju., Trudy RGU Nefti i gaza im. I.M. Gubkina. Avtomatizacija, modelirovanie i jenergoobespe-
chenie, 2012, No 1(266), рр. 129-136. 15. Gizli N., Chemistry& Chemical Technology,
2011, Vol. 5, No 3, рр. 327-331. рр
5. Kotov V.V., Grechkina M.V., Peregonchaja
O.V., Zjablov A.N. , Sorbtsionnye i khromatogra-
ficheskie protsessy2016, Vol. 16, No 1, рр. 118-
122. 16. Pasport № 81 na membrannyj disk MFFK –
0.45, nomer partii 52. data vypuska - 03.2016. OOO NPP «Tehnofil'tr», g. Vladimir. 6. Zajchenko N.A., Vasil'eva V.I., Grigorchuk
O.V., Zjablov A.N. et al., Sorbtsionnye i khroma-
tograficheskie protsessy 2010,Vol. 10, No 5, рр. 745-749. 17. Pasport № 79 na membrannyj disk MPS –
0.45, nomer partii 15. data vypuska - 03.2016. OOO NPP «Tehnofil'tr», g. Vladimir. 18. Skanirujushhij zondovyj mikroskop NanoE-
ducator. Rukovodstvo pol'zovatelja. Zelenograd,
NT-MDT, 2008, 137 р. 7. Powell L.C., Hilal N., Wright C.J., Desalina-
tion, 2017, Vol. 404, рр. 313-321. 19. Krisilova E.V., Eliseeva T.V., Grechkina
M.V., Sorbtsionnye i khromatograficheskie prot-
sessy, 2010, Vol. 10, No 1, рр. 103-107. 8. Johnson D., Hilal N., Desalination, 2015, Vol. 356, рр. 149-164. 9. Boussu K., Van der Bruggen B., Volodin A.,
Van Haesendonck C. et al., Desalination, 2006,
Vol. 191, рр. 245–253. 20. Kapkin V.D., Savineckaja G.A., Chapurin
V.I. Tehnologija
organicheskogo
sinteza. Moskow, Himija, 1987. 400 p. Kovaleva Olga A. Список литературы 10. Vrijenhoek E.M., Hong S., Elimelech M. //
Journal of Membrane Science. 2001. Vol. 188. pp. 115-128. 1. Васильева В.И., Битюцкая Л.А., Зайченко
Н.А., Гречкина М.В. и др. // Сорбционные и
хроматографические процессы. 2008. Т. 8. №
2. С. 260-271. 11. Boussu K., Van der Bruggen B., Volodin A. // Journal of Colloid and Interface Science. 2005. Vol. 286. pp. 632-638. 2. Дьяконова О.В., Соколова С.А., Зяблов
А.Н., Жиброва Ю.А. // Сорбционные и хрома-
тографические процессы. 2008. Т. 8. № 5. C. 863-868. 12. Lee H.S., Im S.J., Kim J.H. // Desalination. 2008. Vol. 219. pp. 48-56. 3. Wyart Y., Georges G., Deumie C., Amra C.,
Moulina P. // Journal of Membrane Science. 2008. Vol. 315. pp. 82-92. 13. Xindong L., Lei W., Wanfu H., Juan L., et
al. // Journal of Engineering Science and Tech-
nology Review. 2016. Vol. 9 (3) pp. 74-79. 14. Quanfu A., Feng L., Yanli J., Huanlin C. //
Journal of Membrane Science. 2011. Vol. 367. No
1-2. pp. 158-165. 4. Калинин В.В., Филиппов А.Н., Ханукаева
Д.Ю. // Труды РГУ Нефти и газа им. И.М. Губ-
кина. Автоматизация, моделирование и энер-
гообеспечение. 2012. № 1(266). С. 129-136. 15. Gizli N. // Chemistry& Chemical Technolo-
gy. 2011. Vol. 5. No 3. pp. 327-331. 5. Котов В.В. , Гречкина М.В. , Перегончая
О.В., Зяблов А.Н. // Сорбционные и хромато-
графические процессы. 2016. Т. 16. № 1. С. 118-122. 16. Паспорт № 81 на мембранный диск
МФФК – 0.45, номер партии 52. дата выпуска -
03.2016. ООО НПП «Технофильтр». г. Влади-
мир. 6. Зайченко Н.А., Васильева В.И., Григорчук
О.В., Зяблов А.Н. и др. // Сорбционные и хро-
матографические процессы. 2010. Т. 10. № 5. C. 745-749 17. Паспорт № 79 на мембранный диск МПС
– 0.45, номер партии 15. дата выпуска -
03.2016. ООО НПП «Технофильтр». г. Влади-
мир. 7. Powell L.C., Hilal N., Wright C.J. // Desalina-
tion. 2017. Vol. 404. pp. 313-321. 18. Сканирующий
зондовый
микроскоп
NanoEducator. Руководство пользователя. Зе-
леноград. НТ-МДТ. 2008. 137 с. 8. Johnson D., Hilal N. // Desalination. 2015. Vol. 356, pp. 149-164. 19. Крисилова Е.В., Елисеева Т.В., Гречкина
М.В. // Сорбционные и хроматографические
процессы. 2010. Т. 10. № 1. С. 103-107. 9. Boussu K., Van der Bruggen B., Volodin A.,
Van Haesendonck C. et al. // Desalination. 2006. Vol. 191. pp. 245-253. Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4 615 20. Список литературы Капкин В.Д., Савинецкая Г.А., Чапурин
В.И. Технология органического синтеза. М. Химия. 1987. 400 с. References – Associate professor of applied
geometry and computer graphics, Ph.D., Tambov State
Technical University, Tambov Ковалева Ольга Александровна – доцент кафед-
ры прикладная геометрия и компьютерная графика,
к.т.н., Тамбовский государственный технический
университет, Тамбов Лазарев Сергей Иванович – профессор кафедры
прикладная геометрия и компьютерная графика,
д.т.н., Тамбовский государственный технический
университет, Тамбов Lazarev Sergey I. – professor of applied geometry and
computer graphics, grand Ph.D, Tambov State Technical
University, Tambov Осипова Ирина Анатольевна – доцент кафедры
физика, к.п.н., Тамбовский государственный техни-
ческий университет, Тамбов Osipova Irina A. – Associate рrofessor of Physics,
Ph.D., Tambov State Technical University, Tambov Kovalev Sergey V. – Associate professor of applied
geometry and computer graphics, grand Ph.D, Tambov
State
Technical
University,
Tambov,
e-
mail: sseedd@mail.ru Ковалев Сергей Владимирович – доцент кафедры
прикладная геометрия и компьютерная графика,
д.т.н., Тамбовский государственный технический
университет, Тамбов Полянский Константин Константинович - про-
фессор кафедры коммерции и товароведения, д.т.н.,
Воронежский филиал “РЭУ им. Г.В. Плеханова”,
Воронеж Polyansky Konstantin K. – рrofessor of the depart-
ment of commerce and commodity, grand Ph.D, Voro-
nezhsky branch "Russian University of Economics. G.V. Plekhanov ", Voronezh Ковалева и др. / Сорбционные и хроматографические процессы. 2017. Т. 17. № 4
|
https://openalex.org/W3010838691
|
https://repozitorij.uni-lj.si/Dokument.php?id=151820&dn=
|
English
| null |
Recent Achievements in Development of TiO2-Based Composite Photocatalytic Materials for Solar Driven Water Purification and Water Splitting
|
Materials
| 2,020
|
cc-by
| 26,838
|
Review
Recent Achievements in Development of TiO2-Based
Composite Photocatalytic Materials for Solar Driven
Water Purification and Water Splitting Klara Perovi´c 1,†, Francis M. dela Rosa 1,†, Marin Kovaˇci´c 1
, Hrvoje Kuši´c 1,*,
Urška Lavrenˇciˇc Štangar 2
, Fernando Fresno 3, Dionysios D. Dionysiou 4
and
Ana Loncaric Bozic 1 Klara Perovi´c 1,†, Francis M. dela Rosa 1,†, Marin Kovaˇci´c 1
, Hrvoje Kuši´c 1,*,
Urška Lavrenˇciˇc Štangar 2
, Fernando Fresno 3, Dionysios D. Dionysiou 4
and
Ana Loncaric Bozic 1 Klara Perovi´c 1,†, Francis M. dela Rosa 1,†, Marin Kovaˇci´c 1
, Hrvoje Kuši´c 1,*,
Urška Lavrenˇciˇc Štangar 2
, Fernando Fresno 3, Dionysios D. Dionysiou 4
and
Ana Loncaric Bozic 1 1
Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 19, HR–10000 Zagreb,
Croatia; kperovic@fkit.hr (K.P.); frosa@fkit.hr (F.M.d.R.); mkovacic@fkit.hr (M.K.); abozic@fkit.hr (A.L.B.)
2 1
Faculty of Chemical Engineering and Technology, University of Zagreb, Marulicev trg 19, HR–10000 Zagreb,
Croatia; kperovic@fkit.hr (K.P.); frosa@fkit.hr (F.M.d.R.); mkovacic@fkit.hr (M.K.); abozic@fkit.hr (A.L.B.)
2
Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia;
Urska.Lavrencic.Stangar@fkkt.uni-lj.si Croatia; kperovic@fkit.hr (K.P.); frosa@fkit.hr (F.M.d.R.); mkovacic@fkit.hr (M.K.); abozic@fkit.hr (A.L.B.)
2
Faculty of Chemistry and Chemical Technology, University of Ljubljana, 1000 Ljubljana, Slovenia;
Urska.Lavrencic.Stangar@fkkt.uni-lj.si 3
Photoactivated Processes Unit, IMDEA Energy, Móstoles, 28935 Madrid, Spain; fernando.fresno@imdea Photoactivated Processes Unit, IMDEA Energy, Móstoles, 28935 Madrid, Spain; fernando.fresno@imdea.org
4
Environmental Engineering and Science Program, University of Cincinnati, Cincinnati, OH 45221–0012,
USA DIONYSDD@UCMAIL UC EDU 4
Environmental Engineering and Science
USA; DIONYSDD@UCMAIL.UC.EDU *
Correspondence: hkusic@fkit.hr †
Equal contribution. Received: 3 February 2020; Accepted: 11 March 2020; Published: 15 March 2020 materials materials 1. Introduction Nowadays, accessible clean water and energy resources are among the highest priorities for
sustainable economic growth and societal wellbeing. Water supports life and is a crucial resource for
humanity; it is also at the core of natural ecosystems and climate regulation. Water stress is primarily a
water quantity issue, but it also occurs as a consequence of a deterioration of water quality and a lack of
appropriate water management [1]. Environmental problems that are associated with water pollution
have been a persistently important issue over recent decades, correlated negatively with the health
and ecosystem. Activities of the Water JPI’s Strategic Research and Innovation Agenda focus on, among
others, new materials and processes, energy efficiency, thus supporting key enabling technologies for
clean water and wastewater treatment [2]. EU Energy Strategies 2020, 2030, and 2050 set increasing
standards for the reduction of greenhouse gas emissions by 20, 40, and 80–95%, respectively, which is
achievable by significant investments in the development and application of new low-carbon and
renewable energy technologies [3]. In light of increased energy demands and the need to reduce
greenhouse gas emissions, the focus has been turned from the fossil fuels toward renewable energy
resources and vectors: solar, wind, tides, waves, geothermal, biomass, biofuels, and hydrogen (H2) [4]. Alternative fuels are required to have as small environmental footprint, and be storable and economical,
whereas H2 satisfies the first two conditions. The research over the last decades has been focused on
fulfilling the third requirement, which triggers its production by solar energy, a largely available and
intrinsically renewable energy resource, through water splitting. It should be emphasized that H2, as a
fuel, possesses higher heat content than gasoline (per unit mass) [5]. The pioneering work of Fujishima and Honda [6] for H2 production by photoelectrochemical water
splitting while using TiO2 photoanode and Pt cathode opened the potential possibilities for generating
this energy vector, i.e., fuel, directly from water and solar energy. Works by Bard and Frank in 1977 [7],
exhibiting photocatalytic oxidation of CN to CNO−, and by Ollis et al. [8], studying the photocatalytic
degradation of organic contaminants in water, practically opened a new research field within new
water purification technologies. H2 production by water splitting and photocatalytic degradation of
organic pollutants in water both rely on the formation of electron/hole (e-/h+) pairs at a semiconducting
material upon its excitation by light with sufficient photon energy [9–12]. Received: 3 February 2020; Accepted: 11 March 2020; Published: 15 March 2020 Abstract: Clean water and the increased use of renewable energy are considered to be two of the main
goals in the effort to achieve a sustainable living environment. The fulfillment of these goals may
include the use of solar-driven photocatalytic processes that are found to be quite effective in water
purification, as well as hydrogen generation. H2 production by water splitting and photocatalytic
degradation of organic pollutants in water both rely on the formation of electron/hole (e−/h+) pairs at
a semiconducting material upon its excitation by light with sufficient photon energy. Most of the
photocatalytic studies involve the use of TiO2 and well-suited model compounds, either as sacrificial
agents or pollutants. However, the wider application of this technology requires the harvesting of a
broader spectrum of solar irradiation and the suppression of the recombination of photogenerated
charge carriers. These limitations can be overcome by the use of different strategies, among which
the focus is put on the creation of heterojunctions with another narrow bandgap semiconductor,
which can provide high response in the visible light region. In this review paper, we report the
most recent advances in the application of TiO2 based heterojunction (semiconductor-semiconductor)
composites for photocatalytic water treatment and water splitting. This review article is subdivided
into two major parts, namely Photocatalytic water treatment and Photocatalytic water splitting,
to give a thorough examination of all achieved progress. The first part provides an overview on
photocatalytic degradation mechanism principles, followed by the most recent applications for
photocatalytic degradation and mineralization of contaminants of emerging concern (CEC), such as
pharmaceuticals and pesticides with a critical insight into removal mechanism, while the second part
focuses on fabrication of TiO2-based heterojunctions with carbon-based materials, transition metal
oxides, transition metal chalcogenides, and multiple composites that were made of three or more
semiconductor materials for photocatalytic water splitting. Keywords: TiO2 heterojunction; semiconductor coupling; water treatment; photocatalytic degradation;
photocatalytic water splitting; H2 production Materials 2020, 13, 1338; doi:10.3390/ma13061338 www.mdpi.com/journal/materials Materials 2020, 13, 1338 2 of 44 1. Introduction These processes, which can
be conducted under environmentally friendly and mild conditions, are economically viable, possessing
a potential of becoming effective methods to produce clean energy and water, owing to their low-cost,
long-term stability, and usage of solar energy [13]. A well-suited model catalyst for photocatalytic studies is TiO2. Its wide application has been
promoted, due to: (i) high photocatalytic activity under the incident photon wavelength of 300 < λ <3
90 nm and (ii) multi-faceted functional properties, such as chemical and thermal stability, resistance to
chemical breakdown, and attractive mechanical properties [14,15]. However, harvesting a broader
spectrum of solar irradiation involves the lowering of the band gap of semiconducting material, whilst
inhibiting the recombination of photogenerated charges. Strategies, including doping with non-metals,
incorporation or deposition of noble metals (ions), and material engineering solutions that are based on
composites formation using transition metals, carbon nanotubes, dye sensitizers, conductive polymers,
graphene (oxide), and semiconducting materials, present viable solutions for set tasks [9,10,15,16]. It is of great importance to combine TiO2 with narrow band gap semiconductors with visible light
response to obtain an effective composite for photocatalytic applications. The obtained synergistic
effect between two or more semiconductors will then promote efficient charge separation, sufficient
visible light response, and high photocatalytic performance. With the dramatic increase of the papers
published related to these topics, a comprehensive review is desirable, providing a general overview on
processes occurring while using TiO2-based heterojunction (semiconductor) systems for photocatalytic
water purification and water splitting. Despite reviews focusing on TiO2–based semiconductor
composites [17,18], those focusing on the removal of contaminants of emerging concern (CECs) are
quite scarce. In addition, this review also summarizes TiO2-based nanocomposites for photocatalytic
water splitting providing insight into effectiveness of a variety of materials groups representing the 3 of 44
zation Materials 2020, 13, 1338
effectiveness of a va alternative for replacing the utilization of expensive, toxic, and non-abundant materials. The first
part of the review focuses on TiO2–based heterojunction (semiconductor-semiconductor coupling)
composites, being selected on the basis of band gap energies suitable to make heterojunctions with
documented applications providing promising results in CECs treatment and stability of prepared
materials, and also respecting their most recent applications for the photocatalytic degradation of
CECs (i.e., demonstrating the current focus within the field), such as pharmaceuticals and pesticides,
with critical insight into the pollutants removal mechanism. 2. Photocatalytic Water Treatment
y
The general mechanism of sem The general mechanism of semiconductor photocatalysis (Figure 1) is composed of three main steps:
1. e−/h+ pairs are generated on the surface of the semiconductor under illumination with the required
wavelength or energy; then, 2.) photogenerated charges (i.e., e−/h+) migrate to the surface of the
semiconductor; and lastly, 3.) e−and h+ induce redox reactions on the surface that facilitate destruction
of organic pollutants [19,20]. As stressed above, TiO2 is still the most studied and widely used material
for photocatalytic degradation reactions. However, TiO2 suffers from fast e−/h+ pair recombination and
large band gap (Eg = 3.1–3.2 eV), which can only be excited under UV light irradiation. The strategies
for improving these issues are provided above, while, among them, semiconductor-coupling presents
a viable structure-properties engineering solution for the enhancement of TiO2 photocatalytic activity
due to the simultaneously reduced e−/h+ recombination rate and enhanced visible light absorption [21]. steps: 1. e−/h+ pairs are generated on the surface of the semiconductor under illumination with the
required wavelength or energy; then, 2.) photogenerated charges (i.e. e−/h+) migrate to the surface of
the semiconductor; and lastly, 3.) e− and h+ induce redox reactions on the surface that facilitate
destruction of organic pollutants [19,20]. As stressed above, TiO2 is still the most studied and widely
used material for photocatalytic degradation reactions. However, TiO2 suffers from fast e−/h+ pair
recombination and large band gap (Eg = 3.1–3.2 eV), which can only be excited under UV light
irradiation. The strategies for improving these issues are provided above, while, among them,
semiconductor-coupling presents a viable structure-properties engineering solution for the
enhancement of TiO2 photocatalytic activity due to the simultaneously reduced e−/h+ recombination
rate and enhanced visible light absorption [21]. Figure 1. Photocatalytic reaction mechanism over semiconductor material. Figure 1. Photocatalytic reaction mechanism over semiconductor material. Figure 1. Photocatalytic reaction mechanism over semiconductor material. Figure 1. Photocatalytic reaction mechanism over semiconductor material. Three main types of heterojunction architectures are reported for TiO2/semiconductor
composites [22]. In Type I heterojunction, the conduction band (CB) of TiO2 is higher in energy
(more negative potential) when compared to the CB of semiconductor 2 and the valence band (VB)
of TiO2 is lower in energy (more positive potential) as compared to the VB of semiconductor 2
[23,24]. This leads to the accumulation of photogenerated h+ and e− in semiconductor 2. 1. Introduction The second part targets the most recent
achievements in the field of fabrication of TiO2-based heterojunctions with carbon based materials,
transition metal oxides, transition metal chalcogenides, and multiple composites that were made of
three or more semiconductor materials for photocatalytic water splitting. heterojunction (semiconductor-semiconductor coupling) composites, being selected on the basis of
band gap energies suitable to make heterojunctions with documented applications providing
promising results in CECs treatment and stability of prepared materials, and also respecting their
most recent applications for the photocatalytic degradation of CECs (i.e. demonstrating the current
focus within the field), such as pharmaceuticals and pesticides, with critical insight into the
pollutants removal mechanism. The second part targets the most recent achievements in the field of
fabrication of TiO2-based heterojunctions with carbon based materials, transition metal oxides,
transition metal chalcogenides, and multiple composites that were made of three or more
semiconductor materials for photocatalytic water splitting. 2 Photocatalytic Water Treatment 2.1.1. TiO2/WO3
/
Tungsten oxi Besides, they studied the mechanisms that are responsible for forming reactive species within
the system and, based on their findings, proposed that, upon forming e−/h+ pairs under solar
irradiation, photogenerated e− from CB of TiO2 are transferred to CB of WO3. Consequently, W6+ was
first reduced to W5+ on WO3 surface, while the W5+ ions are then oxidized to W6+ by adsorbed O2
producing superoxide anion radical (O2●‒). The photogenerated h+ in VB of WO3 are transferred to
VB of TiO2 where they reacted with water (or hydroxyl ions, HO−) forming hydroxyl radicals (•OH)
(Figure 2). The generated reactive oxygen species (ROS) promoted the degradation of 2,4-D and its
intermediates, eventually yielding rather high mineralization extents, while their occurrence in the
system was confirmed through tests with common scavenging agents (e.g., tert-butanol (TB) for
•OH, formic acid (FA) for h+, and p-benzoquinone (BQ) for O2●‒) [24]. Figure 2. Photocatalytic degradation mechanism over TiO2/WO3 composite. Figure 2. Photocatalytic degradation mechanism over TiO2/WO3 composite. Figure 2. Photocatalytic degradation mechanism over TiO2/WO3 composite. Figure 2. Photocatalytic degradation mechanism over TiO2/WO3 composite. The same composite type was used in the degradation of pharmaceuticals. Hence, Mugunthan
et al. [30] treated diclofenac (DCF) while using TiO2/WO3 composites under 4 hrs of visible light
irradiation and reported a maximum of 92% mineralization of overall organic content. They also
elucidated the DCF degradation pathway by LC/MS measurements, which included C-N cleavage in
the DCF molecule forming benzene-ring based intermediates at the first stage, and open-ring
intermediates at the later stage, which were eventually mineralized. Such findings were quite similar
to other studies employing •OH based processes in the degradation of DCF ([33,34]), thus implying
the important role of formed ROS, primarily •OH, in the case of TiO2/WO3 solar driven
photocatalysis as well. Arce-Sarria et al. [35] studied the performance of TiO2/WO3 composite for the
degradation of another pharmaceutical, Amoxicillin (AMX), in pilot scale reactor, where they
achieved 64 4% degradation
The same composite type was used in the degradation of pharmaceuticals. Hence,
Mugunthan et al. [30] treated diclofenac (DCF) while using TiO2/WO3 composites under 4 hrs
of visible light irradiation and reported a maximum of 92% mineralization of overall organic content. They also elucidated the DCF degradation pathway by LC/MS measurements, which included C-N
cleavage in the DCF molecule forming benzene-ring based intermediates at the first stage, and
open-ring intermediates at the later stage, which were eventually mineralized. 2.1.1. TiO2/WO3
/
Tungsten oxi Tungsten oxide (WO3), which is a visible light active photocatalyst with band gap of 2.4–2.8 eV, is a
promising candidate for photocatalytic applications, due to its oxidative properties, nontoxicity, low cost,
and stability in acidic solutions. In addition, WO3 directly matches the band positions of TiO2 to form
a heterojunction (Type II Heterojunction) [29–32]. Several authors studied the application of TiO2/WO3
composites for the degradation of various CECs; either pesticides or pharmaceuticals (Table 1). Hence,
Macias et al. [24] studied the photocatalytic degradation of herbicide 2,4-dichlorophenoxyacetic acid
(2,4-D) while using TiO2/WO3 composites under natural sunlight. They reported the rather high
effectiveness of the studied system: 94.6% degradation of 2,4-D and 88.6% mineralization of overall
organic content under two and four hours of natural sunlight irradiation, respectively. g
g
p
y
g p
is a promising candidate for photocatalytic applications, due to its oxidative properties, nontoxicity,
low cost, and stability in acidic solutions. In addition, WO3 directly matches the band positions of
TiO2 to form a heterojunction (Type II Heterojunction) [29–32]. Several authors studied the application
of TiO2/WO3 composites for the degradation of various CECs; either pesticides or pharmaceuticals
(Table 1). Hence, Macias et al. [24] studied the photocatalytic degradation of herbicide
2,4-dichlorophenoxyacetic acid (2,4-D) while using TiO2/WO3 composites under natural sunlight. They reported the rather high effectiveness of the studied system: 94.6% degradation of 2,4-D and
88.6% mineralization of overall organic content under two and four hours of natural sunlight
irradiation, respectively. Besides, they studied the mechanisms that are responsible for forming reactive species within the
system and, based on their findings, proposed that, upon forming e−/h+ pairs under solar irradiation,
photogenerated e−from CB of TiO2 are transferred to CB of WO3. Consequently, W6+ was first reduced
to W5+ on WO3 surface, while the W5+ ions are then oxidized to W6+ by adsorbed O2 producing
superoxide anion radical (O2•-). The photogenerated h+ in VB of WO3 are transferred to VB of TiO2
where they reacted with water (or hydroxyl ions, HO−) forming hydroxyl radicals (•OH) (Figure 2). The generated reactive oxygen species (ROS) promoted the degradation of 2,4-D and its intermediates,
eventually yielding rather high mineralization extents, while their occurrence in the system was
confirmed through tests with common scavenging agents (e.g., tert-butanol (TB) for •OH, formic acid
(FA) for h+, and p-benzoquinone (BQ) for O2•-) [24]. 2. Photocatalytic Water Treatment
y
The general mechanism of sem In Type II
heterojunction (where TiO2 can be semiconductor 1 or 2), the CB of semiconductor 2 is higher than
the CB position of semiconductor 1 leading to facile transfer of photogenerated e− from CB of
semiconductor 2 to CB of semiconductor 1 [25]. Meanwhile, photogenerated h+ in VB of
semiconductor 1 can travel to the VB of semiconductor 2, which facilitates efficient charge
separation. Type III heterojunction (also known as broken gap situations) [26] shares the same
charge transfer mechanism, like Type II heterojunction. In this case, the CB and VB of semiconductor
2 are higher than CB and VB of TiO2 [27,28]. These heterojunction types are explained in detail in the
context of particular material combinations in the further text and graphically represented through
Fi
2 3 5 6 7 8 9
d 10
Three main types of heterojunction architectures are reported for TiO2/semiconductor
composites [22]. In Type I heterojunction, the conduction band (CB) of TiO2 is higher in energy
(more negative potential) when compared to the CB of semiconductor 2 and the valence band (VB) of
TiO2 is lower in energy (more positive potential) as compared to the VB of semiconductor 2 [23,24]. This leads to the accumulation of photogenerated h+ and e−in semiconductor 2. In Type II heterojunction
(where TiO2 can be semiconductor 1 or 2), the CB of semiconductor 2 is higher than the CB position
of semiconductor 1 leading to facile transfer of photogenerated e−from CB of semiconductor 2 to
CB of semiconductor 1 [25]. Meanwhile, photogenerated h+ in VB of semiconductor 1 can travel
to the VB of semiconductor 2, which facilitates efficient charge separation. Type III heterojunction
(also known as broken gap situations) [26] shares the same charge transfer mechanism, like Type
II heterojunction. In this case, the CB and VB of semiconductor 2 are higher than CB and VB of
TiO2 [27,28]. These heterojunction types are explained in detail in the context of particular material
combinations in the further text and graphically represented through Figures 2, 3 and 5–10. 4 of 44
4 of 39 Materials 2020, 13, 1338
Materials 2020, 13, x FOR 2.1.1. TiO2/WO3
/
Tungsten oxi Such findings were
quite similar to other studies employing •OH based processes in the degradation of DCF ([33,34]),
thus implying the important role of formed ROS, primarily •OH, in the case of TiO2/WO3 solar driven
photocatalysis as well. Arce-Sarria et al. [35] studied the performance of TiO2/WO3 composite for the
degradation of another pharmaceutical, Amoxicillin (AMX), in pilot scale reactor, where they achieved
64.4% degradation. 5 of 44 Materials 2020, 13, 1338 hotocatalytic degradation of contaminants of emerging concern (CECs) over TiO2/WO3 composites. Table 1. Photocatalytic degradation of contaminants of emerging concern (CECs) over TiO2/WO3 composites. Table 1. Photocatalytic degradation of contaminants of emerging concern (CECs) over TiO2/WO3 composites. Catalyst
Target
Pollutant
Initial
Concentration/Working
Volume
((mg L−1) /mL)
Experimental Conditions
Reaction
Time
Removal Extent (%)
Reference
TiO2 - WO3
(0.5 g/L )
2,4-dichlorophenoxy
acetic acid
50
(in 250 mL)
Light Source: natural sunlight
11AM-4PM;
pH = 4
120 min
94.6
(TOC = 88.6)
[24]
TiO2 - WO3
(0.6 g/L)
Diclofenac
25
(in 100 mL)
Light Source: 400 W Metal Halide Lamp;
pH = 5
240 min
TOC = 91
[30]
TiO2 - WO3
(0.1 g/L)
Amoxicillin
100
(in 25,000 mL)
CPC Reactor with accumulated energy
550,000 J/m2
NA
64.4 (@ 550 kJ/m2)
[35]
(WO3/TiO2-C)
(1.0 g/L)
Diclofenac
10
(in 300 mL)
Light Source: 1500 W Xenon Lamp with
filter(λ > 290 nm) ;
pH = 7
NA
100 (@ 250 kJ/m2)
(TOC = 82.4 @ 400
kJ/m2)
[32]
(WO3/TiO2-N)
(1.0 g/L)
Diclofenac
10
Light Source: 1500 W Xenon Lamp with
ID65 solar filter;
pH = 6.5
NA
100 (@ 250 kJ/m2)
(TOC = 100 @400
kJ/m2)
[31]
NA—not available. NA—not available. Materials 2020, 13, 1338 6 of 44 Besides “pure” TiO2/WO3 composite, several authors studied the performance of its enriched analogues
(Table 1). Hence, Cordero-García et al. [32] studied DCF degradation by WO3/C-doped TiO2 and reported
100%DCFdegradationand82.4%mineralizationoftheoverallorganiccontentunder250kJ/m2 and400kJ/m2
of solar-accumulated energy, respectively. They also stated that the WO3/C-doped TiO2 composite showed
superior photocatalytic activity for the complete degradation and mineralization of DCF when comparing to
the pristine TiO2, used as benchmark material. Based on the findings on elucidated mechanisms within the
studied composite and DCF degradation pathway provided, the authors concluded that the incorporation
of elemental carbon to TiO2 crystal structure promoted the formation of a C2p-hybridized valence band
that lowered the band gap of TiO2 by mixing with O2p orbitals. 2.1.2. TiO2/Fe2O3 Iron oxide (α-Fe2O3) is a promising candidate for photocatalytic applications, due to its abundance,
nontoxicity, low cost, stability in aqueous solutions (pH > 3), and narrow band gap (2.0–2.2 eV),
which directly matches the band positions of TiO2 to form heterojunction (Type I Heterojunction) [23,36]. Iron oxide (α-Fe2O3) is a promising candidate for photocatalytic applications, due to its abundance,
nontoxicity, low cost, stability in aqueous solutions (pH > 3), and narrow band gap (2.0–2.2 eV),
which directly matches the band positions of TiO2 to form heterojunction (Type I Heterojunction) [23,36]. Several authors report the photocatalytic degradation of CECs using TiO2/Fe2O3 composites (Table 2). Hence, Mirmasoomi et al. [37] used TiO2/Fe2O3 as a catalyst for photocatalytic degradation of Diazinon
(DZ). The authors reported an optimized system with maximum degradation of DZ equal to 95.07% within
45 min. under visible light irradiation. In another study by Moniz et al. [23], photocatalytic degradation
of 2,4-D while using TiO2/Fe2O3 composites was investigated, reporting 100% 2,4-D degradation and
100% mineralization of overall organic content within 2 h and 3 h, respectively, using irradiation from
a 300 W Xenon Lamp. The authors found out that, when compared to the benchmark TiO2 (P25),
the TiO2/Fe2O3 composite shows superior photocatalytic activity. Based on photoluminescence and
photocurrent studies, the TiO2/Fe2O3 composite exhibits enhanced separation of e−/h+ pairs due to the
formed heterojunction. The proposed mechanism was supported with DFT studies, which firstly involved
the transfer of photogenerated e−from TiO2 CB to Fe2O3 CB. In addition, Fe2O3 binds strongly with
(dissolved) oxygen, thus aiding the photoelectron transfer. This in-situ second stage mechanism facilitates
the facile migration of h+ from the VB of TiO2 [23]. Macías et al. [24] studied the same system, TiO2/Fe2O3
composites for photocatalytic degradation of 2,4-D, but while using natural sunlight. The authors reported
96.8% 2,4-D degradation and 90.0% mineralization of overall organic content under two hours and four
hours, respectively. Contrary to the presented mechanism of Moniz et al. [23], Macias et al. [24] proposed
that the incorporation of Fe2O3 causes the photogenerated e−in CB of TiO2 to be transferred to CB of
Fe2O3, promoting the reduction of Fe3+ to Fe2+. Photogenerated h+ in VB of TiO2 are transferred to VB of
Fe2O3, which leads to the regeneration of Fe3+ and avoids the recombination of e−/h+ pairs at TiO2 surface. 2.1.1. TiO2/WO3
/
Tungsten oxi As a result, upon visible light irradiation,
TiO2 generates e−/h+ pairs, where the photogenerated e−are promoted to the Ti 3d states (VB), thus reducing
Ti4+ to Ti3+. Ti3+ can be easily oxidized by WO3 due to the differences in the reduction potential between
TiO2 (−0.70 V vs NHE) and WO3 (−0.03 V vs NHE). Subsequently, W6+ traps photogenerated e−to form
its reduced state W5+, while the redox reaction occurs further by returning to its original oxidation state
in reaction with adsorbed O2 on the composite catalyst surface (similarly as discussed above in the case
of “pure” TiO2/WO3), thus leading to improved charge separation and the formation of ROS, which
contributed in DCF degradation and mineralization of formed intermediates. The same authors studied the
degradation of DCF with another enhanced WO3/TiO2 composite (N-doped TiO2), and again reported high
degradation and mineralization rates; 100% according to both indicators under 250 kJ/m2 and 400 kJ/m2 of
solar-accumulated energy, respectively [31]. They stressed that the same mechanism that was responsible
for the enhancement of photocatalyst activity in C-doped WO3/TiO2 composite [32] also improved the
performance of N-doped WO3/TiO2 [31]. 2.1.2. TiO2/Fe2O3 In addition, Fe2O3 (Eg = 2.2 eV) can be excited by visible light irradiation producing photogenerated
e−/h+ pairs. Photogenerated e−in CB of Fe2O3 can be transferred to O2 dissolved in water to form O2•-,
while photogenerated h+ in VB of Fe2O3 can facilitate generation of •OH eventually contributing to the
degradation of present organics [24] (Figure 3). The formation of mentioned ROS and their involvement in
degradation of targeted pollutant was confirmed through common scavenging tests using TB, FA, and BQ. 7 of 44 Materials 2020, 13, 1338 Table 2. Photocatalytic degradation of CEC’s over TiO2/Fe2O3 composites. Catalyst
Target Pollutant
Initial
Concentration/Working
Volume
((mg L−1) /mL)
Experimental Conditions
Reaction
Time
Removal Extent (%)
Reference
TiO2/Fe2O3
(0.1 g/L )
Diazinon
10
(in 300 mL)
Light Source: 60 W Philips
Visible lamp;
pH = natural
45 min
95.07
[37]
TiO2/Fe2O3
(10 mg)
2,4-dichlorophenoxy
acetic acid
50
(in 100 mL)
Light Source: 300 W Xenon
Lamp;
pH = natural
120 min
100
(TOC = 100 @ 150
min.)
[23]
TiO2/Fe2O3
(0.5 g/L )
2,4-dichlorophenoxy
acetic acid
50
(in 250 mL)
Light Source: natural sunlight
11AM-4PM;
pH = 4
120 min
96.8
(TOC =90 @ 240 min.)
[24]
TiO2/Fe2O3
(70 mg)
Oxytetracycline
Hydrochloride
60
(in 70 mL)
Light Source: 300 W Iodine
Tungsten Lamp;
pH = 5.5
300 min
75.6
[38]
TiO2/Fe2O3
(1.0 g/L)
Oxytetracycline
60
Light Source: 300 W Iodine
Tungsten Lamp;
pH = 5.5
300 min
~80
[39]
TiO2/Fe2O3/CNT
(100 mg)
Tetracycline
20
(in 100mL)
Light Source: 300 W Xenon
Lamp;
pH = natural
90 min
89.41
[40]
TiO2-coated α-Fe2O3
core-shell
(100 mg)
Tetracycline
Hydrochloride
50
(in 200 mL)
Light Source: 300 W Xenon
Lamp (λ > 420 nm) ;
pH = 5. 45
Oxidant = 120 µL (30% H2O2)
90 min
100
[41] 8 of 44
hrough Materials 2020, 13, 1338
mentioned ROS and
common scavenging Figure 3. Photocatalytic degradation mechanism over TiO2/Fe2O3 composite. Figure 3. Photocatalytic degradation mechanism over TiO2/Fe2O3 composite. TiO /F
O
it Figure 3. Photocatalytic degradation mechanism over TiO2/Fe2O3 composite. Figure 3. Photocatalytic degradation mechanism over TiO2/Fe2O3 composite. The photocatalytic degradation of the pharmaceutical tetracycline (TC) and its derivatives, such
as oxytetracycline (OTC), using TiO2/Fe2O3 materials has also been reported. 2.1.2. TiO2/Fe2O3 Hence, it was found out
that, under visible light irradiation (λ = 400–750 nm), α-Fe2O3 was activated and generated e−/h+ pairs,
and then photogenerated e− from CB of α-Fe2O3 moved to TiO2 trapping sites for atmospheric O2 to
form O2●‒, which was proven to largely contribute to the degradation of OTC. On the other hand, the
photogenerated h+ from VB of α-Fe2O3 primarily reacted with OH−, resulting in the generation of
•OH, which also contributed to the degradation of OTC. When compared to TiO2 reference material,
the TiO2/Fe2O3 composite exhibited an enhanced photocatalytic activity under visible light due to
efficient e−/h+ separation, as stated above [38]. The same authors [38,39] also studied the degradation
mechanism of OTC while using LC/MS TOF analysis and, based on the formed intermediates,
established the OTC degradation pathway, and concluded that •OH mainly mediated degradation. Besides, “pure” TiO2/Fe2O3, enriched analogue with carbon nanotubes (CNTs) was also studied
(Table 2). Hence, TiO2/Fe2O3/CNTs was used as the catalyst for photocatalytic degradation of TC,
under visible light illumination [40]. It was found that the effectiveness of photocatalytic
degradation of TC within 90 min. treatment using TiO2/Fe2O3/CNTs was almost twice higher when
comparing to that achieved by benchmark TiO2; 89.41% and 47.64%, respectively. The authors
attributed the improved photocatalytic efficiency to the presence of the CNT, which acted as a
photogenerated e− acceptor, thereby suppressing e−/h+ recombination [40]. In another study, the
core-shell structured α-Fe2O3 (with TiO2 shell of around 15 nm) exhibited 100% TC removal in 90
min. [41]. The degradation improvement was ascribed to the addition of H2O2 in the system, which
generated more ROS than by the common photocatalytic mechanisms described above [41]. Hence,
The photocatalytic degradation of the pharmaceutical tetracycline (TC) and its derivatives, such as
oxytetracycline (OTC), using TiO2/Fe2O3 materials has also been reported. Hence, it was found out
that, under visible light irradiation (λ = 400–750 nm), α-Fe2O3 was activated and generated e−/h+
pairs, and then photogenerated e−from CB of α-Fe2O3 moved to TiO2 trapping sites for atmospheric
O2 to form O2•-, which was proven to largely contribute to the degradation of OTC. On the other
hand, the photogenerated h+ from VB of α-Fe2O3 primarily reacted with OH−, resulting in the
generation of •OH, which also contributed to the degradation of OTC. 2.1.2. TiO2/Fe2O3 When compared to TiO2
reference material, the TiO2/Fe2O3 composite exhibited an enhanced photocatalytic activity under
visible light due to efficient e−/h+ separation, as stated above [38]. The same authors [38,39] also
studied the degradation mechanism of OTC while using LC/MS TOF analysis and, based on the formed
intermediates, established the OTC degradation pathway, and concluded that •OH mainly mediated
degradation. Besides, “pure” TiO2/Fe2O3, enriched analogue with carbon nanotubes (CNTs) was also
studied (Table 2). Hence, TiO2/Fe2O3/CNTs was used as the catalyst for photocatalytic degradation
of TC, under visible light illumination [40]. It was found that the effectiveness of photocatalytic
degradation of TC within 90 min. treatment using TiO2/Fe2O3/CNTs was almost twice higher when
comparing to that achieved by benchmark TiO2; 89.41% and 47.64%, respectively. The authors attributed
the improved photocatalytic efficiency to the presence of the CNT, which acted as a photogenerated e−
acceptor, thereby suppressing e−/h+ recombination [40]. In another study, the core-shell structured
α-Fe2O3 (with TiO2 shell of around 15 nm) exhibited 100% TC removal in 90 min. [41]. The degradation
improvement was ascribed to the addition of H2O2 in the system, which generated more ROS than by
the common photocatalytic mechanisms described above [41]. Hence, the contribution of H2O2 in such
a system can be described through restraining e−/h+ recombination and increasing HO• generation in
the system, as in Equation (1) [15]: (1) H2O2 + H+ + e−→HO• + H2O
(1) H2O2 + H+ + e−→HO• + H2O 2.1.3. TiO2/Spinel Ferrite Spinel ferrites (MFe2O4) are metal oxides, where M is a divalent ion (i.e., Mg2+, Ca2+, Sr2+, Ni2+,
Zn2+, etc.), serving as promising candidates for photocatalytic applications due to their narrow band
gap range (1.3–2.2 eV) and magnetic properties [42,43]. Spinel ferrites band positions match TiO2,
thus possessing compatibility to form a heterojunction (Type II Heterojunction) [44–47]. Spinel ferrites (MFe2O4) are metal oxides, where M is a divalent ion (i.e., Mg2+, Ca2+, Sr2+, Ni2+,
Zn2+, etc.), serving as promising candidates for photocatalytic applications due to their narrow band
gap range (1.3–2.2 eV) and magnetic properties [42,43]. Spinel ferrites band positions match TiO2,
thus possessing compatibility to form a heterojunction (Type II Heterojunction) [44–47]. The literature provides applications of MFe2O4/TiO2 materials as photocatalysts in treatment
of CECs, as in the case of previously discussed TiO2-based composites, however, it should be
noted that authors within such composites used modified TiO2 (Table 3). Hence, Chen et al. [44]
studied photocatalytic degradation of TC and its derivatives using N-doped TiO2/CaFe2O4/diatomite,
and reported 91.7% removal of TC within 150 min. under visible light irradiation. The authors studied
the composite stability and reusability; the results obtained after five cycles indicates that employed 9 of 44 Materials 2020, 13, 1338 composite is rather stable, enabling 89.2% removal of TC. They also proposed the photocatalytic
mechanism occurring within the composite; the excitation of both N-TiO2 and CaFe2O4 by visible light
leads to the formation of e−/h+ pairs (Figure 4). The photogenerated e−in CB of N-TiO2 can directly
react to adsorbed O2 generating O2•-, while photogenerated h+ in VB of N-TiO2 directly react with
H2O and OH−producing •OH. Simultaneously, photogenerated e−in CB of CaFe2O4 can undergo
the same mechanism (i.e., reaction with O2 to produce O2•-). In addition, the formed heterojunction
helps the migration of e−from CB of CaFe2O4 to CB of N-TiO2, and h+ from VB of N-TiO2 to VB
of CaFe2O4 (Figure 4). Such a transfer of charge carriers between the two semiconductors hinders
the recombination process and enhances the photocatalytic activity of the composite, thus leading to
more efficient generation of ROS (O2•- and •OH) [44]; the existence of formed ROS was confirmed
through scavenging tests while using isopropyl alcohol (IPA), ammonium oxalate (AO), and BQ for
•OH, h+ and O2•-, respectively. 2.1.3. TiO2/Spinel Ferrite Such behavior is confirmed by studying the degradation pathway of
TC; it was found that the TC intermediates match those that formed through radical driven reactions
undergoing in the first step demethylation and hydroxylation. The second step considered the removal
of functional groups (amino, hydroxyl, and methyl) and further ring opening reactions that are mainly
mediated by photogenerated h+, yielding small fragments that were eventually mineralized [44]. Such a pathway confirmed the dual role of photogenerated h+, as a promotor •OH generation and as
sites for the direct oxidation of adsorbed organics. There are several other studies investigating the
application of different MFe2O4/TiO2 materials (N-doped TiO2/SrFe2O4 diatomite [46]; Ce/N-co-doped
TiO2/NiFe2O4/ diatomite and ZnFe2O4/TiO2 [47]) for the photocatalytic degradation of CECs, such as
TC, OTC, and bisphenol A (BPA). Interestingly, the same mechanisms responsible for charge transfer
and consequent generation of ROS were reported, regardless of the different M type within the spinel
ferrite part of composite and/or TiO2 (non-doped or doped with metal and/or non-metal ions). Materials 2020, 13, x FOR PEER REVIEW
8 of 39
ammonium oxalate (AO), and BQ for •OH, h+ and O2●‒, respectively. Such behavior is confirmed by
studying the degradation pathway of TC; it was found that the TC intermediates match those that
formed through radical driven reactions undergoing in the first step demethylation and
hydroxylation. The second step considered the removal of functional groups (amino, hydroxyl, and
methyl) and further ring opening reactions that are mainly mediated by photogenerated h+, yielding
small fragments that were eventually mineralized [44]. Such a pathway confirmed the dual role of
photogenerated h+, as a promotor •OH generation and as sites for the direct oxidation of adsorbed
organics. There are several other studies investigating the application of different MFe2O4/TiO2
materials (N-doped TiO2/SrFe2O4 diatomite [46]; Ce/N-co-doped TiO2/NiFe2O4/ diatomite and
ZnFe2O4/TiO2 [47]) for the photocatalytic degradation of CECs, such as TC, OTC, and bisphenol A
(BPA). Interestingly, the same mechanisms responsible for charge transfer and consequent
generation of ROS were reported, regardless of the different M type within the spinel ferrite part of
i
d/
TiO (
d
d
d
d
i h
l
d/
l i
) p
/
(
p
p
/
)
Figure 4. Proposed mechanism for the tetracycline (TC) photodegradation process using N-doped
TiO2/CaFe2O4/ diatomite [44]. Figure 4. Proposed mechanism for the tetracycline (TC) photodegradation process using N-doped
TiO2/CaFe2O4/ diatomite [44]. Figure 4. 2.1.3. TiO2/Spinel Ferrite Proposed mechanism for the tetracycline (TC) photodegradation process using N-doped
TiO2/CaFe2O4/ diatomite [44]. Figure 4. Proposed mechanism for the tetracycline (TC) photodegradation process using N-doped
TiO2/CaFe2O4/ diatomite [44]. Materials 2020, 13, 1338 10 of 44 Table 3. Photocatalytic degradation of CEC’s over TiO2/MFe2O4 composites. Catalyst
Target
Pollutant
Initial Concentration/
Working Volume
((mg L−1) /mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
reference
N-TiO2/ CaFe2O4
/diatomite
(2.0 g/L)
Tetracycline
10
(in 200 mL)
Light Source: 150 W Xenon
Lamp with UV light filter
150 min
91.7
(TOC =~80 @ 2h)
[44]
N-TiO2/ SrFe2O4
/diatomite
(2.0 g/L)
Tetracycline
10
(in 200 mL)
Light Source: 150 W Xenon
Lamp with UV light filter
150 min
92
(TOC = ~80 @ 2h)
[46]
Ce/N co-doped TiO2 /
NiFe2O4 diatomite
(0.5 g/L)
Tetracycline
20
(in 200 mL)
Light Source: 150 W Xenon
Lamp with UV light filter
180 min
98.2
(TOC = ~95)
[45]
ZnFe2O4 / TiO2
(1.0 g/L)
Bisphenol A
10
(in 200 mL)
Light Source: 300 W Xenon Lamp
pH= 7
30 min
100
[47] Table 3. Photocatalytic degradation of CEC’s over TiO2/MFe2O4 composites. Table 3. Photocatalytic degradation of CEC’s over TiO2/MFe2O4 composites. Materials 2020, 13, 1338 11 of 44 11 of 44 2.1.4. TiO2/Cu2O Cu2O, a p-type semiconductor (Eg = 2.0–2.2 eV), is also a good candidate for making heterojunctions
with TiO2 (Type II Heterojunction). Hence, the photocatalytic degradation of various CECs (TC [48],
and tetrabromodiphenyl ethers [49]) using TiO2/Cu2O composite materials was reported under solar
light irradiation (Table 4). Based on the findings in the mentioned studies, the photocatalytic mechanism
of TiO2/Cu2O under solar light illumination involves the activation of both Cu2O and TiO2 to generate
e−/h+ pairs (Figure 5). Photogenerated e−in CB of Cu2O then can migrate to CB of TiO2 and, along with
photogenerated e−in CB of TiO2, react with O2 to form O2•_. Simultaneously, photogenerated h+ in
VB of Cu2O can be directly involved in the oxidation of adsorbed organics, while photogenerated
h+ in VB of TiO2 can directly oxidize adsorbed organics or react with H2O (i.e., OH−) and generate
•OH. Besides, these h+ can also directly migrate to the VB of Cu2O, thus leading to effective charge
separation that improves the overall photocatalytic activity of the composite [48]. Materials 2020, 13, x FOR PEER REVIEW
9 of 39 Figure 5. Photocatalytic degradation mechanism over TiO2/Cu2O composite. Figure 5. Photocatalytic degradation mechanism over TiO2/Cu2O composite. Figure 5. Photocatalytic degradation mechanism over TiO2/Cu2O composite. Figure 5. Photocatalytic degradation mechanism over TiO2/Cu2O composite. 2.1.5. TiO2/Bi2O3 2.1.5. TiO2/Bi2O3 Catalyst
Target Pollutant
Initial
Concentration/
Working
Volume
((mg L−1) /mL)
Experimental
Conditions
Reaction
Time
Removal
Extent (%)
reference
N-TiO2/ CaFe2O4
/diatomite
(2.0 g/L)
Tetracycline
10
(in 200 mL)
Light Source: 150
W Xenon Lamp
with UV light
filter
150 min
91.7
(TOC =~80 @ 2h)
[44]
N-TiO2/ SrFe2O4
/diatomite
Tetracycline
10
Light Source: 150
W Xenon Lamp
150 min
92
[46]
Bi2O3, a semiconductor with band gap range in the visible region (2.5–2.8 eV), is also a good
candidate for making heterojunctions with TiO2 (Type II Heterojunction). Studies including its application
in photocatalytic degradation of CECs (quinalphos [50] and ofloxacin [51]) under solar light irradiation
(Table 5) revealed the occurring photocatalytic mechanism. Both of the composite phases can be
activated under solar irradiation generating e−/h+ pairs (Figure 6). Accordingly, photogenerated h+ in
VB of TiO2 are involved in the production of •OH (through reactions with H2O, i.e., OH−) as of e−/h+
pairs. In addition, h+ in VB of Bi2O3 can be transferred to VB of TiO2 that contributes to the direct
oxidation of adsorbed organics or the generation of •OH [51]. Materials 2020, 13, x FOR PEER REVIEW
10 of 39 (2.0 g/L)
(in 200 mL)
with UV light
filter
(TOC 80 @ 2h)
Ce/N co-doped
TiO2 / NiFe2O4
diatomite
(0.5 g/L)
Tetracycline
20
(in 200 mL)
Light Source: 150
W Xenon Lamp
with UV light
filter
180 min
98.2
(TOC = ~95)
[45]
ZnFe2O4 / TiO2
(1.0 g/L)
Bisphenol A
10
(in 200 mL)
Light Source: 300
W Xenon Lamp
pH= 7
30 min
100
[47]
2.1.5. TiO2/Bi2O3
Bi2O3, a semiconductor with band gap range in the visible region (2.5–2.8 eV), is also a good
candidate for making heterojunctions with TiO2 (Type II Heterojunction). Studies including its
application in photocatalytic degradation of CECs (quinalphos [50] and ofloxacin [51]) under solar
light irradiation (Table 5) revealed the occurring photocatalytic mechanism. Both of the composite
phases can be activated under solar irradiation generating e−/h+ pairs (Figure 6). Accordingly,
photogenerated h+ in VB of TiO2 are involved in the production of •OH (through reactions with H2O,
Figure 6. Photocatalytic degradation mechanism over TiO2/Bi2O3 composite. Figure 6. Photocatalytic degradation mechanism over TiO2/Bi2O3 composite. 2.2. Coupling of TiO2 with Metal Sulfides Cadmium sulfide (CdS), a metal sulfide semiconductor with a visible light range band gap
(Eg = 2.1–2.4 eV), has been proven to be compatible with TiO2, due to its higher position of CB than that
of TiO2 (Type II Heterojunction) (Figure 7) [25,52]. However, one should be aware that its application can
lead to adverse effects due to its instability, resulting in the leaching of toxic Cd2+ during treatment [53]. Although its CB and VB positions are thermodynamically favorable for photocatalytic application,
CdS as a photocatalytic material faces serious problems. Next to the above-mentioned promotion
of toxic effects, issues, like poor stability due to photocorrosion and limited separation efficiency of
photogenerated charge carriers, do not speak in favor of CdS application [54,55]. Photocorrosion is not
only related to the photogenerated h+ in semiconductor itself that oxidizes S2– and release Cd2+ to the
solution, but also with newly formed O2, where higher solubility in water leads to more dramatic levels
of photocorrosion of CdS [54,56]. However, CdS was widely investigated in photocatalytic purposes,
even in recent studies that focused on the degradation of CECs (ofloxacin, ciprofloxacin, tetracycline,
and 17α-ethynylestradiol), where it was used in various forms (nano-rods, nano-belts) [25,52,57,58]
(Table 6). Generally, upon visible light illumination, CdS is excited and generates the e−/h+ pair,
where photogenerated e−in CB of CdS migrates to CB of TiO2 and is consumed in reactions with O2 to
produce O2•-, while h+ remain in the VB of CdS. Materials 2020, 13, x FOR PEER REVIEW
11 of 39
is excited and generates the e−/h+ pair, where photogenerated e− in CB of CdS migrates to CB of TiO2
and is consumed in reactions with O to produce O ●while h+ remain in the VB of CdS Figure 7. General photocatalytic degradation mechanism over TiO2/MS (M = Cd or Cu) or MS2 (M =
Mo and Sn) composite. Figure 7. General photocatalytic degradation mechanism over TiO2/MS (M = Cd or Cu) or MS2 (M =
Mo and Sn) composite. Figure 7. General photocatalytic degradation mechanism over TiO2/MS (M = Cd or Cu) or MS2 (M =
Mo and Sn) composite. Figure 7. General photocatalytic degradation mechanism over TiO2/MS (M = Cd or Cu) or MS2 (M =
Mo and Sn) composite. 2.2. Coupling of TiO2 with Metal Sulfides Copper sulfide (CuS), which is another metal sulfide semiconductor with narrow band gap of
2.0 eV, has also been reported to be coupled with TiO2 (Type II Heterojunction) [59,60]. Jiang et al. [59]
reported a 85.5% degradation of enrofloxacin and 27.7% mineralization of overall organic content
using immobilized CuS/TiO2 nanobelts (Table 6). They elucidated the mechanisms occurring in the
composite upon excitation by solar irradiation. Hence, such broad wavelengths excited both
composite phases (CuS and TiO2) and resulted in e−/h+ pairs, while the transfer of charges was
analogous, as in the case of the CdS/TiO2 composite. Photogenerated e− in CB (−0.33 eV) of CuS
underwent transfer to CB (−0.19 eV) of TiO2 and were consumed in reactions with O2 forming O2●‒. Photogenerated h+ in VB of CuS remained there and present potential active sites for direct
degradation of organics that were adsorbed at the CuS surface, since they cannot be involved in
generation of •OH due to too high energy band positioning. On the other hand, photogenerated h+ in
VB of TiO2 can directly react with adsorbed organics and OH− generating •OH. Chen et.al [60],
incorporated Au nanoparticles to CuS/TiO2 nanobelts structure to enhance the photocatalytic
degradation ability of the composite by capturing e− and, consequently, suppressing the
recombination of photogenerated charges. As a result, they obtained 96% degradation of OTC and
68% mineralization of the overall organic content within one hour under artificial sunlight
illumination. Accordingly, the mechanism of such enriched CuS/TiO2 composite involves, besides
the above discussed mechanism, the path considering the transfer of e− to Au, which leads to
Copper sulfide (CuS), which is another metal sulfide semiconductor with narrow band gap of
2.0 eV, has also been reported to be coupled with TiO2 (Type II Heterojunction) [59,60]. Jiang et al. [59]
reported a 85.5% degradation of enrofloxacin and 27.7% mineralization of overall organic content
using immobilized CuS/TiO2 nanobelts (Table 6). They elucidated the mechanisms occurring in the
composite upon excitation by solar irradiation. Hence, such broad wavelengths excited both composite
phases (CuS and TiO2) and resulted in e−/h+ pairs, while the transfer of charges was analogous, as in
the case of the CdS/TiO2 composite. Photogenerated e−in CB (−0.33 eV) of CuS underwent transfer to
CB (−0.19 eV) of TiO2 and were consumed in reactions with O2 forming O2•-. 2.1.5. TiO2/Bi2O3 2.0 g/L)
filter
N co-doped
2 / NiFe2O4
iatomite
0.5 g/L)
Tetracycline
20
(in 200 mL)
Light Source: 150
W Xenon Lamp
with UV light
filter
180 min
98.2
(TOC = ~95)
[45]
e2O4 / TiO2
1.0 g/L)
Bisphenol A
10
(in 200 mL)
Light Source: 300
W Xenon Lamp
pH= 7
30 min
100
[47]
2.1.5. TiO2/Bi2O3
Bi2O3, a semiconductor with band gap range in the visible region (2.5–2.8 eV), is also a good
candidate for making heterojunctions with TiO2 (Type II Heterojunction). Studies including its
application in photocatalytic degradation of CECs (quinalphos [50] and ofloxacin [51]) under solar
light irradiation (Table 5) revealed the occurring photocatalytic mechanism. Both of the composite degradat
ealed the uinalphos [
tocatalytic uinalphos [
ocatalytic n be activated under solar irradiation generating e−/h+ pairs (Figure 6). Ac
erated h+ in VB of TiO2 are involved in the production of •OH (through reactions
Figure 6. Photocatalytic degradation mechanism over TiO2/Bi2O3 composite. Figure 6. Photocatalytic degradation mechanism over TiO2/Bi2O3 composite. 12 of 44 Materials 2020, 13, 1338 Table 4. Photocatalytic degradation of CEC’s over TiO2/Cu2O composites. Table 4. Photocatalytic degradation of CEC’s over TiO2/Cu2O composites. Catalyst
Target Pollutant
Initial
Concentration/
Working Volume
((mg L−1) /mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
Cu2O-TiO2 supported
palygorskite
(1.0 g/L)
Tetracycline
Hydrochloride
30
(in 50mL)
Light Source: 500 Xe Lamp;
pH = 8.7
240 min
88.81
[48]
TiO2-Cu2O film
Tetrabromodiphenyl
Ethers
5
(in 100 mL)
Light Source: 300 W Xenon Lamp;
pH = natural
solvent CH3OH:H2O (50:50 v/v)
150 min
90
[49]
Table 5. Photocatalytic degradation of CEC’s over TiO2/Bi2O3 composites. Catalyst
Target Pollutant
Initial
Concentration/
Working Volume
((mg L−1) /mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
Bi2O3–TiO2
(50 mg)
Quinalphos
25
(in 50 mL)
Light Source: Visible light with
1.56µmol/m2/s; pH = 8
100 min
92
[50]
Bi2O3–TiO2
(0.5 g/L)
Ofloxacin
25
Light Source: 70.3 K lux;
pH = 7
120 min
92
[51] 13 of 44 Materials 2020, 13, 1338 13 of 44 2.2. Coupling of TiO2 with Metal Sulfides Photogenerated h+ in
VB of CuS remained there and present potential active sites for direct degradation of organics that
were adsorbed at the CuS surface, since they cannot be involved in generation of •OH due to too
high energy band positioning. On the other hand, photogenerated h+ in VB of TiO2 can directly react
with adsorbed organics and OH−generating •OH. Chen et.al [60], incorporated Au nanoparticles to
CuS/TiO2 nanobelts structure to enhance the photocatalytic degradation ability of the composite by
capturing e−and, consequently, suppressing the recombination of photogenerated charges. As a result,
they obtained 96% degradation of OTC and 68% mineralization of the overall organic content within
one hour under artificial sunlight illumination. Accordingly, the mechanism of such enriched CuS/TiO2
composite involves, besides the above discussed mechanism, the path considering the transfer of
e−to Au, which leads to enhanced charge separation, thus delaying recombination. In such a case Materials 2020, 13, 1338 14 of 44 scenario, photogenerated h+ would have higher probability to react either with adsorbed organics or
with HO- in order to generate •OH (exclusively those in VB of TiO2), thus contributing to the overall
system efficiency. The involvement of formed ROS into reaction mechanisms for OTC degradation
was confirmed by scavenging tests using TB, AO, and BQ. Molybdenum disulfide (MoS2), a two-dimensional (2D) layered metal chalcogenide with an
indirect band gap of 1.1 eV and 1.9 eV direct band gap in monolayered form, with unique structure,
low-cost, high thermal stability, and electrostatic integrity, is also a suitable candidate for forming
heterojunction with TiO2 (Type II Heterojunction) [61–63]. Hence, Kumar et al. [64] reported its
application in the photocatalytic degradation of paracetamol. Furthermore, Irandost et al. [61] applied
the modified MoS2/TiO2 composite (they used N,S-co-doped TiO2) in the photocatalytic degradation
of DCF under visible LED lamp irradiation (Table 6). Hence, the synergistic effect of dopants in TiO2
promoted its visible light activity, yielding the formation of e−/h+ pairs in both composite phases. The mechanism of charge formation and consequent transfer was similar, as described above for
CuS/TiO2, which was excited by solar irradiation. Hence, photogenerated e−in CB of N,S-co doped
TiO2 and CB of MoS2 were able to undergo reactions with O2 forming O2•-, while h+ in VB of TiO2
promoted •OH formation in reactions with HO- and provide the direct oxidation of adsorbed organics,
while, again, h+ in MoS2 were able to do only the latter. 2.2. Coupling of TiO2 with Metal Sulfides The importance of •OH and h+ in DCF
degradation was confirmed by trapping agents used in scavenging tests: TB and potassium iodide
(KI), respectively. Tin sulfide (SnS2), which is a metal sulfide semiconductor with band gap of 2.2 eV [65], has also
been reported to be coupled with TiO2 (Type II Heterojunction) [66,67]. Hence, Kovaˇci´c et al. [66]
reported improved the degradation of 17β-estradiol (E2), for 51%, using SnS2/TiO2 when comparing
to the benchmark material (P25) TiO2 under solar irradiation. A similar improvement was obtained
by comparing performances of the same materials in the case of DCF degradation [67] (Table 6). The reason for such improvement relies on the potential of photogenerated e−in CB of SnS2 to migrate
to CB of TiO2, while h+ remained at the VB of SnS2. In such case, the efficient separation of charges is
achieved, thus facilitating the improved redox reactions, enabling effective degradation of adsorbed
organics directly on the surface by h+, in spite of the limited ability of such a composite to generate
•OH. Accordingly, the adsorption has been shown as an important step in the effectiveness of the
SnS2/TiO2 composite. Kovaˇci´c et al. [67] utilized DFT calculations to study the surface interaction of
polar compounds (DCF) and non-polar compounds (memantine) at SnS2/TiO2 composite and found
that DCF was more efficiently degraded due to much higher adsorption ability in comparison to
memantine, which is one of its structure feature limitations (amine functionality). 2.3. Coupling of TiO2 with Silver- Based Semiconductors Silver Phosphate (Ag3PO4), a promising semiconductor with narrow band gap (Eg ≥2.4 eV),
showed good photocatalytic performance in the degradation of organic pollutants under visible
light irradiation [68,69]. Namely, Ag3PO4 exhibits a quantum efficiency of up to 90% [68] and it
can absorb wavelengths that are shorter than ~530 nm [69]. Despite the qualities of Ag3PO4 as a
potential photocatalyst, it still suffers from limitations, such as photocorrosion, small but not negligible
solubility in water (Ksp = 1.6 × 10−16), and particle agglomeration upon synthesis [70]. To overcome
these limitations, constructing a heterojunction between Ag3PO4 and a compatible semiconductor
has attracted attention due to the increase in charge separation and production of more ROS [71]. The positions of VB and CB in TiO2 directly match the Ag3PO4 band positions, thus providing the
compatibility to form a heterojunction. Hence, Wang et al. [72] investigated the performance of TiO2 nanotubes/Ag3PO4 quantum dots
for the degradation of TC under visible light illumination, and reported a high removal rate within a
short treatment period; 90% TC removal within 8 min (Table 7). 15 of 44 Materials 2020, 13, 1338 Table 6. Photocatalytic degradation of CEC’s over TiO2 /Metal sulfide composites. Table 6. Photocatalytic degradation of CEC’s over TiO2 /Metal sulfide composites. 2.3. Coupling of TiO2 with Silver- Based Semiconductors y
g
p
Catalyst
Target Pollutant
Initial
Concentration/
Working Volume
((mg L−1)/mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
CdS –TiO2
(50 mg)
Tetracycline
Hydrochloride
50
(in 50mL)
Light Source: 500 W Xenon
Lamp with filter (λ > 400 nm);
pH = natural
480 min
87.0
[58]
Au-CdS/TiO2 nanowire
(20 mg)
Ciprofloxacin
20
Average solar light intensity =
100, 000
60 min
99
[57]
CdS/TiO2
(450 mg)
Ofloxacin
10
(in 100mL)
Light Source: 85 W Oreva bulb
with 4150 lumens (λ = 450-650
nm); pH = natural
180 min
86
[52]
CdS nano-rod/TiO2
nano-belt
( 0.50 g/L)
17α-ethynylestradiol
3
(in 10 mL)
Light Source: 500 W Xenon
Lamp with filter (λ > 420 nm);
pH = natural
120 min
92
[25]
CuS/TiO2 nanobelts
Enrofloxacin
5
(in 35 mL)
Light Source: 35 W Xenon Lamp;
pH = natural
120 min
85.5
(TOC = 27.7)
[59]
Au-CuS-TiO2 nanobelts
Oxytetracycline
5
( in 35 mL)
Light Source: 35 W Xenon Lamp;
pH = natural
60 min
96
(TOC = 68)
[60]
MoS2 /TiO2
(25 mg/L)
Acetaminophen
302
Light Source: Sunlight;
pH = natural
25 min
40
[64]
N,S co-doped TiO2 @MoS2
(0.98g/L)
Diclofenac
0.15
( in 100 mL)
Light Source: 60 W LED lamp;
pH = 5.5
150 min
98
[61]
TiO2/SnS2 films
17β-estradiol
1.36
(in 90 mL)
Light Source: 450 W Xenon Arc
Lamp
90 min
51.0
[66]
TiO2/SnS2 films
Diclofenac
31.8
( in 90mL)
Light Source: 450 W Xenon Arc
Lamp; pH = 4
60 min
76.21
[67] 16 of 44 16 of 44 Materials 2020, 13, 1338 Table 7. Photocatalytic degradation of CEC’s over TiO2/Silver-Based Semiconductor composites. 2.3. Coupling of TiO2 with Silver- Based Semiconductors y
g
p
Catalyst
Target
Pollutant
Initial
Concentration/
Working Volume
((mg L−1)/mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
Ti3+ -doped TiO2
nanotubes/ Ag3PO4
quantum dots
(0.5 g/L)
Tetracycline
10
(NA)
Light Source: 400 W Xenon Lamp;
pH = natural
8 min
90
[72]
TiO2 nanotube/ Ag3PO4
nanoparticles
(40 mg)
Ciprofloxacin
10
(in 40 mL)
Light Source: 300 W Xenon Lamp
60 min
85.3
[73]
TiO2-x / Ag3PO4
(100 mg)
Bisphenol A
10
(in 100 mL)
Light Source: 500 W Xenon Lamp
with filter (λ = 420 nm);
pH = natural
16 min
95
[74]
Ag2O/ TiO2 quantum dots
(0.25 g/L)
Levofloxacin
10
(in 100 mL)
Light Source: 85 W Oreva CFL (4150
lumens)
(λ = 380–700 nm)
pH=4
90 min
81
[27]
Ag2O /TiO2 –zeolite
(50 mg)
Norfloxacin
5
(in 100 mL)
Light Source: 35 W Xenon Lamp
60 min
98.7
(TOC = 83.1)
[28] Materials 2020, 13, 1338
for the degradation
short treatment per 17 of 44
within a The conventional heterojunction transfer mechanism (Figure 8a) explains that the photogenerated
h+ in the composite would be promoted from the VB of Ag3PO4 to VB of Ti3+-doped TiO2 nanotubes,
where can react with H2O or HO−forming •OH. Simultaneously, photogenerated e−from the
Ti3+-doped TiO2 nanotubes CB can react with O2 forming O2•- or may transfer to the CB of Ag3PO4. However, O2•- are not formed in Ag3PO4, due to the fact that the position of its CB is lower than the
standard reduction potential of O2•-/O2. Wang et al. [72] concluded that TC was primarily degraded
by O2•−and photogenerated h+ based on the results of the conducted electron trapping experiments. Accordingly, they have extended the study by proposing a Z-scheme heterojunction transfer mechanism
(Figure 8b). Under this mechanism, Ag(0) acts a recombination center, “collecting” photogenerated
e−from CB of Ag3PO4, where they undergo recombination with the photogenerated h+ from VB of
Ti3+-doped TiO2 nanotubes. In such case, photogenerated h+ on VB of Ag3PO4 might participate
in the direct oxidation reactions with adsorbed organics, while the photogenerated e−in the CB of
Ti3+-doped TiO2 nanotubes can be involved in forming desired ROS, O2•-, thus contributing to the
enhanced performance of composite photocatalyst. Du et al. [73] applied analogue TiO2/Ag3PO4
composite employing TiO2 nanotube arrays for the degradation of ciprofloxacin (CIP) under solar
irradiation and reported that 85.3% removal of CIP within 60 min. was facilitated through the
above-mentioned mechanisms. 2.3. Coupling of TiO2 with Silver- Based Semiconductors Furthermore, Liu et al. [74] reported 95% degradation of BPA in 16 min. using TiO2−X/Ag3PO4 under visible light irradiation (Table 7). They reported that both composite
phases, TiO2−X and Ag3PO4, were excited and generated e−/h+ pairs. Hence, photogenerated h+ in VB
of TiO2−X are promoted to VB of Ag3PO4 and contributed to the direct oxidation of adsorbed organics,
similarly as reported in the study by Wang et al. [72]. Photogenerated e−from the CB of Ag3PO4 are
transferred to oxygen vacancies (Vo) of TiO2 and contributed in reactions with adsorbed O2 generating
O2•- (Figure 9). They also investigated the role of these species in the degradation of BPA and found,
based on monitoring BPA degradation pathway by LC/MS analysis, that intermediates are formed
through two pathways: 1) hydroxylation, through reactions with O2•- yielding BPA-o-catechol; and,
2) direct oxidation by h+ forming isopropenylphenol and phenol, which was further oxidized by h+
yielding hydroquinone and its dehydrated form benzoquinone. photogenerated h+ in the composite would be promoted from the VB of Ag3PO4 to VB of Ti3+-doped
TiO2 nanotubes, where can react with H2O or HO− forming •OH. Simultaneously, photogenerated e−
from the Ti3+-doped TiO2 nanotubes CB can react with O2 forming O2●‒ or may transfer to the CB of
Ag3PO4. However, O2●‒ are not formed in Ag3PO4, due to the fact that the position of its CB is lower
than the standard reduction potential of O2●‒/O2. Wang et al. [72] concluded that TC was primarily
degraded by O2●− and photogenerated h+ based on the results of the conducted electron trapping
experiments. Accordingly, they have extended the study by proposing a Z-scheme heterojunction
transfer mechanism (Figure 8b). Under this mechanism, Ag(0) acts a recombination center,
“collecting” photogenerated e− from CB of Ag3PO4, where they undergo recombination with the
photogenerated h+ from VB of Ti3+-doped TiO2 nanotubes. In such case, photogenerated h+ on VB of
Ag3PO4 might participate in the direct oxidation reactions with adsorbed organics, while the
photogenerated e− in the CB of Ti3+-doped TiO2 nanotubes can be involved in forming desired ROS,
O2●‒, thus contributing to the enhanced performance of composite photocatalyst. Du et al. [73]
applied analogue TiO2/Ag3PO4 composite employing TiO2 nanotube arrays for the degradation of
ciprofloxacin (CIP) under solar irradiation and reported that 85.3% removal of CIP within 60 min. was facilitated through the above-mentioned mechanisms. Furthermore, Liu et al. [74] reported 95%
degradation of BPA in 16 min. Materials 2020, 13, 1338 Materials 2020, 13, 1338 18 of 44 Silver oxide (Ag2O), a visible light active photocatalyst with band gap of 1.2 eV, is another silver-based
compound with semiconducting properties. Based on the band positions (VB and CB), it represents
a promising matching candidate to form heterojunctions with TiO2 (Type III Heterojunction). Hence,
photocatalytic degradation of levofloxacin (LEV) using Ag2O/TiO2 quantum dots is reported with the
maximum of 81% LEV degradation within 90 min. of visible light irradiation [27]. Based on the proposed
mechanism under visible light illumination (Figure 10), upon excitation of Ag2O, e−/h+ pairs are formed,
whereas TiO2 is not activated due to its wide band gap. Photogenerated e−in the CB of Ag2O were
transferred to CB of TiO2 and involved in reactions with adsorbed O2 forming O2•- that participated
in LEV degradation. In addition, photogenerated h+ in VB of Ag2O yielded the formation of •OH,
through reactions with OH−, and participated in LEV degradation as well. The authors employed
LC-MS analysis to elucidate LEV degradation pathway and, as such, establish the role of formed ROS. Hence, parent compound LEV underwent decarboxylation of the acetyl group; hydroxylation resulting
in the formation of quinolone moieties; demethylation and the subsequent addition of hydrogen atom
generating modifications at piperazine ring; while successive •OH attack resulted in multi-hydroxylated
intermediates. Such findings confirmed the dominant role of •OH in LEV degradation. Silver oxide (Ag2O), a visible light active photocatalyst with band gap of 1.2 eV, is another
silver-based compound with semiconducting properties. Based on the band positions (VB and CB),
it represents a promising matching candidate to form heterojunctions with TiO2 (Type III
Heterojunction). Hence, photocatalytic degradation of levofloxacin (LEV) using Ag2O/TiO2 quantum
dots is reported with the maximum of 81% LEV degradation within 90 min. of visible light
irradiation [27]. Based on the proposed mechanism under visible light illumination (Figure 10), upon
excitation of Ag2O, e−/h+ pairs are formed, whereas TiO2 is not activated due to its wide band gap. Photogenerated e− in the CB of Ag2O were transferred to CB of TiO2 and involved in reactions with
adsorbed O2 forming O2●‒ that participated in LEV degradation. In addition, photogenerated h+ in VB
of Ag2O yielded the formation of •OH, through reactions with OH−, and participated in LEV
degradation as well. The authors employed LC-MS analysis to elucidate LEV degradation pathway
and, as such, establish the role of formed ROS. 2.4. Coupling of TiO2 with Graphene and Graphene-Like Materials 2.4. Coupling of TiO2 with Graphene and Graphene-Like Materials 2.4. Coupling of TiO2 with Graphene and Graphene-Like Materials 2.4. Coupling of TiO2 with Graphene and Graphene-Like Materials 2.3. Coupling of TiO2 with Silver- Based Semiconductors using TiO2−X/Ag3PO4 under visible light irradiation (Table 7). They
reported that both composite phases, TiO2−X and Ag3PO4, were excited and generated e−/h+ pairs. Hence, photogenerated h+ in VB of TiO2−X are promoted to VB of Ag3PO4 and contributed to the
direct oxidation of adsorbed organics, similarly as reported in the study by Wang et al. [72]. Photogenerated e− from the CB of Ag3PO4 are transferred to oxygen vacancies (Vo) of TiO2 and
contributed in reactions with adsorbed O2 generating O2●‒ (Figure 9). They also investigated the role
of these species in the degradation of BPA and found, based on monitoring BPA degradation
pathway by LC/MS analysis, that intermediates are formed through two pathways: 1)
hydroxylation, through reactions with O2●‒ yielding BPA-o-catechol; and, 2) direct oxidation by h+
forming isopropenylphenol and phenol, which was further oxidized by h+ yielding hydroquinone
and its dehydrated form benzoquinone. Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z
h
h
j
i
Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z-scheme heterojunction. erials 2020, 13, x FOR PEER REVIEW
14 of Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z
h
h t
j
ti
Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z-scheme heterojunction. erials 2020, 13, x FOR PEER REVIEW
14 of Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z
h
h t
j
ti
Figure 8. Photocatalytic mechanisms Ti3+-TNTs/Ag3PO4 (a) conventional heterojunction, and (b)
Z-scheme heterojunction. rials 2020, 13, x FOR PEER REVIEW
14 o j j
Figure 9. Photocatalytic reaction mechanism of TiO2−X/Ag3PO4 under visible light irradiation. Figure 9. Photocatalytic reaction mechanism of TiO2−X/Ag3PO4 under visible light irradiation. Figure 9. Photocatalytic reaction mechanism of TiO2−X/Ag3PO4 under visible light irradiation. Figure 9. Photocatalytic reaction mechanism of TiO2−X/Ag3PO4 under visible light irradiation. Materials 2020, 13, 1338 Hence, parent compound LEV underwent
decarboxylation of the acetyl group; hydroxylation resulting in the formation of quinolone moieties;
demethylation and the subsequent addition of hydrogen atom generating modifications at
piperazine ring; while successive •OH attack resulted in multi-hydroxylated intermediates. Such
findings confirmed the dominant role of •OH in LEV degradation. Figure 10. Photocatalytic degradation mechanism over TiO2/Ag2O composite. Figure 10. Photocatalytic degradation mechanism over TiO2/Ag2O composite. Figure 10. Photocatalytic degradation mechanism over TiO2/Ag2O composite. Figure 10. Photocatalytic degradation mechanism over TiO2/Ag2O composite. In another study, Gou et al. [28] investigated the application of Ag2O/TiO2/zeolite composite for
solar-driven degradation of norfloxacin (NOR) (Table 7). Besides high effectiveness (98.7% NOR
degradation and 83.1% mineralization of organic content within 60 min. treatment), they elucidated
the NOR degradation pathway, involving in the initial stage decarboxylation, defluorination or
In another study, Gou et al. [28] investigated the application of Ag2O/TiO2/zeolite composite
for solar-driven degradation of norfloxacin (NOR) (Table 7). Besides high effectiveness (98.7% NOR
degradation and 83.1% mineralization of organic content within 60 min. treatment), they elucidated
the NOR degradation pathway, involving in the initial stage decarboxylation, defluorination or
hydroxylation of parent compound (NOR), which confirmed the involvement of both formed ROS
(O2•- and •OH). 2.4.1. TiO2/Graphene Composites Graphene is a zero bandgap semiconductor with a sheet-like structure (i.e., it is considered as a
2D monolayer material) consisting of sp2 hybridized carbon atoms with excellent thermal conductivity,
optical transmittance, high mechanical strength, large surface area (2600 m2/g), and appreciable
charge carrier transport [75]. Under light illumination, it can achieve a reverse saturation state with
high density (˜1013 cm2) of hot electrons above the Fermi level, which can be used as a powerful
agent in redox reactions [76]. It was also found that the incorporation of graphene-based materials
(i.e., graphene oxide and its reduced form; GO and rGO, respectively) with TiO2 might suppress e−/h+
pairs recombination. As such, TiO2/graphene-based composites were employed in the photocatalytic
degradation of CECs (Table 8). 19 of 44 Materials 2020, 13, 1338 Table 8. Photocatalytic degradation of CEC’s over TiO2/Semiconductor/graphene composites. Catalyst
Target Pollutant
Initial Concentration/
Working Volume
((mg L−1)/mL)
Experimental Conditions
Reaction Time
Removal
Extent (%)
Reference
TiO2/ WO3/GO
(2 mg)
Bisphenol A
20
( in 50 mL)
Light Source: sunlight
Ph = 7
7 h
93.2
[77]
Graphene-WO3 /TiO2
nanotube
(photoelectrodes )
Dimethyl Phthalate
10
(in 40 mL)
Light Source:
150W Xe lamps
120 min
75.9
[78]
TiO2 /ZnO/GO
(0.5 g/L)
Bisphenol A
10
(in 50 mL)
Light Source: 18 UV lamps (λ
=365 nm) ;Visible metal halide
lamps(λ = 400–800 nm) pH = 6
120 min. (UV)
180 min. (Vis)
99.7 (UV)
94.9 (Vis)
[79]
TiO2 /ZnO/GO
(0.5 g/L)
Ibuprofen
10
(in 50 mL)
Light Source: 18 UV lamps (λ =
365 nm) ;Visible metal halide
lamps(λ = 400–800 nm) pH = 6
120 min. (UV)
180 min. (Vis)
98.5 (UV)
79.6 (Vis)
[79]
TiO2 /ZnO/GO
(0.5 g/L)
Flurbiprofen
10
( in 50 mL)
Light Source: 18 UV lamps
(λ=365 nm) ;Visible metal halide
lamps(λ= 400–800 nm) pH= 6
120 min. (UV)
180 min. 2.4.1. TiO2/Graphene Composites (Vis)
98.1(UV)
82.2 (Vis)
[79]
ZnFe2O4/rGO/TiO2
(0.1 g)
Fulvic Acid
20
(in 50 mL)
Light Source: 300 W (λ=420 nm);
Vol H2O2 = 0.8 mL, pH= 7
180 min
95.4%
[80]
TiO2 /MoS2 /rGO
(0.5 g/L)
Bisphenol A
10
(in 50 mL)
Light Source:
20 W (λ = 254 nm);
300 min
62.4
[81]
TiO2/BiVO4/rGO
Tetracycline
10 µg/L
(NA)
Light Source: 1000 W Xe Lamp (λ
= 420 nm) with filter
120 min
96.2
[82]
TiO2/BiVO4/rGO
Chlorotetracycline
10 µg/L
(NA)
Light Source: 1000 W Xe Lamp (λ
= 420 nm) with filter
120 min
97.5
[82]
TiO2/BiVO4/rGO
Oxytetracycline
10 µg/L
(NA)
Light Source: 1000 W Xe Lamp (λ
= 420 nm) with filter
120 min
98.7
[82]
TiO2/BiVO4/rGO
Doxycycline
10 µg/L
(NA)
Light Source: 1000 W Xe Lamp (λ
= 420 nm) with filter
120 min
99.6
[82] Table 8. Photocatalytic degradation of CEC’s over TiO2/Semiconductor/graphene composites. Materials 2020, 13, 1338 20 of 44 20 of 44 2.4.2. TiO2/Semiconductor/Graphene Composites As described in above sections, the coupling of TiO2 with other semiconductors promotes
efficient charge transfer, eventually yielding improved photocatalytic activity. However, in most cases,
the recombination is still an existing issue that needs to be suppressed. Such a double effect can be
obtained by combining composite concept involving two semiconductors (even “pure” TiO2, which
cannot be active under visible light) with graphene-based materials. For instance, Hao et al. [77]
reported 93.2% degradation of BPA in seven hours of sunlight irradiation while using the TiO2/WO3/GO
composite. The mechanism occurring in such combined composite involved the excitation of both
TiO2 and WO3 under solar light irradiation (TiO2 utilized UV-A fraction), yielding the generation e−/h+
in both semiconductors. Hence, photogenerated e−in CB of TiO2 can directly react with absorbed O2,
producing O2•−, or it can be transferred to CB of WO3, and then further migrate to GO enhancing charge
separation. Since the amount of adsorbed O2 is quite limited, the tendency of e−to recombine with h+
is rather favored; ~90% of pairs recombine rapidly after excitation [14]. Hence, the charge separation
represents an important factor in the evaluation of photocatalyst performance. Accordingly, in the case
of effective separation and recombination suppression, as in the case with GO, photogenerated h+ in
VB of activated composite components, e.g., of TiO2 and WO3 in the case of TiO2/WO3/GO, can be
involved in a larger amount, either directly or indirectly (through formation of •OH) in the degradation
of present organics. It should be noted that, in composites with two semiconductors, GO could also
act as redox site, attracting photogenerated e−and h+, thus promoting improved surface migration
of charges [77]. Table 8 summarizes several works regarding TiO2/semiconductor/GO composites
employed for the degradation of CECs with analogous mechanism, as mentioned above. 2.4.3. TiO2/g-C3N4 Graphitic carbon nitride (g-C3N4), a two-dimensional, metal-free polymeric π-conjugated
semiconductor material, which has attracted a lot of attention [83–91] since the pioneering work
of Wang et al. [92] in 2009, due to its high stability, visible light response with the bandgap of 2.7 eV
and non-toxicity [93], thus representing a viable candidate to be applied in photocatalytic water
treatment [80], has certainly been one of the most investigated photocatalysts inside carbon-based
nanomaterials. It can be easily synthesized through the direct pyrolysis of nitrogen-rich precursors,
such as melamine, cyanamide, dicyandiamide, and urea, but its practical application and principle
drawback is low specific surface area and high rate of electron-hole recombination [83,94,95]. Therefore, g-C3N4 modification to address shortcomings are needed, e.g., as an excellent candidate to
form heterojunction with TiO2 (Type II Heterojunction), due to their matched band positions (VB and
CB). Hence, several studies employing g-C3N4/TiO2 were focused on photocatalytic degradation of
CECs (Table 9). For instance, Yang et al. [96] reported 88.1% degradation of CIP within 180 min. under
visible light irradiation. The authors ascribed the improved photocatalytic activity to multiple effects:
(i) an increase in the surface area of the composite; (ii) good dispersity of TiO2 in g-C3N4 enabling the
intimately coupling of composite phases; and, (iii) extension of light absorption of the composite due
to low band gap of g-C3N4. Trapping experiments that were conducted revealed that photogenerated
h+ were the major reactive site involved in CIP degradation. In another study, Li et al. [97] reported the 100% degradation of Acyclovir in 90 min. using
g-C3N4/TiO2 under visible light irradiation. However, after seven hours of continuous irradiation, any
TOC removal was not noticed, implying the formation of rather recalcitrant intermediates with high
resistance to degradation by ROS that formed within the studied system. Trapping experiments for
formed reactive species elucidated that g-C3N4/TiO2 under visible light irradiation only produced h+
and O2•-, and not the most reactive •OH, explaining limited oxidation capability and none TOC removal
in the case of acyclovir degradation. This significant contribution proves that the use of g-C3N4/TiO2
under visible light irradiation must undergo careful laboratory tests regarding the susceptibility of
targeted organics and their intermediates to degradation by h+ and O2•- prior to considering real scale
application [97]. Materials 2020, 13, 1338 21 of 44 Several studies also showed that the tailoring of composite morphology promotes improved
photocatalytic efficiency. For instance, Yu et al. 2.4.3. TiO2/g-C3N4 [98] prepared a mesoporous g-C3N4/TiO2 that was
applied to polysulfone ultrafiltration membranes for sulfamethoxazole (SMX) removal. It was found
that mpg-C3N4/TiO2 exhibit 69% SMX degradation within 30 hours of sunlight irradiation. On the
other hand, TiO2 nanosheets with exposed facets (001) (core)-g-C3N4 (shell) composite exhibit a
higher degradation rate of 2.2 mg/min., which is 36% faster when compared to TiO2 and g-C3N4
physically-mixed composite. The improved effect is ascribed to the close interaction of TiO2 and
g-C3N4 core-shell structure, whereas, in physically mixed composite the formed heterojunction is
random and non-uniform [99]. The use of support materials, such as clays [100] and polymers [101], has been also utilized
for improved adsorption capacity and the stability of g-C3N4/TiO2 composites. For instance,
Chen et al. [101] used g-C3N4–shielding polyester fiber (PET)/TiO2 for photocatalytic degradation
of sulfaquinoxaline and thiamethoxam. Interestingly, the composite removal efficiency for
sulfaquinoxaline reached 97%, after 10 consequent cycles. Furthermore, the introduction of kaolinite
with g-C3N4/TiO2 improved the surface area and adsorption capacity of the composite, leading to 92%
degradation of CIP in 240 min. of visible light irradiation [100]. An additional approach considers doping of metals and non-metals in TiO2, enhancing its light
absorption capacity from UV absorption to visible light absorption. Thus, incorporating doped TiO2
with g-C3N4 structures has also attracted great attention for the degradation of CECs. For instance,
S-Ag/TiO2 @g-C3N4 [102] was employed for the degradation of Triclosan (TS) and yielded 92.3%
degradation of TS within 60 min. under visible light irradiation. Song et al. [103] fabricated a
nanofibrous Co-TiO2 coated with g-C3N4, which was applied to TC removal; the authors reported a
consistent stability of composite photocatalyst during five consecutive cycles. Besides doping, sensitization with dyes [104] and carbon dots [105] was also found to enhance the
light absorption capacity of g-C3N4/TiO2 composite. For example, D35 organic dye was applied next to
g-C3N4/TiO2 and it was found that the light absorption range was enhanced up to 675 nm [104]. On the
other hand, Su et al. [105] studied the application of C dots decorated/g-C3N4/TiO2 for the degradation
of enrofloxacin under visible light and assigned the observed enhancement to the upconversion
photoluminescence properties of C dots, which convert near-infrared light wavelength into visible light
wavelength [106]. 2.4.3. TiO2/g-C3N4 As effective solutions for improving g-C3N4/TiO2 performance, the incorporation of
graphene quantum dots [107] and another semiconductor (i.e., MoS2 [108], WO3 [109]) is also reported;
such systems resulted in enhanced separation of charges and the suppression of their recombination,
thus leading to improved photocatalytic activity in the degradation of CECs. 22 of 44 Materials 2020, 13, 1338 Table 9. Photocatalytic degradation of CEC’s over TiO2 /g-C3N4 composites. Table 9. Photocatalytic degradation of CEC’s over TiO2 /g-C3N4 composites. Table 9. Photocatalytic degradation of CEC’s over TiO2 /g-C3N4 composites. Catalyst
Target Pollutant
Initial Concentration/
Working Volume
((mg L−1)/mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
g-C3N4/TiO2
(30 mg)
Ciprofloxacin
10
(in 80 mL)
Light Source: 300 W Xe Lamp with
filter (λ > 400 nm)
pH = natural
180 min
88.1
[96]
g-C3N4/TiO2
(30 mg)
Acyclovir
10
(in 100 mL)
Light Source: 300 W Xe Lamp with
filter (λ > 420 nm)
pH = natural
90 min
100
[97]
mpg-C3N4/TiO2
(membrane)
Sulfamethoxazole
10
(in 50 mL)
Light Source: 300 W Xe Lamp
pH = natural
Flow rate = 13 mL/min. Membrane flux = 918 L /m2 h
1800 min
69
[98]
TiO2@g-C3N4
core-shell
(100 mg)
Tetracycline
20
(in 100 mL)
Light Source: Xenon Lamp with full
spectrum
pH = natural
9 min
(2.2
mg/min.)
[99]
g-C3N4 –shielding
polyester/ TiO2
(130 mg)
sulfaquinoxaline
2 × 10−5 mol/L
(30 mL)
Light Source:
Q-Sun Xe-1 test, pH = 7
90 min
97
[101]
g-C3N4 –shielding
polyester/ TiO2
(130 mg)
thiamethoxam
2 × 10−5 mol/L
(30 mL)
Light Source:
Q-Sun Xe-1 test, pH = 7
180 min
~95
[101]
g-C3N4/TiO2/kaolinite
(200 mg)
Ciprofloxacin
10
(in 100 mL)
Light Source: Ave. light intensity
=90 mW/cm2 ; Xe Lamp with filter (λ
> 400 nm), pH = natural
240 min
92
[100]
S-Ag/ TiO2 @ g-C3N4
(0.20 g/L)
Triclosan
10
(in 100 mL)
Light Source: 250 W Xe Lamp with
filter (λ > 420 nm), pH = 7.8
60 min
92.3
(Detoxification
Efficiency=
64.3± 3.9)
[102]
Co-TiO2 @g-C3N4
(5 mg ; 2 × 2 cm2
membranes)
Tetracycline
Hydrochloride
20
(in 10 mL)
Light Source: 300 W Xe Lamp with
filter (λ > 420 nm), pH = 7
60 min. 90.8
[103] 23 of 44 Materials 2020, 13, 1338 Table 9. Cont. Table 9. Cont. 3. Photocatalytic Water Splitting Such a negative tendency can be improved by controllin
the recombination rate [75], as also described in detail in the case of the composite materials used fo
water purification. The second process is the separation of photogenerated charge carriers that favo
H2 evolution, also mentioned above in the case of water purification, but here with more importan
Generally speaking, there are two competitive processes that occur inside the photocatalyst and
affect H2 evolution. Similarly as in the case of water purification, first is the charge recombination
process. Such a process reduces the excited charges for >90%, as mentioned above [14]; according
to some authors, even less than 1% of photoexcited charge carriers are able to participate in the
photo-redox reactions forming H2 [111]. Such a negative tendency can be improved by controlling
the recombination rate [75], as also described in detail in the case of the composite materials used for
water purification. The second process is the separation of photogenerated charge carriers that favor
H2 evolution, also mentioned above in the case of water purification, but here with more important
role [111]. H2 evolution, also mentioned above in the case of water purification, but here with more important
role [111]. The positions of CB and VB define the redox potential of photogenerated charge carriers. A CB
minimum (CBmin) that is smaller than 0 V vs. standard hydrogen electrode (SHE) is required for H2
generation, while the maximum of VB (VBmax) has to be higher than O2/H2O reduction potential, by
definition, in order to enable O2 evolution [112]. As mentioned above, H2 generation through this
process is non-spontaneous, needing the standard Gibbs free energy change of +237 kJ/mol or 1.23
eV, and to accomplish water splitting under visible light irradiation, the bandgap of the
photocatalyst should be more than 1.23 eV and less than 3.0 eV [111]. The electronic structures of
diverse semiconductors fulfill the necessary conditions for the water splitting reaction, as can be
The positions of CB and VB define the redox potential of photogenerated charge carriers. A CB
minimum (CBmin) that is smaller than 0 V vs. standard hydrogen electrode (SHE) is required for H2
generation, while the maximum of VB (VBmax) has to be higher than O2/H2O reduction potential,
by definition, in order to enable O2 evolution [112]. 3. Photocatalytic Water Splitting Photocatalytic water splitting implies a non-spontaneous process, where the light photons are used
to break the water molecules assisted by a photocatalyst, which generates photoexcited charge carriers,
i.e., e−/h+ pairs, delivering them to the solid-liquid interface, where the redox half-reactions of water
oxidation and reduction are catalyzed [110,111], analogously, as described above for photocatalytic
water treatment. The difference in water splitting is that photogenerated charges (i.e., e−and h+)
need to react with H+ as the electron acceptor adsorbed on the photocatalyst surface or within the
surrounding electrical double layer of the charged particles in order to generate H2 [112], instead of
O2 generating O2•−, as in photocatalytic water treatment (Figure 1). The donors are the same; H2O,
however, desired the product of such reaction is O2. Figure 11 shows the principal mechanism of
photocatalytic water splitting with the use of TiO2 semiconductor nanoparticle. The VB and CB of
semiconductor or their composites have to have favorable positions in order to enable occurrence of
such reactions. Materials 2020, 13, x FOR PEER REVIEW
19 of 39
e− and h+) need to react with H+ as the electron acceptor adsorbed on the photocatalyst surface or
within the surrounding electrical double layer of the charged particles in order to generate H2 [112],
instead of O2 generating O2●−, as in photocatalytic water treatment (Figure 1). The donors are the
same; H2O, however, desired the product of such reaction is O2. Figure 11 shows the principal
mechanism of photocatalytic water splitting with the use of TiO2 semiconductor nanoparticle. The
VB and CB of semiconductor or their composites have to have favorable positions in order to enable
occurrence of such reactions. Figure 11. The principle photocatalytic water splitting mechanism over illuminated TiO2
Figure 11. The principle photocatalytic water splitting mechanism over illuminated TiO2 nanoparticle. Figure 11. The principle photocatalytic water splitting mechanism over illuminated TiO
Figure 11. The principle photocatalytic water splitting mechanism over illuminated TiO2 nanoparticle. nanoparticle. Generally speaking, there are two competitive processes that occur inside the photocatalyst and
affect H2 evolution. Similarly as in the case of water purification, first is the charge recombination
process. Such a process reduces the excited charges for >90%, as mentioned above [14]; according t
some authors, even less than 1% of photoexcited charge carriers are able to participate in th
photo-redox reactions forming H2 [111]. 2.4.3. TiO2/g-C3N4 Catalyst
Target Pollutant
Initial Concentration/
Working Volume
((mg L−1)/mL)
Experimental Conditions
Reaction
Time
Removal
Extent (%)
Reference
D35-TiO2/g-C3N4
(0.5g/L)
Bisphenol A
10
(in 100 mL)
Light Source: 300 W Metal Halide
pH = 7, Oxidant = 2mM Persulfate
15 min
100
(TOC= 50)
[104]
C dots decorated
g-C3N4/ TiO2
(1.0 g/L)
Enrofloxacin
4
( in 50 mL)
Light Source: 350 W Xe Lamp with
filter (λ > 420 nm)
pH = natural
60 min
91.6
[105]
graphene quantum
dots/ Mn-N-TiO2
/g-C3N4
(45 mg)
Ciprofloxacin
10
(in 80 mL)
Light Source: 300 W Xe Lamp
(320 ≤λ ≤780 nm), pH = 7
120 min
89
[107]
graphene quantum
dots/ Mn-N-TiO2
/g-C3N4
(45 mg)
Diethyl Phthalate
10
(in 80mL)
Light Source: 300 W Xe Lamp
(320 ≤λ ≤780 nm), pH = 7
120 min
70.4
[107]
MoS2 supported
TiO2/g-C3N4
(30 mg)
Atrazine
10
(in 100 mL)
Light Source: 500 W Xe Lamp
(λ > 420 nm), pH = 7
300 min
86.5
[108]
WO3–TiO2 @g-C3N4
Acetylsalicylate
10
(in 100 mL)
Light Source: 500 W Metal Halide
pH = natural
90 min
98
[109]
WO3–TiO2 @g-C3N4
Methyl-theobromine
10
(in 100 mL)
Light Source: 500 W Metal Halide
pH = natural
90 min
97
[109] Materials 2020, 13, 1338 24 of 44 24 of 44 3. Photocatalytic Water Splitting As mentioned above, H2 generation through
this process is non-spontaneous, needing the standard Gibbs free energy change of +237 kJ/mol
or 1.23 eV, and to accomplish water splitting under visible light irradiation, the bandgap of the
photocatalyst should be more than 1.23 eV and less than 3.0 eV [111]. The electronic structures of
diverse semiconductors fulfill the necessary conditions for the water splitting reaction, as can be seen
from Figure 12. 25 of 44 25 of 44 Materials 2020, 13, 1338 Figure 12. Valence band (VB) and conduction band (CB) band positions of various (a) n-type
semiconductors; (b) p-type semiconductors [111]. Figure 12. Valence band (VB) and conduction band (CB) band positions of various (a) n-type
semiconductors; (b) p-type semiconductors [111]. Figure 12. Valence band (VB) and conduction band (CB) band positions of various (a) n-type
semiconductors; (b) p-type semiconductors [111]. Figure 12. Valence band (VB) and conduction band (CB) band positions of various (a) n-type
semiconductors; (b) p-type semiconductors [111]. Within the scope of this review are recent achievements in TiO2-heterojunction systems for
photocatalytic H2 generation. It is important to explain the separation mechanisms of charge carriers
that occur in such hybrid materials: (i) Schottky junctions—photogenerated e− migration from
semiconductor to metal surface due to a higher work function of metal than those of semiconductor,
thus forming a Schottky junction (Figure 13a); (ii) Type II heterojunction (represented in details in the
case of water purification) (Figure 13b); and, (iii) p-n Heterojunction—supply of an additional electric
field to accelerate the charge carrier transfer (Figure 13c); and, (iv) Direct Z-scheme heterojunction—e−
in the CB of second semiconductor recombined with the photogenerated h+ in the VB of the first
semiconductor, leaving the photogenerated e− in first semiconductor and the photogenerated h+ in
second semiconductor for photocatalysis (Figure 13d) [112]. Within the scope of this review are recent achievements in TiO2-heterojunction systems for
photocatalytic H2 generation. 3. Photocatalytic Water Splitting It is important to explain the separation mechanisms of charge
carriers that occur in such hybrid materials: (i) Schottky junctions—photogenerated e−migration from
semiconductor to metal surface due to a higher work function of metal than those of semiconductor,
thus forming a Schottky junction (Figure 13a); (ii) Type II heterojunction (represented in details in the
case of water purification) (Figure 13b); and, (iii) p-n Heterojunction—supply of an additional electric
field to accelerate the charge carrier transfer (Figure 13c); and, (iv) Direct Z-scheme heterojunction—e−
in the CB of second semiconductor recombined with the photogenerated h+ in the VB of the first
semiconductor, leaving the photogenerated e−in first semiconductor and the photogenerated h+ in
second semiconductor for photocatalysis (Figure 13d) [112]. The process efficiency is determined through the Quantum yield (QY) and Apparent Quantum Yield
(AQY), as described with Equations (2) and (3) [93]. The overall quantum yield is predicted to be
higher than the apparent one since the number of absorbed photons is usually less than that of incident
photons [111]. QY (%) = Number of reacted electrons
Number of absorbed photons×100 = 2 x Nubmer of hydrogen molecules
Number of absorbed photons
× 10
(2)
AQY (%) = Nubmer of reacted electrons
Number of incident photons×
(3) QY (%) = Number of reacted electrons
Number of absorbed photons×100 = 2 x Nubmer of hydrogen molecules
Number of absorbed photons
× 10
(2)
AQY (%) = Nubmer of reacted electrons
Number of incident photons×
(3) (2) (3) H2 generation can also be realized in the presence of sacrificial agents, which, in this case,
serve as electron donors that accept photogenerated h+ of the VB, thus enhancing the separation
of photogenerated charge carriers, which results in higher quantum efficiency [113]. Alcohols are
generally used as a h+ scavenger, and the more α-H atoms the alcohol has, the higher H2 production 26 of 44 26 of 44 Materials 2020, 13, 1338 rate is achieved due to more efficient consumption of h+ in the photoreaction. The number of α-H atoms
in the alcohols can serve as the reference when selecting an appropriate scavenger for photocatalytic
reaction [112]. Materials 2020, 13, x FOR PEER REVIEW
21 of 39 Figure 13. Separation mechanisms of charge carriers in hybrid materials: (a) Schottky junction; (b)
Type II Heterojunction; (c) p-n Heterojunction; and, (d) Direct Z-scheme Heterojunction [112]. Figure 13. AQY (%) =
3.1. Carbon-Based/TiO2 Q
(
)
ே௨ ௗ௧ ௧௦
ே௨ ௗ௧ ௧௦
( )
H2 generation can also be realized in the presence of sacrificial agents, which, in this case, serve
as electron donors that accept photogenerated h+ of the VB, thus enhancing the separation of
photogenerated charge carriers, which results in higher quantum efficiency [113]. Alcohols are
generally used as a h+ scavenger, and the more α-H atoms the alcohol has, the higher H2 production
rate is achieved due to more efficient consumption of h+ in the photoreaction. The number of α-H
h
l
h l
h
f
h
l
f
Among a variety of materials that are selected for the preparation of TiO2-based nanocomposites to
increase their photocatalytic efficiency, nanostructured carbon materials, such as carbon nanotubes and
graphene family nanomaterials (e.g., GO, rGO, g-C3N4), are of particular interest [114]. The advantages,
such as chemical stability, structural diversity with prominent light-absorptive, and electron transport
properties, make them promising materials for use in photocatalytic H2 generation by the water
splitting processes [115]. 3. Photocatalytic Water Splitting Separation mechanisms of charge carriers in hybrid materials: (A) Schottky junction; (B)
Type II Heterojunction; (C) p-n Heterojunction; and, (D) Direct Z-scheme Heterojunction [112]. Figure 13. Separation mechanisms of charge carriers in hybrid materials: (a) Schottky junction; (b)
Type II Heterojunction; (c) p-n Heterojunction; and, (d) Direct Z-scheme Heterojunction [112]. Figure 13. Separation mechanisms of charge carriers in hybrid materials: (A) Schottky junction; (B)
Type II Heterojunction; (C) p-n Heterojunction; and, (D) Direct Z-scheme Heterojunction [112]. The process efficiency is determined through the Quantum yield (QY) and Apparent Quantum
Yield (AQY), as described with Equations (2) and (3) [93]. The overall quantum yield is predicted to
be higher than the apparent one since the number of absorbed photons is usually less than that of
incident photons [111]. QY (%) =
ே௨ ௧ௗ ௧௦ൈ100 =
ଶ ௫ ே௨ ௬ௗ ௨௦ൈ10
(2)
After briefly providing the basic principles to fully understand the H2 evolution through
photocatalytic water splitting, following sections are more focused on the recent achievements
in fabrication and the evaluation of different TiO2-based heterojunctions with different families of
materials, including carbon-based, transition metal oxides and chalcogenides, and multiple-based
composites consisting of three or more semiconductor materials for H2 generation. photocatalytic react
3.1.1. TiO2/g-C3N4 After briefly providing the basic principles to fully understand the H2 evolution through
photocatalytic water splitting, following sections are more focused on the recent achievements in
fabrication and the evaluation of different TiO2-based heterojunctions with different families of
materials, including carbon-based, transition metal oxides and chalcogenides, and multiple-based
composites consisting of three or more semiconductor materials for H2 generation. 3 1 Carbon Based/TiO2
The advantages and limitations of g-C3N4 are already mentioned above in the case of water
treatment. The limitations referring to low light utilization efficiency and insufficient surface area can
be easily broken by the preparation of 2D nanomaterials, especially g-C3N4 nanosheets (CNNS) [84]. The self-assembly method of construction 2D/2D TiO2/CNNS heterojunction composites achieved a
hydrogen evolution rate (HER) of 350 µmol/h/g under visible light, in comparison with the produced
H2 with the use of pure TiO2 nanosheets (20 µmol/h/g) and g-C3N4 nanosheets (130 µmol/h/g) [85]. 3.1. Carbon-Based/TiO2
Among a variety of materials that are selected for the preparation of TiO2-based
h
h
l
ff
d
b
l
h
Liu et al. recorded another use of CNNS [84], who synthesized partially reduced TiO2−x
through NaBH4 treatments with the formation of an additional mid-gap band state (Ti3+ and oxygen Materials 2020, 13, 1338 27 of 44 vacancies—Ovs) to extend absorption edge. The implementation of novel design tactic in the form of a
protective carbon layer that was coated onto TiO2−x/CNNS hetero-junction photocatalyst enhanced
the photocatalytic efficiency. The H2 evolution was tested under visible and simulated solar light
with the use of triethanolamine (TEOA) as a sacrificial agent and Pt as a co-catalyst. In the case
of visible light irradiation, the highest HER was 417.24 µmol/h/g, while, under AM 1.5 irradiation,
the obtained amount was 1830.93 µmol/h/g. The enhanced photocatalytic activity that was ascribed
to the formation of Ti3+ defects was also noticed with the use of g-C3N4/Ti3+-doped TiO2 Z-scheme
system that was synthesized via the polycondensation of urea with TiO2, followed by hydrogenation
treatment [86]. UV-Vis diffuse reflectance spectroscopy, X-ray photoelectron spectroscopy (XPS), and
electron paramagnetic resonance (EPR) have shown that hydrogenation treatment conferred Ti3+ defect
states that were below the CBmin of TiO2 and improved the visible light absorption of the composite
with the obtained HER of 1938 µmol/h/g under solar light. Although special efforts are being made to synthesize noble-metal free nanocomposites, there is
still widespread use of Pt as a co-catalyst in H2 evolution reactions. photocatalytic react
3.1.1. TiO2/g-C3N4 Except for the already mentioned
TiO2−x/CNNS photocatalyst [84], TiO2/g-C3N4 composites with the use of photodeposited Pt as
co-catalyst reached HER of 4128 µmol/h/g [87] and 1041 µmol/h/g [83] under solar and visible light
irradiation, respectively. Pan et al. [88] also exhibited a high HER of 13800 µmol/h/m2 by the use of
Pt as a co-catalyst with g-C3N4/TiO2 nanofilm. Enhanced activity is also attributed to the use of a
magnetic-driven rotating frame, which was developed to enhance the mass transfer process during the
photocatalytic reaction. The charge transfer efficiency between TiO2/g-C3N4 composite can be enhanced by the doping of
different heteroatoms, like C and K atoms. Hence, Zou et al. [89] synthesized C-doped TiO2@g-C3N4
core-shell hollow nanospheres with enhanced visible-light photocatalytic activity for H2 evolution of
35.6 µmol/h/g, which was 22.7 and 10.5 times higher than that of C-TiO2 and g-C3N4. The structure of
TiO2 hollow spheres resulted in the reflection of light within the interior cavity, thus increasing the
utilization of the light energy. Ma et al. [90] prepared a series of K intercalated g-C3N4 modified TiO2
nanobelts with enhanced light absorption, transfer efficiency, and H2 evolution efficiency of 50 µmol/h,
which is 6.4 times greater than that of pristine g-C3N4. The use of carbon atoms in the form of carbon
quantum dots (CQDs) as electron reservoirs improves the efficiency of separating the photogenerated
charge carriers. CQDs present an important class of carbon materials since their discovery in 2004 by
Xu et al. [116], with varying sizes in the range of 1–10 nm. They are good materials for photocatalytic
applications due to features, like superiority in chemical stability and low toxicity [117]. Pan et al. synthesized he 2D carbon quantum dots modified porous g-C3N4/TiO2 nano-heterojunction [91] and
reached 6.497 µmol/h/g of produced H2 with the full spectrum absorption. 3.1.2. TiO2-G/GO/rGO Finally, the photocatalytic activity of the nanocomposite was enhanced towards the production of
H2, reaching 6500 mol/g in 8 h, which was much higher amount when comparing to that obtained by
TiO2-P25 (460 mol/g) at the same irradiation time. The reduced form of GO, rGO, is a two-dimensional carbon material with the role of an electron
mediator that is much superior in chemical stability and morphological diversity than GO [120]
GO/TiO2 nanocomposites have recently been used for H2 production via photocatalytic water
splitting under visible light through the formation of Ti-O-C bonding by unpaired π electron of GO
with TiO2 surface [114,119]. GO acts as an e−acceptor, promoting the separation of the photogenerated
e−/h+ pairs in TiO2. These nanocomposites can be synthesized by photo assisted reduction via mixing
or sonication and by sol-gel [114]. Hernández-Majalca et al. [114] enhanced the synthesis for the
GO-TiO2 nanocomposite using photoassisted anchoring and modifying GO oxidation method through
the use of microwaves. The obtained nonporous product had a specific surface area of 45 m2/g and
absorption onset of 477 nm, which made it active under visible light. Finally, the photocatalytic
activity of the nanocomposite was enhanced towards the production of H2, reaching 6500 mol/g in 8 h,
which was much higher amount when comparing to that obtained by TiO2-P25 (460 mol/g) at the same
irradiation time. mediator that is much superior in chemical stability and morphological diversity than GO [120]. Iwase et al. published the very first report using rGO as a e−mediator in 2011 [121]. Since then, a
number of published works were recorded for the use of rGO-based composites in photocatalytic H2
production [75,122,123]. Recent achievements in the synthesis of TiO2/rGO composites for the
purpose of H2 generation include work from Reedy et al. [75] and Samal et al. [122]. They obtained
rather high HERs while using TiO2/rGO composites under solar and visible light: 24880 µmol/h/g
The reduced form of GO, rGO, is a two-dimensional carbon material with the role of an electron
mediator that is much superior in chemical stability and morphological diversity than GO [120]. Iwase et al. published the very first report using rGO as a e−mediator in 2011 [121]. Since then,
a number of published works were recorded for the use of rGO-based composites in photocatalytic
H2 production [75,122,123]. 3.1.2. TiO2-G/GO/rGO Following above-mentioned hot-electron mechanism, which can promote redox reactions, Lu et al. explored 3D graphene materials (3DG) coupled with TiO2 [76] for efficient photocatalytic H2 production
under UV-visible light. TiO2/3DG with a 5 wt.% graphene loading that was annealed at 650 ◦C exhibited
the highest H2 evolution rate of 1205 µmol/h/g. Yi et al. [118] synthesized a composite in which TiO2 nanobelts were supported by N-doped
graphene (NG) coordinated with a single Co atom to replace noble metals with a cost-effective
photocatalyst. Under simulated solar irradiation (Figure 14), e−/h+ pairs are formed. The transfer of
photogenerated e−from the CB of TiO2 to Co-NG was energetically favorable since the Fermi energy
level of graphene (−0.08V vs. NHE) is lower than the CB of TiO2 (−0.39 V vs. NHE). NG, with a large
specific surface area, acted as “freeway” for e−transportation, delivering e−from TiO2 to Co single-atom,
where they were trapped catalyzing H+ reduction to form H2 due to lower the overpotential needed
for Co-NG when comparing to that of NG. Co-NG/TiO2 showed HER of 677.44 µmol/h/g under the
illumination of AM 1.5 G simulated sunlight. 28 of 44
Co
the Materials 2020, 13, 1338
specific surface
single-atom, wh p
p
g
Figure 14. Schematic diagram of proposed photocatalytic mechanism in the CO-NG/TiO2 system. Figure 14. Schematic diagram of proposed photocatalytic mechanism in the CO-NG/TiO2 system. Figure 14. Schematic diagram of proposed photocatalytic mechanism in the CO-NG/TiO2 system. Figure 14. Schematic diagram of proposed photocatalytic mechanism in the CO-NG/TiO2 system. GO/TiO2 nanocomposites have recently been used for H2 production via photocatalytic water
splitting under visible light through the formation of Ti-O-C bonding by unpaired π electron of GO
with TiO2 surface [114,119]. GO acts as an e− acceptor, promoting the separation of the
photogenerated e−/h+ pairs in TiO2. These nanocomposites can be synthesized by photo assisted
reduction via mixing or sonication and by sol-gel [114]. Hernández-Majalca et al. [114] enhanced the
synthesis for the GO-TiO2 nanocomposite using photoassisted anchoring and modifying GO
oxidation method through the use of microwaves. The obtained nonporous product had a specific
surface area of 45 m2/g and absorption onset of 477 nm, which made it active under visible light. 3.1.2. TiO2-G/GO/rGO Recent achievements in the synthesis of TiO2/rGO composites for the
purpose of H2 generation include work from Reedy et al. [75] and Samal et al. [122]. They obtained
rather high HERs while using TiO2/rGO composites under solar and visible light: 24880 µmol/h/g and
2700 µmol/h/g of produced H2, respectively. Ida et al. undertook further investigation on TiO2/rGO
composites [123], managing to enhance the photocatalytic activity of the obtained composite by the
simultaneous doping of nitrogen on TiO2 and rGO. The following values for the HER are obtained:
TiO2 (1585 µmol/h/g) < N-TiO2 (6179 µmol/h/g) < TiO2/RGO (12244 µmol/h/g) < N-TiO2/N-RGO
(15,028 µmol/h/g). 3.1.3. TiO2/CNT Recently, TiO2/carbon nanotubes (CNT) have been of great interest due to their high-quality active
sites, large specific surface area, and retention of charge recombination, where CNTs can act as a p-type 29 of 44 Materials 2020, 13, 1338 semiconductor, having a role as a powerful electron sink [119]. The coupling of CNT with TiO2 forms
an advanced nanocomposite with enhanced quantum efficiency that forms heterojunction acting as
an impurity by forming Ti-O-C or Ti-C defects that enable visible light absorption and, consequently,
the creation of e−/h+ pairs and hindering e−/h+ recombination [124]. CNTs, such as single-wall carbon
nanotubes (SWCNTs) and multiwall carbon nanotubes (MWCNTs), have attracted much interest due to
their unique chemical, electrical, and optical properties [125]. Olowoyo et al. [124] prepared a series of
TiO2 nanoparticles that were modified with MWCNTs by a combined sonothermal-hydrothermal method. The synthesized photocatalysts were examined for water splitting under batch conditions at different pH
ranges. The highest rate of H2 yield, amounting 69.41 µmol/h/g, was obtained using 2 wt.% CNT-TiO2
under visible light at pH 2. Hence, the acidic medium improved the photocatalytic feasibility of the
system due to a higher concentration of H+ ions, serving as the reactants, thus increasing the reaction rate. Bellamkonda et al. [126] used a different approach in synthesizing CNT-G-TiO2 composites and
prepared nanocomposites via the solution-based method, in which nanocrystalline anatase TiO2 was
grown onto graphene nanosheets and carbon nanotubes. Spectroscopic and photocatalytic studies
revealed that graphene acts as an electron reservoir, while the role of CNTs is to prevent the restacking
of graphene nanosheets and provide additional electron transport channels, thereby suppressing the
recombination rate of e−/h+ pairs in the obtained composite. The combination of all these factors
resulted in increasing the HER from 19000 µmol/h/g (obtained by anatase TiO2) to 22000 µmol/h/g
(obtained by G-TiO2), and finally to 29000 µmol/h/g (obtained by CNT-G-TiO2), which is 8-fold higher
than obtained by the commercial TiO2 (Degussa P25). semiconductor, having a role as a powerful electron sink [119]. The coupling of CNT with TiO2 forms
an advanced nanocomposite with enhanced quantum efficiency that forms heterojunction acting as
an impurity by forming Ti-O-C or Ti-C defects that enable visible light absorption and, consequently,
the creation of e−/h+ pairs and hindering e−/h+ recombination [124]. 3.1.3. TiO2/CNT CNTs, such as single-wall carbon
nanotubes (SWCNTs) and multiwall carbon nanotubes (MWCNTs), have attracted much interest due to
their unique chemical, electrical, and optical properties [125]. Olowoyo et al. [124] prepared a series of
TiO2 nanoparticles that were modified with MWCNTs by a combined sonothermal-hydrothermal method. The synthesized photocatalysts were examined for water splitting under batch conditions at different pH
ranges. The highest rate of H2 yield, amounting 69.41 µmol/h/g, was obtained using 2 wt.% CNT-TiO2
under visible light at pH 2. Hence, the acidic medium improved the photocatalytic feasibility of the
system due to a higher concentration of H+ ions, serving as the reactants, thus increasing the reaction rate. Bellamkonda et al. [126] used a different approach in synthesizing CNT-G-TiO2 composites and
prepared nanocomposites via the solution-based method, in which nanocrystalline anatase TiO2 was
grown onto graphene nanosheets and carbon nanotubes. Spectroscopic and photocatalytic studies
revealed that graphene acts as an electron reservoir, while the role of CNTs is to prevent the restacking
of graphene nanosheets and provide additional electron transport channels, thereby suppressing the
recombination rate of e−/h+ pairs in the obtained composite. The combination of all these factors
resulted in increasing the HER from 19000 µmol/h/g (obtained by anatase TiO2) to 22000 µmol/h/g
(obtained by G-TiO2), and finally to 29000 µmol/h/g (obtained by CNT-G-TiO2), which is 8-fold higher
than obtained by the commercial TiO2 (Degussa P25). The photocatalytic performance of TiO2 under visible light can be promoted by coupling both
MWCNTs and SWCNTs, as presented by Umer et al. [125]. Such an effect occurs due to their
dual natural behavior, such as reducing rapid recombination of e−/h+ pairs and providing support
in harvesting visible light. The maximum H2 evolution rate of 5486 µmol/h/g was achieved over
MWCNT/TiO2/SWCNT, which is 1.24– and 1.42–fold higher than using single CTN-TiO2 composites
(SWCNT/TiO2 and MWCNT/TiO2, respectively). 3.2. Transition-Metal Oxides/TiO2 Excellent chemical stability has opened the possibility of the application of transition metal oxides
(TMO) in the field of clean energy production. The above displayed Figure 12 contains main TMOs,
like p-type (CuO, V2O5) and n-type (TiO2, WO3, MoO3, ZnO, Fe2O3) semiconductors that are used in
photocatalytic H2 production with pertaining VB and CB energy levels. Visible-light driven TMOs with
narrow band gaps are highly desired. The most used materials within this group, such as CuO, Fe2O3,
and WO3, have the bandgap energies that allow for them to be active in the visible light region, but the low
energy levels of CB position disable them from consuming photoinduced electrons in reactions yielding
H2. By changing the morphologies of desired components and co-doping with different elements, their
CB and VB edges can be shifted toward a H2 reduction and O2 oxidation potential [127]. Some TMOs, specifically WO3, have been loaded with a different co-catalyst, like Rh, to effectively
produce H2 from water, to control the desired morphology in the form of nanorods, nanotubes, and
nanowires. Camposeco et al. [128] focused on the use of Rh-WO3 photocatalyst that was supported on
TiO2 nanotubes (Rh-WO3/NT) for H2 production via the water splitting process. WO3 alone cannot take
part in H2 production since the CB energy level of WO3 is lower than H2 reduction potential. However,
by loading with Rh nanoparticles, the enhancement in H2 production was noticed. An analysis of
energy band levels for the VB and CB that were determined by UV-Vis results and XPS spectra showed
that the presence of WO3 and Rh in the titanate nanotubes simultaneously shift the VBmax and CBmin,
thus reducing the bandgap of titanate nanotubes. 0.5 wt.% Rh– 3 wt.% WO3/NT nanocomposite
under visible light irradiation yielded HER of 87 µmol/h, while 3 wt.% WO3/NT showed much lower
effectiveness (only 13 µmol/h). In another work, Ren et al. [129] constructed cooperative Schottky and p-n (SPN) heterojunction by
forming a NiO/Ni/TiO2 heterostructure that showed a narrower band gap, higher photocurrent density, Materials 2020, 13, 1338 30 of 44 30 of 44 and ability to absorb light in the visible region. High HER is obtained for the observed composite,
amounting 4653 µmol/h/g, which is approx. 2.3 times higher than obtained with NiO/TiO2 with a p-n
junction (2059 µmol/h/g), under visible light irradiation and by the use of Pt as a co-catalyst. 3.2. Transition-Metal Oxides/TiO2 j
g
g
y
y
A representative example of TMOs is also hematite (Fe2O3), already referred above to as promising
visible light active photocatalyst for water treatment. The coupling of Fe2O3 with TiO2 efficiently
inhibits the recombination of photogenerated charge carriers and enhances the absorption of solar
light [130]. By the use of thermal decomposition of FeCl3 and TiCl4 as precursors, Bhagya et al. [113]
synthesized the Fe2O3-TiO2 composite and investigated its photocatalytic activity for H2 production
under the influence of different proton sources. Besides focusing on the use of TMOs for improving
photocatalytic efficiency, this work also highlights the influence of different sacrificial agents as electron
donors that consume photogenerated h+, yielding H2 production. Under simulated solar irradiation,
a very high H2 rate of 880 µmol/h, with an apparent quantum efficiency of 19.39%, is achieved while
using Fe2O3-TiO2 and diethylamine hydrogen chloride (DAH), which is much higher than that obtained
in the case without DAH (323 µmol/h). Madhumitha et al. [130] also explored the influence of different
sacrificial reagents on H2 production under a visible light source. With the optimization of parameters,
i.e., catalyst dosage, flow rate, incident light irradiation, and type of sacrificial agent, they achieved
the maximum of HER, amounting 2700 µmol/h. The increase in photoactivity was attributed to the
effective charge transfer from TiO2 to Fe2O3 and the use of EDTA, which suppressed the recombination
of photogenerated charge carriers. Easy preparation, environmental friendliness, and good re-utilization enable the wide use of
ZnO/TiO2-based composites. Xie et al. [131] achieved high rates of H2 evolution while using ZnO/TiO2
composites with Pt as a co-catalyst. Under visible light irradiation, 2150 µmol/h/g of H2 is achieved. Additionally, high carrier mobility can be achieved by the use of ZnO in the form of quantum dots (QDs),
which presents ideal heavy-metal free “green” modification of TiO2 composites. Chen et al. obtained the
fabrication of ZnO QDs decorated TiO2 nanowires via a facile calcination method [132]. They used the
obtained composite under solar irradiation next to Pt as a co-catalyst and achieved HER of 313.5 µmol/h. The use of TMO QDs is also recorded in the work of Liu et al. [133], who decorated two-dimensional
TiO2 nanobelts with zero-dimensional Co3O4 quantum dots. 3.2. Transition-Metal Oxides/TiO2 When compared with bulk materials, 0D
Co3O4 QDs have attracted considerable attention due to their small size (<10 nm), providing a large
specific surface area with more active sites and shorter charges transport paths. Due to the decoration
of Co3O4 QDs, the bandgap of the obtained composite was also narrowed, and in application next to Pt
as co-catalyst, they obtained a rather high HER of 1735.1 µmol/h/g. In comparison, Zhang et al. [134],
with the use of bulk p-Co3O4/n-TiO2, achieved a smaller HER of 8.16 µmol/h/g. Among the TMOs that appear as promising candidates for coupling with TiO2, CuO is one of such,
especially due to its narrow bandgap (1.4–1.6 eV) and the promotion of effective charge separation [5,135],
as already reported as promising water treatment photocatalyst. Hasan et al. [5] conducted solar H2
production using a TiO2/CuO nanofiber composite that was synthesized by electrospinning technique. Fabricated nanofibers were annealed in different atmospheres to determine the crystalline phase and
photocatalytic performance. For the nanofibers that were crystallized in the anatase phase, EPR and XRD
analysis referred to the substitution of some Ti4+ ions by Cu2+ ions, leading to the formation of some
defects below the CB of TiO2, which led to a narrow band gap, yielding enhanced HER in the amount of
2715 µmol/h/g. For comparison, without annealing in a different atmosphere, Wang et al. [135] only
achieved 47 µmol/h/g of produced H2 while using the TiO2/CuO composite that was irradiated by solar
light. Other oxidation states of Cu inside the oxides are investigated for coupling with TiO2, such as
Cu2O [136]. With all of the benefits, such as environmental compatibility, high visible light activity and
earth abundance, wider applications of Cu2O in water splitting are still limiting, since the redox potential
of monovalent copper lies within its band gap, thus photogenerated charge carriers thermodynamically
prefer the transformation of Cu2O into CuO and Cu, rather than to be used in redox reactions with
water constituents forming H2 [136]. Wei et al. [136] stabilized the Cu2O by modulating the defects
in faceted Cu2O/TiO2 heterostructures to suppress this disproportionation process. Hence, Cu2O was 31 of 44 Materials 2020, 13, 1338 arranged onto 101-faceted TiO2 and it was found that oxygen vacancies in {101}-faceted TiO2 can create
a unique channel for Z-scheme charge transfer in Cu2O/TiO2 heterostructures. 3.2. Transition-Metal Oxides/TiO2 Composite that was
obtained by such an approach showed the maximum HER of 32600 µmol/h/g, with a quantum efficiency
of 53.5% at an irradiation wavelength of 350 nm. 3.3. Transition Metal Chalcogenides-TiO2 As already mentioned, various techniques have been applied to modify the TiO2 photocatalysts
with a purpose of wider application in the field of solar-driven H2 production through water splitting. Sensitization with narrow bandgap semiconductors was found to be an effective method for enhancing
all of the deficiencies that occur during the sole use of TiO2. Regarding an appropriate band gaps,
another group of semiconductors with great application potential in photocatalytic H2 generation are
transition metal chalcogenides. Recently, CdS is one of the most studied materials [54,55,137–140];
however, the toxic effects due to potential leaching of Cd2+ have to be strongly considered, followed
by others (MoS2, ZnS, ZnSe, CdSe) indicated in Figure 12, as mentioned in part related to water
treatment [127]. 3.3.1. TiO2/CdS CdS is the most important chalcogenides semiconductor as a hydrogen production catalyst due to its
narrow bandgap (2.4 eV), which enables its visible light response [56]. Its drawbacks described above in
Section 2.2. can be alleviated; susceptibility to photocorrosion can be suppressed by the use of sacrificial
agents (sodium sulfite/sulfide) that effectively consume photogenerated h+, while the limited separation
efficiency of photogenerated charge carriers can be solved either by using CdS in the form of QDs due to
a shorter transportation path or by incorporating CdS onto support materials, such as TiO2 [55,56]. Rao et al. [137] synthesized CdS/TiO2 core/shell nanorods with tunable shell thickness to minimize
charge carriers recombination and limit photocorrosion. The investigation of photocatalytic activity
performed under UV-vis light irradiation confirmed that optimized concentration of sacrificial agents
(0.3 M Na2S and Na2SO4 aqueous solution), shell thickness of 6.3 nm, and solution pH of 8.0 enhance the
H2 production rate of 5791 mL/h/g. Du et al. [138], who fabricated pyramid-like CdS nanoparticles that
were grown on porous TiO2, obtained the same type of composite, but with the different morphology. Under UV-vis irradiation and without noble-metal co-catalysts, 5 mol% CdS-TiO2 achieved an H2
production rate of 1048.7 µmol/h/g, which is almost six times and 1.5 times higher than that of pure
TiO2 and CdS, respectively. Table 10 provides further examples of TiO2/CdS-based composites with
their respective photocatalytic activities for H2 production. Table 10. The photocatalytic performance of H2 generation in some related TiO2/CdS-based nanocomposites. Photocatalyst
Light Source
HER
Reference
CdS/Pt/TiO2 film
300 W Xe lamp
3.074 µmol/h/g
[54]
CdS/Pt/TiO2 nanosheets
350 W Xe arc lamp
265 µmol/h
[141]
CdS/Pt/TiO2 nanotubes
300 W Xe lamp
402 µmol/h
[142]
CdS/TiO2 nanotubes
350 W Xe lamp
2585 µL/h/g
[143]
CdS-Ti-MCM-48-21-25
300 W Xe lamp
2726 µmol/h
[139] Table 10. The photocatalytic performance of H2 generation in some related TiO2/CdS-based nanocomposites. The photocatalytic performance of H2 generation in some related TiO2/CdS-based nanocomposites. 3.3.2. TiO2/CuS CuS has emerged as an alternative co-catalyst for H2 production, which is abundant, cheap,
and nonhazardous. With its VB and CB at positions of −1.56 eV vs. NHE, and −0.09 eV, CuS shows
low reflectance in the visible and relatively high reflectance in the near-infrared region, which makes it
a good candidate for solar energy absorption [144]. Chandra et al. [144] investigated the photocatalytic
activity for H2 generation of synthesized CuS/TiO2 (CT) heterostructured nanocomposite under UV-vis
and only visible light. Under irradiation, the highest HER of 12362 µmol/h/g was achieved while Materials 2020, 13, 1338 32 of 44 using 0.4 mol.% CuS/TiO2, while only 155 µmol/h/g of H2 was produced under visible light and
comparable conditions. In comparison, Dang et al. [145] produced a series of CuxS (x = 1 or 2) co-modified TiO2
nanocomposites while using a one-step precipitation approach. The EDS, XPS, and XRF results
confirmed the existence of three different phases: CuS, Cu2S, and TiO2. The results have shown
that CuS and Cu2S dual co-catalysts under simulated solar light exhibited high H2 production of
5620 µmol/h/g, which is about 58 times higher than that of the unloaded TiO2. The enhancement in
H2 production can be contributed to the co-deposition of CuS and Cu2S onto the TiO2 nanoparticle
surface that efficiently extended visible light absorption and facilitated the separation of charge carriers. The CuxS/TiO2 composite showed high stability; after three consecutive cycles the photocatalytic
efficiency for H2 production decreased for only 11.7%. 3.4. Multiple TiO2-Based Composites This section is dedicated to multiple ternary and quaternary TiO2-based composites. Recently, significant emphasis was laid on the formation of Z-scheme structured photocatalysts. In such
materials, redox reactions occur in each semiconductor, allowing for the combination of a semiconductor
with strong reduction power with another semiconductor with strong oxidation power [93]. All of the solid Z-scheme heterojunctions are usually composed of two photocatalysts and an
electron mediator, which enable efficient H2 production through the synergistic action between
two isolated photosystems and electron mediator cleverly arranged in a nano-platform [147]. Reversible redox couples (e.g., IO3−/I−, Fe3+/Fe2+) are usually applied as electron mediators in
the Z-scheme system. Solid electron mediators are more suitable for the application. Noble metals
(Au, Ag) and carbon-based materials (MWCNT, rGO, CQDs) are commonly used as solid electron
mediators for photocatalytic H2 generation [148]. Ng et al. [147], who synthesized Zn0.5Cd0.5S-MWCNT-TiO2 ternary nanocomposite, where MWCNT
acted as an electron mediator, fabricated a solid Z-scheme system. The obtained material efficiently
suppressed charge recombination and promoted water reduction, achieving HER of 21.9 µmol/h. Furthermore, Liu et al. [149] have used carbon quantum dots (QDs) as an electron mediator between
TiO2 and Zn0.5Cd0.5S film and achieved 38740 µmol/h/m2 of produced H2 under solar light. Lv et al. investigated the use of rGO as an electron mediator [120], synthesizing sandwich-like TiO2/rGO/LaFeO3
ternary heterostructure, which, under solar light, obtained HER of 893 µmol/h/g, which is almost 3.2,
14.4, and 11.4 times superior to the direct Z-scheme components TiO2/LaFeO3 composite, pure TiO2,
and LaFeO3, respectively. The photocatalytic mechanism of H2 production is the same for all three
above mentioned solid electron mediators: MWCNT, rGO, and CQDs (Figure 16). Upon excitation
by light, e−/h+ are formed in Z-scheme semiconducting components, depending on the ability of each
component to absorb emitted light. Hence, photogenerated e−from semiconductor I can be easily
recombined with h+ from semiconductor II through the electron mediator, leaving more oxidative holes
and reductive electrons to participate in the redox reactions in the corresponding active sites [148]. In Z-type heterojunction, noble metals can also perform the role of electron mediators. Zou et al. performed the construction of g-C3N4/Au/C-TiO2 hollow spheres with Au nanoparticles (NPs) as the
electron mediator [150]. 3.3.3. TiO2/MoS2 MoS2 represents two-dimensional transition metal dichalcogenide (TMD) that can be prepared into
ultrathin-layered structures and, thus, participates in the H2 evolution reaction as an effective non-noble
metal alternative [117,146]. Without the use of any sacrificial agent and co-catalyst, Huang et al. [146]
observed photocatalytic H2 generation while using MoS2 quantum dots@TiO2 nanotube arrays
nanocomposite in pure water under visible light. The photocatalytic activity was influenced by
the amount of MoS2 QDs coated on TiO2 NTAs. Hence, the maximum of 53.9 µmol/cm2/h of H2 was
produced, which was ascribed to the decreased bandgap and the surface plasmonic properties of the
obtained composite promoting charge carrier separation and the absorption capacity to visible light. Du et al. [63] grew in situ two transition metal chalcogenides—MoS2 and CdS—on porous TiO2 by using
the sol-gel method, followed by the calcination and hydrothermal method. Under visible light irradiation
and without the use of noble metals as the co-catalyst, 3% MoS2-CdS-TiO2 produced 4146 µmol/h/g of
H2. In this ternary composite (Figure 15), the porous structure of TiO2 accepts generated e−from CdS
and provides surface area for H2 production, while MoS2, as a conductive medium, enabled the transfer
of e−between CB of CdS and TiO2, simultaneously inhibiting the photocorrosion of CdS as h+ collector. Materials 2020, 13, x FOR PEER REVIEW
28 of 39 Figure 15. The proposed photocatalytic mechanism in MoS2-CdS-TiO2 photocatalyst. Figure 15. The proposed photocatalytic mechanism in MoS2-CdS-TiO2 photocatalyst. Figure 15. The proposed photocatalytic mechanism in MoS2-CdS-TiO2 photocatalyst. Figure 15. The proposed photocatalytic mechanism in MoS2-CdS-TiO2 photocatalyst. Materials 2020, 13, 1338 33 of 44 33 of 44 3.4. Multiple TiO2-Based Composites The as-prepared composite showed high HER of 129 µmol/h/g under visible
light, which can be attributed to the efficient charge separation in the constructed Z-scheme system,
the broadened visible-light response range, owing to the surface plasmon resonance (SPR) effects on
Au nanoparticles, and the hollow structure of C-TiO2 that gives photocatalyst unique properties of low
density and high light-harvesting efficiency. In another work presented by Yang et al. [151], Au NPs
were applied as a solid electron mediator in the ternary urchin-like ZnIn2S4-Au-TiO2 nanocomposite,
which, at an optimal ratio of 24 wt.% Au NPs and 60 wt.% ZnIn2S4, achieved HER of 186.3 µmol/h/g
under solar light irradiation. Table 11 lists other multiple composites, except ones with the Z-scheme heterojunction that are
already mentioned, as well as reaction conditions in the photocatalytic system and the obtained
hydrogen evolution rates. Materials 2020, 13, 1338 34 of 44 Table 11. Multiple TiO2-based composites for the use in photocatalytic hydrogen generation. Photocatalyst
Reaction Conditions
HER
Reference
CdSQDs/WC/TiO2
Photocatalyst dispersed in 20 vol.% lactic acid as an electron donor under visible
light. 624.9 µmol/h/
[55]
ZnO/ZnCr2O4@TiO2-NTA
Photocatalyst dispersed in aqueous methanol solution under simulated solar light. 1680 µmol/cm2
[152]
F-TiO2/CdSe-DETA
Photocatalyst was dispersed in a mixed solution of Na2S and Na2SO3 as a
sacrificial agents under visible light with the use of Pt as a cocatalyst. 12381 µmol/h/g
[153]
g-C3N4/TiO2/RGO
5 mg of the g-C3N4-TiO2/RGO nano-composite was dispersed in 50 mL
glycerol-water solution under UV-vis light. 19610 µmol/h/g
[13]
CDS/CNF/Pt-TiO2
Photocatalyst was dispersed in a mixed solution of Na2S and Na2SO3 as a
sacrificial agents under visible light. 16.34 µmol for 3 h
[154]
F-TiO2/CdS-DETA, Pt as a
cocatalyst
50 mg of the photocatalyst was dispersed in 100 mL of
mixed aqueous solution containing 0.35 mg/L Na2S and 0.25 mg/L Na2SO3 with
the use of Pt as a cocatalyst. 5342.86 µmol/h/g
[155]
CdS@TiO2@Au
20 mg of the
photocatalyst was dispersed in 40 ml of aqueous solution containing 0.1 M Na2S
and 0.1 M Na2SO3 as the sacrificial agents under visible light. 1720 µmol/h/g
[156]
TiO2-Au-CdS
0.1 g of the sample was immersed in an aqueous solution containing 0.1 M Na2S
and 0.1 M Na2SO3 as the sacrificial agents under visible light. 1810 µmol/h/g
[157]
N-TiO2/g-C3N4@NixP
50 mg photocatalyst was suspended in a 100 mL solution containing 10 vol.%
triethanolamine (TEOA) under 300 W Xe lamp irradiation. In Z-typ
performed th
4. Conclusions p
g
/
/
p
p
(
)
electron mediator [150]. The as-prepared composite showed high HER of 129 µmol/h/g under visible
light, which can be attributed to the efficient charge separation in the constructed Z-scheme system,
the broadened visible-light response range, owing to the surface plasmon resonance (SPR) effects on
Au nanoparticles, and the hollow structure of C-TiO2 that gives photocatalyst unique properties of
low density and high light-harvesting efficiency. In another work presented by Yang et al. [151], Au
NPs were applied as a solid electron mediator in the ternary urchin-like ZnIn2S4-Au-TiO2
nanocomposite which at an optimal ratio of 24 wt % Au NPs and 60 wt % ZnIn S achieved HER of
TiO2-based photocatalytic technology represents a promising up-coming technology for both
renewable energy generation and water purification applications. It is necessary to work on
developing TiO2-based semiconductor materials, which are active under broader spectrum of solar light,
overcoming the indicated disadvantages of the solely TiO2 utilization, since TiO2 as a photocatalytic
material cannot be used alone due to its limitations such as activity only in UV light region and rapid
recombination of photogenerated charge carriers. nanocomposite, which, at an optimal ratio of 24 wt.% Au NPs and 60 wt.% ZnIn2S4, achieved HER of
186.3 µmol/h/g under solar light irradiation. Table 11 lists other multiple composites, except ones with the Z-scheme heterojunction that are
already mentioned, as well as reaction conditions in the photocatalytic system and the obtained
hydrogen evolution rates. TiO2-semiconductor coupling offers promising results in water purification, particularly
for the degradation and mineralization of CECs. However, it is necessary to evaluate the
toxicities of degradation intermediates of CEC to check the real efficiency of such composites. Furthermore, the immobilization of TiO2–Semiconductor composites to photocatalytic reaction
membranes must be envisaged for further upscale opportunities. Table 11. Multiple TiO2-based composites for the use in photocatalytic hydrogen generation. Photocatalyst
Reaction Conditions
HER
Reference
CdSQDs/WC/TiO2
Photocatalyst dispersed in 20 vol.% lactic acid as an
electron donor under visible light. 624.9 µmol/h/
[55]
ZnO/ZnCr2O4@TiO2-NTA
Photocatalyst dispersed in aqueous methanol solution
under simulated solar light. 1680 µmol/cm2
[152]
F-TiO2/CdSe-DETA
Photocatalyst was dispersed in a mixed solution of Na2S
and Na2SO3 as a sacrificial agents under visible light with
the use of Pt as a cocatalyst. 12381 µmol/h/g
[153]
g-C3N4/TiO2/RGO
5 mg of the g-C3N4-TiO2/RGO nano-composite was
dispersed in 50 mL glycerol-water solution under UV-vis
light. 3.4. Multiple TiO2-Based Composites 5438 µmol/h/g
[158]
TiO2/CdS/CNT
0.1 g of photocatalyst was dispersed in solution containing 70 mL of distilled
water or seawater and 30 mL of sacrificial agent. The photocatalyst were
irradiated using three visible-light sunlamps of each 100 W and UV lamp of 8 W. 3502 µmol/h from pure water
and 1373 µmol/h from seawater
[159]
WS2/ C-TiO2/ g-C3N4
50 mg of photocatalyst was added in to 80 mL TEOA aqueous solution with the
use of Pt as a cocatalyst. 17726 for DI water and 29978
µmol/g for seawater
[148]
TiO2/La2O2CO3/rGO
0.05 g of powder catalyst was dispersed in 80 Ml ethylene-glycol water (5/95, v/v)
solution under UV-vis light. 583 µmol/h
[160]
TiO2-Cu@C
Photocatalyst was dispersed in methanol aqueous solution under UV-vis light. 3911 µmol/g/h
[161] 35 of 44
ron Materials 2020, 13, 1338
semiconductor I
ediato
lea i in the corresponding active sites [148]. Figure 16. The type Z-heterojunction with the use of different solid electron mediators (multiwall
carbon nanotubes (MWCNTs), carbon quantum dots (CQDs), rGO). Figure 16. The type Z-heterojunction with the use of different solid electron mediators (multiwall
carbon nanotubes (MWCNTs), carbon quantum dots (CQDs), rGO). Figure 16. The type Z-heterojunction with the use of different solid electron mediators (multiwall
carbon nanotubes (MWCNTs), carbon quantum dots (CQDs), rGO). Figure 16. The type Z-heterojunction with the use of different solid electron mediators (multiwall
carbon nanotubes (MWCNTs), carbon quantum dots (CQDs), rGO). Author Contributions: Conceptualization, H.K., F.F., and A.L.B.; writing—original draft preparation, K.P.,
F.M.d.R., H.K.; writing—review and editing, M.K., U.L.Š., D.D.D., F.F. and A.L.B. All authors have read and agreed
to the published version of the manuscript. Funding:
This research was funded by projects:
“Nano-sized Solar-active Catalysts for Environmental
Technologies” (NaSCEnT, IP-2018-01-1982), Croatian Science Foundation, and “Water Purification and Energy References Photocatalytic water splitting—The
untamed dream: A review of recent advances. Molecules 2016, 21, 900. [CrossRef] 12. Jing, D.; Guo, L.; Zhao, L.; Zhang, X.; Liu, H.; Li, M.; Shen, S.; Liu, G.; Hu, X.; Zhang, X.; et al. Efficient solar
hydrogen production by photocatalytic water splitting: From fundamental study to pilot demonstration. Int. J. Hydrogen Energy 2010, 35, 7087–7097. [CrossRef] 13. Hafeez, H.Y.; Lakhera, S.K.; Bellamkonda, S.; Rao, G.R.; Shankar, M.V.; Bahnemann, D.W.; Neppolian, B. Construction of ternary hybrid layered reduced graphene oxide supported g-C3N4-TiO2 nanocomposite and
its photocatalytic hydrogen production activity. Int. J. Hydrogen Energy 2018, 43, 3892–3904. [CrossRef] 13. Hafeez, H.Y.; Lakhera, S.K.; Bellamkonda, S.; Rao, G.R.; Shankar, M.V.; Bahnemann, D.W.; Neppolian, B. Construction of ternary hybrid layered reduced graphene oxide supported g-C3N4-TiO2 nanocomposite and
its photocatalytic hydrogen production activity. Int. J. Hydrogen Energy 2018, 43, 3892–3904. [CrossRef]
14. Schneider, J.; Matsuoka, M.; Takeuchi, M.; Zhang, J.; Horiuchi, Y.; Anpo, M.; Bahnemann, D.W. Understanding
TiO2 photocatalysis: Mechanisms and materials. Chem. Rev. 2014, 114, 9919–9986. [CrossRef] [PubMed]
15. Pichat, P. Photocatalysis and Water Purification: From Fundamentals to Recent Applications; Wiley: Weinheim, 14. Schneider, J.; Matsuoka, M.; Takeuchi, M.; Zhang, J.; Horiuchi, Y.; Anpo, M.; Bahnemann, D.W. Understanding
TiO2 photocatalysis: Mechanisms and materials. Chem. Rev. 2014, 114, 9919–9986. [CrossRef] [PubMed] 15. Pichat, P. Photocatalysis and Water Purification: From Fundamentals to Recent Applications; Wiley: Weinheim,
Germany, 2013. 16. Fagan, R.; McCormack, D.E.; Dionysiou, D.D.; Pillai, S.C. A review of solar and visible light active TiO2
photocatalysis for treating bacteria, cyanotoxins and contaminants of emerging concern. Mater. Sci. Semicond. Process. 2016, 42, 2–14. [CrossRef] 17. Qi, K.; Cheng, B.; Yu, J.; Ho, W. A review on TiO2 -based Z-scheme photocatalysts. Chin. J. Catal. 2017, 38, 1936–1955. [CrossRef] 18. Calvete, M.J.F.; Piccirillo, G.; Vinagreiro, C.S.; Pereira, M.M. Hybrid materials for heterogeneous photocatalytic
degradation of antibiotics. Coord. Chem. Rev. 2019, 395, 63–85. [CrossRef] 19. Tong, H.; Ouyang, S.; Bi, Y.; Umezawa, N.; Oshikiri, M.; Ye, J. Nano-photocatalytic materials: Possibilities
and challenges. Adv. Mater. 2012, 24, 229–251. [CrossRef] [PubMed] 19. Tong, H.; Ouyang, S.; Bi, Y.; Umezawa, N.; Oshikiri, M.; Ye, J. Nano-p
and challenges. Adv. Mater. 2012, 24, 229–251. [CrossRef] [PubMed] 20. Pasternak, S.; Paz, Y. On the similarity and dissimilarity between photocatalytic water splitting and
photocatalytic degradation of pollutants. ChemPhysChem 2013, 14, 2059–2070. [CrossRef] 21. Wang, H.; Zhang, L.; Chen, Z.; Hu, J.; Li, S.; Wang, Z.; Liu, J.; Wang, X. In Z-typ
performed th
4. Conclusions 19610 µmol/h/g
[13]
Great progress has also been recorded in the development of TiO2-based heterojunction for the
application in solar-driven photocatalytic hydrogen generation. By morphological control of the
obtained composites, higher H2 generation, as well as better light harvesting can be achieved. It is
noticed that still a great deal of research is being conducted by the use of different noble-metal co-catalysts
and sacrificial agents, which, although increasing the efficiency of the process, reduces its environmental
friendliness and increases the performance cost. Accordingly, to allow for the practical deployment
of such units, as well as commercialization, it is necessary to produce cost-effective systems that will
consider economic impact assessment, including operation cost and energy consumption, and that will
not require the use of costly co-catalysts and other substances that promote system performance. Author Contributions: Conceptualization, H.K., F.F., and A.L.B.; writing—original draft preparation, K.P.,
F.M.d.R., H.K.; writing—review and editing, M.K., U.L.Š., D.D.D., F.F. and A.L.B. All authors have read and agreed
to the published version of the manuscript. Funding:
This research was funded by projects:
“Nano-sized Solar-active Catalysts for Environmental
Technologies” (NaSCEnT, IP-2018-01-1982), Croatian Science Foundation, and “Water Purification and Energy 36 of 44 36 of 44 Materials 2020, 13, 1338 Conversion using Novel Composite Materials and Solar Irradiation” (KK.01.1.1.04.0001), European Structural and
Investment Funds (Croatia) Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References 1. European Commission. Directive 2000/60/EC of the European Parliament and of the Council, of 23 October
2000. Off. J. Eur. Communities 2000, 327, 1–72. 1. European Commission. Directive 2000/60/EC of the European Parliament and of the Council, of 23 October
2000. Off. J. Eur. Communities 2000, 327, 1–72. . Water JPI. Available online: http://www.waterjpi.eu/mapping-agenda/strategic-re (accessed on 12 December 2 2. Water JPI. Available online: http://www.waterjpi.eu/mapping-agenda/strategic-re (accessed on 12 December 2019). 3. European Commission. Energy Strategy. Available online: https://ec.europa.eu/energy/en/topics/energy-
strategy (accessed on 12 December 2019). 3. European Commission. Energy Strategy. Available online: https://ec.europa.eu/energy/en/topics/energy-
strategy (accessed on 12 December 2019). 3. European Commission. Energy Strategy. Available online: https://ec.europa.eu/energy/en/topics/energy-
strategy (accessed on 12 December 2019). 4. Martin, M. Alternative Energy Sources and Technologies; Springer: Basel, Switzerland, 2016. . Martin, M. Alternative Energy Sources and Technologies; Springer: Basel, Switzerland, 2016. Hasan, M.M.; Allam, N.K. Unbiased spontaneous solar hydrogen production using stable TiO2-CuO
composite nanofiber photocatalysts. RSC Adv. 2018, 8, 37219–37228. [CrossRef] 6. Fujishima, A.; Honda, K. Electrochemical Photolysis of Water at a Semiconductor Electrode. Nature
1972, 238, 37–38. [CrossRef] [PubMed] 6. Fujishima, A.; Honda, K. Electrochemical Photolysis of Water at a Semiconductor Electrode. Nature
1972, 238, 37–38. [CrossRef] [PubMed] 7. Frank, S.N.; Bard, A.J. Heterogeneous photocatalytic oxidation of cyanide ion in aqueous solutions at TiO2. J. Am. Chem. Soc. 1977, 99, 303–304. [CrossRef] 7. Frank, S.N.; Bard, A.J. Heterogeneous photocatalytic oxidation of cyanide ion in aqueous solutions at TiO2. J. Am. Chem. Soc. 1977, 99, 303–304. [CrossRef] 8. Turchi, C.S.; Ollis, D.F. Photocatalytic degradation of organic water contaminants: Mechanisms involving
hydroxyl radical attack. J. Catal. 1990, 122, 178–192. [CrossRef] 8. Turchi, C.S.; Ollis, D.F. Photocatalytic degradation of organic water contaminants: Mechanisms involving
hydroxyl radical attack. J. Catal. 1990, 122, 178–192. [CrossRef] 9. Chong, M.N.; Jin, B.; Chow, C.W.K.; Saint, C. Recent developments in photocatalytic water treatment
technology: A review. Water Res. 2010, 44, 2997–3027. [CrossRef] [PubMed] 9. Chong, M.N.; Jin, B.; Chow, C.W.K.; Saint, C. Recent developments in photocatalytic water treatment
technology: A review. Water Res. 2010, 44, 2997–3027. [CrossRef] [PubMed] 10. Pelaez, M.; Nolan, N.T.; Pillai, S.C.; Seery, M.K.; Falaras, P.; Kontos, A.G.; Dunlop, P.S.M.; Hamilton, J.W.J.;
Byrne, J.A.; O’Shea, K.; et al. A review on the visible light active titanium dioxide photocatalysts for
environmental applications. Appl. Catal. B Environ. 2012, 125, 331–349. [CrossRef] 11. Jafari, T.; Moharreri, E.; Amin, A.S.; Miao, R.; Song, W.; Suib, S.L. References Semiconductor heterojunction
photocatalysts: Design, construction, and photocatalytic performances. Chem. Soc. Rev. 2014, 43, 5234–5244. [CrossRef] 22. Moniz, S.J.A.; Shevlin, S.A.; Martin, D.J.; Guo, Z.X.; Tang, J. Visible-light driven heterojunction photocatalysts
for water splitting-a critical review. Energy Environ. Sci. 2015, 8, 731–759. [CrossRef] 37 of 44 Materials 2020, 13, 1338 37 of 44 23. Moniz, S.J.A.; Shevlin, S.A.; An, X.; Guo, Z.X.; Tang, J. Fe2O3-TiO2 nanocomposites for enhanced charge
separation and photocatalytic activity. Chem. A Eur. J. 2014, 20, 15571–15579. [CrossRef] 24. Macías-Tamez,
R.;
Villanueva-Rodríguez,
M.;
Ramos-Delgado,
N.A.;
Maya-Treviño,
L.;
Hernández-Ramírez, A. Comparative Study of the Photocatalytic Degradation of the Herbicide
2,4-D Using WO3/TiO2 and Fe2O3/TiO2 as Catalysts. Water Air Soil Pollut. 2017, 228, 379. [CrossRef] 25. Luo, L.; Long, J.; Zhao, S.; Dai, J.; Ma, L.; Wang, H.; Xia, L.; Shu, L.; Jiang, F. Effective visible-light-driven
photocatalytic degradation of 17α-ethynylestradiol by crosslinked CdS nano-rod/TiO2 (B) nano-belt composite. Process Saf. Environ. Prot. 2019, 130, 77–85. [CrossRef] 26. Marschall, R. Semiconductor composites: Strategies for enhancing charge carrier separation to improve
photocatalytic activity. Adv. Funct. Mater. 2014, 24, 2421–2440. [CrossRef] 27. Kaur, A.; Salunke, D.B.; Umar, A.; Mehta, S.K.; Sinha, A.S.K.; Kansal, S.K. Visible light driven photocatalytic
degradation of fluoroquinolone levofloxacin drug using Ag2 O/TiO2 quantum dots: A mechanistic study
and degradation pathway. New J. Chem. 2017, 41, 12079–12090. [CrossRef] 28. Gou, J.; Ma, Q.; Deng, X.; Cui, Y.; Zhang, H.; Cheng, X.; Li, X.; Xie, M.; Cheng, Q. Fabrication of
Ag2O/TiO2-Zeolite composite and its enhanced solar light photocatalytic performance and mechanism for
degradation of norfloxacin. Chem. Eng. J. 2017, 308, 818–826. [CrossRef] 9. Dong, P.; Hou, G.; Xi, X.; Shao, R.; Dong, F. WO3-based photocatalysts: Morphology control, acti
enhancement and multifunctional applications. Environ. Sci. Nano 2017, 4, 539–557. [CrossRef] 30. Mugunthan, E.; Saidutta, M.B.; Jagadeeshbabu, P.E. Visible light assisted photocatalytic degradation of
diclofenac using TiO2-WO3 mixed oxide catalysts. Environ. Nanotechnol. Monit. Manag. 2018, 10, 322–330. 31. Cordero-García, A.; Turnes Palomino, G.; Hinojosa-Reyes, L.; Guzmán-Mar, J.L.; Maya-Teviño, L.;
Hernández-Ramírez, A. Photocatalytic behaviour of WO3/TiO2-N for diclofenac degradation using simulated
solar radiation as an activation source. Environ. Sci. Pollut. Res. 2017, 24, 4613–4624. [CrossRef] 32. Cordero-García, A.; Guzmán-Mar, J.L.; Hinojosa-Reyes, L.; Ruiz-Ruiz, E.; Hernández-Ramírez, A. Effect
of carbon doping on WO3/TiO2 coupled oxide and its photocatalytic activity on diclofenac degradation. Ceram. Int. 2016, 42, 9796–9803. [CrossRef] 33. Pérez-Estrada, L.A.; Malato, S.; Gernjak, W.; Agüera, A.; Thurman, E.M.; Ferrer, I.; Fernández-Alba, A.R. References Photo-fenton degradation of diclofenac: Identification of main intermediates and degradation pathway. Environ. Sci. Technol. 2005, 39, 8300–8306. [CrossRef] 34. Salaeh, S.; Juretic Perisic, D.; Biosic, M.; Kusic, H.; Babic, S.; Lavrencic Stangar, U.; Dionysiou, D.D.; Loncaric
Bozic, A. Diclofenac removal by simulated solar assisted photocatalysis using TiO2-based zeolite catalyst;
mechanisms, pathways and environmental aspects. Chem. Eng. J. 2016, 304, 289–302. [CrossRef] 35. Arce-Sarria, A.; Machuca-Martínez, F.; Bustillo-Lecompte, C.; Hernández-Ramírez, A.; Colina-Márquez, J. Degradation and loss of antibacterial activity of commercial amoxicillin with TiO2/WO3-assisted solar
photocatalysis. Catalysts 2018, 8, 222. [CrossRef] 36. Mishra, M.; Chun, D.M. α-Fe2O3 as a photocatalytic material: A review. Appl. Catal. A Gen. 2015, 498, 126–141. [CrossRef] 37. Mirmasoomi, S.R.; Mehdipour Ghazi, M.; Galedari, M. Photocatalytic degradation of diazinon under visible
light using TiO2/Fe2O3 nanocomposite synthesized by ultrasonic-assisted impregnation method. Sep. Purif. Technol. 2017, 175, 418–427. [CrossRef] 8. Li, R.; Liu, J.; Jia, Y.; Zhen, Q. Photocatalytic degradation mechanism of oxytetracyclines using Fe2O3-T
nanopowders. J. Nanosci. Nanotechnol. 2017, 17, 3010–3015. [CrossRef] 39. Li, R.; Jia, Y.; Wu, J.; Zhen, Q. Photocatalytic degradation and pathway of oxytetracycline in aqueous solution
by Fe2O3-TiO2 nanopowder. RSC Adv. 2015, 5, 40764–40771. [CrossRef] 40. Lu, C.; Guan, W.; Zhang, G.; Ye, L.; Zhou, Y.; Zhang, X. TiO2 /Fe2O3/CNTs magnetic photocatalyst: A fast
and convenient synthesis and visible-light-driven photocatalytic degradation of tetracycline. Micro Nano
Lett. 2013, 8, 749–752. [CrossRef] 41. Zheng, X.; Fu, W.; Kang, F.; Peng, H.; Wen, J. Enhanced photo-Fenton degradation of tetracycline using TiO2
-coated α-Fe2O3 core–shell heterojunction. J. Ind. Eng. Chem. 2018, 68, 14–23. [CrossRef] 41. Zheng, X.; Fu, W.; Kang, F.; Peng, H.; Wen, J. Enhanced photo-Fenton degradation of tetracycline using TiO2
-coated α-Fe2O3 core–shell heterojunction. J. Ind. Eng. Chem. 2018, 68, 14–23. [CrossRef] 42. Casbeer, E.; Sharma, V.K.; Li, X.Z. Synthesis and photocatalytic activity of ferrites under visible light: A
review. Sep. Purif. Technol. 2012, 87, 1–14. [CrossRef] 42. Casbeer, E.; Sharma, V.K.; Li, X.Z. Synthesis and photocatalytic activity of ferrites under visible light: A
review. Sep. Purif. Technol. 2012, 87, 1–14. [CrossRef] 43. Garcia-Muñoz, P.; Fresno, F.; de la Peña O’Shea, V.A.; Keller, N. Ferrite Materials for Photoassisted
Environmental and Solar Fuels Applications. Top. Curr. Chem. 2020, 378, 6. [CrossRef] Materials 2020, 13, 1338 38 of 44 38 of 44 44. Chen, Y.;
Wu, Q.;
Wang, J.;
Song, Y. The fabrication of magnetic recyclable nitrogen-doped
titanium dioxide/calcium ferrite/diatomite heterojunction nanocomposite for improved visible-light-driven
degradation of tetracycline. References J. Chem. Technol. Biotechnol. 2019, 94, 2702–2712. [CrossRef] 45. Chen, Y.; Liu, K. Fabrication of magnetically recyclable Ce/N co-doped TiO2/NiFe2O4/diatomite ternary
hybrid: Improved photocatalytic efficiency under visible light irradiation. J. Alloys Compd. 2017, 697, 161–173. [CrossRef] 46. Wu, Q.; Zhang, Z. The fabrication of magnetic recyclable nitrogen modified titanium dioxide/strontium
ferrite/diatomite heterojunction nanocomposite for enhanced visible-light-driven photodegradation of
tetracycline. Int. J. Hydrogen Energy 2019, 44, 8261–8272. [CrossRef] 47. Nguyen, T.B.; Doong, R.A. Heterostructured ZnFe2O4/TiO2 nanocomposites with a highly recyclable
visible-light-response for bisphenol a degradation. RSC Adv. 2017, 7, 50006–50016. [CrossRef] 48. Shi, Y.; Yang, Z.; Wang, B.; An, H.; Chen, Z.; Cui, H. Adsorption and photocatalytic degradation of tetracycline
hydrochloride using a palygorskite-supported Cu2O-TiO2 composite. Appl. Clay Sci. 2016, 119, 311–320. [CrossRef] 49. Hu, Z.; Wang, X.; Dong, H.; Li, S.; Li, X.; Li, L. Efficient photocatalytic degradation of tetrabromodiphenyl
ethers and simultaneous hydrogen production by TiO2-Cu2O composite films in N2 atmosphere: Influencing
factors, kinetics and mechanism. J. Hazard. Mater. 2017, 340, 1–15. [CrossRef] 50. Sud, D.; Syal, A. Investigations on the Phase Transformation, Optical Characteristics, and Photocatalytic
Activity of Synthesized Heterostructured Nanoporous Bi2O3–TiO2. J. Chin. Chem. Soc. 2016, 63, 776–783. [CrossRef] 51. Sood, S.; Mehta, S.K.; Sinha, A.S.K.; Kansal, S.K. Bi2O3/TiO2 heterostructures: Synthesis, characterization and
their application in solar light mediated photocatalyzed degradation of an antibiotic, ofloxacin. Chem. Eng. J. 2016, 290, 45–52. [CrossRef] 52. Kaur, A.; Umar, A.; Anderson, W.A.; Kansal, S.K. Facile synthesis of CdS/TiO2 nanocomposite and their
catalytic activity for ofloxacin degradation under visible illumination. J. Photochem. Photobiol. A Chem. 2018, 360, 34–43. [CrossRef] 53. Kuši´c, H.; Leszczynska, D. Altered toxicity of organic pollutants in water originated from simultaneous
exposure to UV photolysis and CdSe/ZnS quantum dots. Chemosphere 2012, 89, 900–906. [CrossRef] 54. Ning, X.; Li, J.; Yang, B.; Zhen, W.; Li, Z.; Tian, B.; Lu, G. Inhibition of photocorrosion of CdS via assembling
with thin film TiO2 and removing formed oxygen by artificial gill for visible light overall water splitting. Appl. Catal. B Environ. 2017, 212, 129–139. [CrossRef] 55. Pan, Y.X.; Zhou, T.; Han, J.; Hong, J.; Wang, Y.; Zhang, W.; Xu, R. CdS quantum dots and tungsten carbide
supported on anatase-rutile composite TiO2 for highly efficient visible-light-driven photocatalytic H2
evolution from water. Catal. Sci. Technol. 2016, 6, 2206–2213. [CrossRef] 56. Zhu, R.; Yang, R.; Hu, L.; Chen, B. Preparation of Z-Scheme system of CdS-RGO-BiVO4 and its activity for
hydrogen production. Int. J. References Hydrogen Energy 2019, 44, 25119–25128. [CrossRef] 57. Kandi, D.; Behera, A.; Martha, S.; Naik, B.; Parida, K.M. Quantum confinement chemistry of CdS QDs plus
hot electron of Au over TiO2 nanowire protruding to be encouraging photocatalyst towards nitrophenol
conversion and ciprofloxacin degradation. J. Environ. Chem. Eng. 2019, 7, 102821. [CrossRef] p
g
J
g
58. Li, W.; Ding, H.; Ji, H.; Dai, W.; Guo, J.; Du, G. Photocatalytic degradation of tetracycline hydrochloride via a
CdS-TiO2 heterostructure composite under visible light irradiation. Nanomaterials 2018, 8, 415. [CrossRef] 59. Jiang, Y.; Zhang, M.; Xin, Y.; Chai, C.; Chen, Q. Construction of immobilized CuS/TiO2 nanobelts heterojunction
photocatalyst for photocatalytic degradation of enrofloxacin: Synthesis, characterization, influencing factors
and mechanism insight. J. Chem. Technol. Biotechnol. 2019, 94, 2219–2228. 60. Chen, Q.; Wu, S.; Xin, Y. Synthesis of Au-CuS-TiO2 nanobelts photocatalyst for efficient photocatalytic
degradation of antibiotic oxytetracycline. Chem. Eng. J. 2016, 302, 377–387. [CrossRef] 61. Irandost, M.; Akbarzadeh, R.; Pirsaheb, M.; Asadi, A.; Mohammadi, P.; Sillanpää, M. Fabrication of highly
visible active N, S co-doped TiO2@MoS2 heterojunction with synergistic effect for photocatalytic degradation
of diclofenac: Mechanisms, modeling and degradation pathway. J. Mol. Liq. 2019, 291, 111342. [CrossRef] 62. Wu, L.; Shi, S.; Li, Q.; Zhang, X.; Cui, X. TiO2 nanoparticles modified with 2D MoSe2 for enhanced
photocatalytic activity on hydrogen evolution. Int. J. Hydrogen Energy 2019, 44, 720–728. [CrossRef] 39 of 44 Materials 2020, 13, 1338 39 of 44 63. Du, J.; Wang, H.; Yang, M.; Zhang, F.; Wu, H.; Cheng, X.; Yuan, S.; Zhang, B.; Li, K.; Wang, Y.; et al. Highly
efficient hydrogen evolution catalysis based on MoS2/CdS/TiO2 porous composites. Int. J. Hydrogen Energy
2018, 43, 9307–9315. [CrossRef] 64. Kumar, N.; Bhadwal, A.S.; Mizaikoff, B.; Singh, S.; Kranz, C. Electrochemical detection and photocatalytic
performance of MoS2/TiO2 nanocomposite against pharmaceutical contaminant: Paracetamol. Sens. Bio-Sens. Res. 2019, 24, 100288. [CrossRef] 65. Deng, L.; Liu, H.; Gao, X.; Su, X.; Zhu, Z. SnS2/TiO2 nanocomposites with enhanced visible light-driven
photoreduction of aqueous Cr(VI). Ceram. Int. 2016, 42, 3808–3815. [CrossRef] 66. Kovacic, M.; Kopcic, N.; Kusic, H.; Bozic, A.L. Solar driven degradation of 17β-estradiol using composite
photocatalytic materials and artificial irradiation source: Influence of process and water matrix parameters. J. Photochem. Photobiol. A Chem. 2018, 361, 48–61. [CrossRef] 67. Kovacic, M.; Papac, J.; Kusic, H.; Karamanis, P.; Loncaric Bozic, A. Degradation of polar and non-polar
pharmaceutical pollutants in water by solar assisted photocatalysis using hydrothermal TiO2-SnS2. Chem. Eng. References J. 2019, 382, 122826. [CrossRef] 68. Yan, T.; Guan, W.; Li, W.; You, J. Ag3PO4 photocatalysts loaded on uniform SiO2 supports for efficient
degradation of methyl orange under visible light irradiation. RSC Adv. 2014, 4, 37095–37099. [CrossRef]
69. Yi, Z.; Ye, J.; Kikugawa, N.; Kako, T.; Ouyang, S.; Stuart-Williams, H.; Yang, H.; Cao, J.; Luo, W.; Li, Z.; 68. Yan, T.; Guan, W.; Li, W.; You, J. Ag3PO4 photocatalysts loaded on uniform SiO2 supports for efficient
degradation of methyl orange under visible light irradiation. RSC Adv. 2014, 4, 37095–37099. [CrossRef] 69. Yi, Z.; Ye, J.; Kikugawa, N.; Kako, T.; Ouyang, S.; Stuart-Williams, H.; Yang, H.; Cao, J.; Luo, W.; Li, Z.;
et al. An orthophosphate semiconductor with photooxidation properties under visible-light irradiation. Nat. Mater. 2010, 9, 559–564. [CrossRef] 70. Ren, J.; Chai, Y.; Liu, Q.; Zhang, L.; Dai, W.L. Intercorrelated Ag3PO4 nanoparticles decorated with graphic
carbon nitride: Enhanced stability and photocatalytic activities for water treatment. Appl. Surf. Sci. 2017, 403, 177–186. [CrossRef] 71. Meng, S.; Ning, X.; Zhang, T.; Chen, S.F.; Fu, X. What is the transfer mechanism of photogenerated carriers
for the nanocomposite photocatalyst Ag3PO4/g-C3N4, band-band transfer or a direct Z-scheme? Phys. Chem. Chem. Phys. 2015, 17, 11577–11585. [CrossRef] 72. Wang, Y.; Yu, H.; Lu, Y.; Qu, J.; Zhu, S.; Huo, M. A nano-composite comprised of Ti3+ -doped TiO2 nanotubes and
Ag3PO4 quantum dots with enhanced photocatalytic activity under visible light. Mater. Lett. 2019, 240, 35–38. [CrossRef] 73. Du, J.; Ma, S.; Yan, Y.; Li, K.; Zhao, F.; Zhou, J. Corn-silk-templated synthesis of TiO2 nanotube arrays with
Ag3PO4 nanoparticles for efficient oxidation of organic pollutants and pathogenic bacteria under solar light. Colloids Surf. A Physicochem. Eng. Asp. 2019, 572, 237–249. [CrossRef] 74. Liu, J.; Xie, F.; Li, R.; Li, T.; Jia, Z.; Wang, Y.; Wang, Y.; Zhang, X.; Fan, C. TiO2-x /Ag3PO4 photocatalyst: Oxygen
vacancy dependent visible light photocatalytic performance and BPA degradative pathway. Mater. Sci. Semicond. Process. 2019, 97, 1–10. [CrossRef] 75. Police, A.K.R.; Chennaiahgari, M.; Boddula, R.; Vattikuti, S.V.P.; Mandari, K.K.; Chan, B. Single-step
hydrothermal synthesis of wrinkled graphene wrapped TiO2 nanotubes for photocatalytic hydrogen
production and supercapacitor applications. Mater. Res. Bull. 2018, 98, 314–321. [CrossRef] 76. Lu, Y.; Ma, B.; Yang, Y.; Huang, E.; Ge, Z.; Zhang, T.; Zhang, S.; Li, L.; Guan, N.; Ma, Y.; et al. High activity of
hot electrons from bulk 3D graphene materials for efficient photocatalytic hydrogen production. Nano Res. References 2017, 10, 1662–1672. [CrossRef] 77. Hao, X.; Li, M.; Zhang, L.; Wang, K.; Liu, C. Photocatalyst TiO2/WO3/GO nano-composite with high efficient
photocatalytic performance for BPA degradation under visible light and solar light illumination. J. Ind. Eng. Chem. 2017, 55, 140–148. [CrossRef] 78. Wang, G.; Chen, Q.; Xin, Y.; Liu, Y.; Zang, Z.; Hu, C.; Zhang, B. Construction of graphene-WO3/TiO2 nanotube
array photoelectrodes and its enhanced performance for photocatalytic degradation of dimethyl phthalate. Electrochim. Acta 2016, 222, 1903–1913. [CrossRef] 79. Bilgin Simsek, E.; Kilic, B.; Asgin, M.; Akan, A. Graphene oxide based heterojunction TiO2–ZnO catalysts
with outstanding photocatalytic performance for bisphenol-A, ibuprofen and flurbiprofen. J. Ind. Eng. Chem. 2018, 59, 115–126. [CrossRef] 80. Feng, J.; Wang, Y.; Hou, Y.; Li, L. Hierarchical structured ZnFe2O4@RGO@TiO2 composite as powerful visible
light catalyst for degradation of fulvic acid. J. Nanopart. Res. 2017, 19, 178. [CrossRef] Materials 2020, 13, 1338 40 of 44 40 of 44 81. Luo, L.j.; Li, J.; Dai, J.; Xia, L.; Barrow, C.J.; Wang, H.; Jegatheesan, J.; Yang, M. Bisphenol A removal on
TiO2–MoS2–reduced graphene oxide composite by adsorption and photocatalysis. Process Saf. Environ. Prot. 2017, 112, 274–279. [CrossRef] 82. Wang, W.; Han, Q.; Zhu, Z.; Zhang, L.; Zhong, S.; Liu, B. Enhanced photocatalytic degradation performance
of organic contaminants by heterojunction photocatalyst BiVO4/TiO2/RGO and its compatibility on four
different tetracycline antibiotics. Adv. Powder Technol. 2019, 30, 1882–1896. [CrossRef] 83. Alcudia-Ramos, M.A.; Fuentez-Torres, M.O.; Ortiz-Chi, F.; Espinosa-González, C.G.; Hernández-Como, N.;
García-Zaleta, D.S.; Kesarla, M.K.; Torres-Torres, J.G.; Collins-Martínez, V.; Godavarthi, S. Fabrication of
g-C3N4/TiO2 heterojunction composite for enhanced photocatalytic hydrogen production. Ceram. Int. 2020, 46, 38–45. [CrossRef] 84. Liu, C.; Wu, P.; Wu, J.; Hou, J.; Bai, H.; Liu, Z. Effective protect of oxygen vacancies in carbon layer coated
black TiO2-x/CNNS hetero-junction photocatalyst. Chem. Eng. J. 2019, 359, 58–68. [CrossRef] 85. Yang, Y.; Li, X.; Lu, C.; Huang, W. G-C3N4 Nanosheets Coupled with TiO2 Nanosheets as 2D/2D Heterojunction
Photocatalysts Toward High Photocatalytic Activity for Hydrogen Production. Catal. Lett. 2019, 140, 2930–2939. [CrossRef] 86. Kong, L.; Zhang, X.; Wang, C.; Xu, J.; Du, X.; Li, L. Ti3+ defect mediated g-C3N4 /TiO2 Z-scheme system for
enhanced photocatalytic redox performance. Appl. Surf. Sci. 2018, 448, 288–296. [CrossRef] 87. Wang, J.; Wang, G.; Wang, X.; Wu, Y.; Su, Y.; Tang, H. 3D/2D direct Z-scheme heterojunctions of hierarchical
TiO2 microflowers/g-C3N4 nanosheets with enhanced charge carrier separation for photocatalytic H2
evolution. Carbon 2019, 149, 618–626. [CrossRef] 88. References Pan, C.; Jia, J.; Hu, X.; Fan, J.; Liu, E. In situ construction of g-C3N4 /TiO2 heterojunction films with enhanced
photocatalytic activity over magnetic-driven rotating frame. Appl. Surf. Sci. 2018, 430, 283–292. [CrossRef] 89. Zou, Y.; Shi, J.W.; Ma, D.; Fan, Z.; Lu, L.; Niu, C. In situ synthesis of C-doped TiO2@g-C3N4 core-shell
hollow nanospheres with enhanced visible-light photocatalytic activity for H2 evolution. Chem. Eng. J. 2017, 322, 435–444. [CrossRef] 90. Ma, J.; Zhou, W.; Tan, X.; Yu, T. Potassium ions intercalated into g-C3N4-modified TiO2 nanobelts for the
enhancement of photocatalytic hydrogen evolution activity under visible-light irradiation. Nanotechnology
2018, 29, 215706. [CrossRef] 91. Pan, J.; You, M.; Chi, C.; Dong, Z.; Wang, B.; Zhu, M.; Zhao, W.; Song, C.; Zheng, Y.; Li, C. The two
dimension carbon quantum dots modified porous g-C3N4/TiO2 nano-heterojunctions for visible light
hydrogen production enhancement. Int. J. Hydrogen Energy 2018, 43, 6586–6593. [CrossRef] 92. Wang, X.; Maeda, K.; Thomas, A.; Takanabe, K.; Xin, G.; Carlsson, J.M.; Domen, K.; Antonietti, M. A metal-free
polymeric photocatalyst for hydrogen production from water under visible light. Nat. Mater. 2009, 8, 76–80. [CrossRef] 93. Corredor, J.; Rivero, M.J.; Rangel, C.M.; Gloaguen, F.; Ortiz, I. Comprehensive review and future perspectives
on the photocatalytic hydrogen production. J. Chem. Technol. Biotechnol. 2019, 94, 3049–3063. [CrossRef] 93. Corredor, J.; Rivero, M.J.; Rangel, C.M.; Gloaguen, F.; Ortiz, I. Comprehensive review and future perspectives
on the photocatalytic hydrogen production. J. Chem. Technol. Biotechnol. 2019, 94, 3049–3063. [CrossRef]
94. Wen, J.; Xie, J.; Chen, X.; Li, X. A review on g-C3N4 -based photocatalysts. Appl. Surf. Sci. 2017, 391, 72–123. [CrossRef] 94. Wen, J.; Xie, J.; Chen, X.; Li, X. A review on g-C3N4 -based photocatalysts. Appl. Surf. Sci. 2017, 391, 72–123. [CrossRef] 95. Huang, H.; He, M.; Yang, X.; Tian, Z.; Hu, J.; Wen, B. One-pot hydrothermal synthesis of TiO2/RCN
heterojunction photocatalyst for production of hydrogen and rhodamine B degradation. Appl. Surf. Sci. 2019, 493, 202–211. [CrossRef] 96. Yang, Z.; Yan, J.; Lian, J.; Xu, H.; She, X.; Li, H. g-C3N4/TiO2 Nanocomposites for Degradation of Ciprofloxacin
under Visible Light Irradiation. ChemistrySelect 2016, 1, 5679–5685. [CrossRef] 97. Li, G.; Nie, X.; Gao, Y.; An, T. Can environmental pharmaceuticals be photocatalytically degraded and
completely mineralized in water using g-C3N4/TiO2 under visible light irradiation?-Implications of persistent
toxic intermediates. Appl. Catal. B Environ. 2016, 180, 726–732. [CrossRef] 98. Yu, S.; Wang, Y.; Sun, F.; Wang, R.; Zhou, Y. References Novel mpg-C3N4/TiO2 nanocomposite photocatalytic membrane
reactor for sulfamethoxazole photodegradation. Chem. Eng. J. 2018, 337, 183–192. [CrossRef] 99. Wang, W.; Fang, J.; Shao, S.; Lai, M.; Lu, C. Compact and uniform TiO2@g-C3N4 core-shell quantum
heterojunction for photocatalytic degradation of tetracycline antibiotics. Appl. Catal. B Environ. 2017, 217, 57–64. [CrossRef] 41 of 44 41 of 44 Materials 2020, 13, 1338 100. Li, C.; Sun, Z.; Zhang, W.; Yu, C.; Zheng, S. Highly efficient g-C3N4/TiO2/kaolinite composite with novel
three-dimensional structure and enhanced visible light responding ability towards ciprofloxacin and S. aureus. Appl. Catal. B Environ. 2018, 220, 272–282. [CrossRef] 101. Chen, Y.; Lu, W.; Shen, H.; Gu, Y.; Xu, T.; Zhu, Z.; Wang, G.; Chen, W. Solar-driven efficient degradation
of emerging contaminants by g-C3N4-shielding polyester fiber/TiO2 composites. Appl. Catal. B Environ. 2019, 258, 117960. [CrossRef] 102. Xie, X.; Chen, C.; Wang, X.; Li, J.; Naraginti, S. Efficient detoxification of triclosan by a S-Ag/TiO2@g-C3N4
hybrid photocatalyst: Process optimization and bio-toxicity assessment. RSC Adv. 2019, 9, 20439–20449. [CrossRef] 103. Song, J.; Wu, X.; Zhang, M.; Liu, C.; Yu, J.; Sun, G.; Si, Y.; Ding, B. Highly flexible, core-shell heterostructured,
and visible-light-driven titania-based nanofibrous membranes for antibiotic removal and E. coil inactivation. Chem. Eng. J. 2020, 379, 122269. [CrossRef] 104. Yang, L.; Bai, X.; Shi, J.; Du, X.; Xu, L.; Jin, P. Quasi-full-visible-light absorption by D35-TiO2/g-C3N4 for
synergistic persulfate activation towards efficient photodegradation of micropollutants. Appl. Catal. B
Environ. 2019, 256, 117759. [CrossRef] 105. Su, Y.; Chen, P.; Wang, F.; Zhang, Q.; Chen, T.; Wang, Y.; Yao, K.; Lv, W.; Liu, G. Decoration of TiO2/g-C3N4
Z-scheme by carbon dots as a novel photocatalyst with improved visible-light photocatalytic performance
for the degradation of enrofloxacin. RSC Adv. 2017, 7, 34096–34103. [CrossRef] 106. Wang, F.; Chen, P.; Feng, Y.; Xie, Z.; Liu, Y.; Su, Y.; Zhang, Q.; Wang, Y.; Yao, K.; Lv, W.; et al. Facile synthesis
of N-doped carbon dots/g-C3N4 photocatalyst with enhanced visible-light photocatalytic activity for the
degradation of indomethacin. Appl. Catal. B Environ. 2017, 207, 103–113. [CrossRef] 107. Nie, Y.C.; Yu, F.; Wang, L.C.; Xing, Q.J.; Liu, X.; Pei, Y.; Zou, J.P.; Dai, W.L.; Li, Y.; Suib, S.L. Photocatalytic
degradation of organic pollutants coupled with simultaneous photocatalytic H2 evolution over graphene
quantum dots/Mn-N-TiO2/g-C3N4 composite catalysts: Performance and mechanism. Appl. Catal. B Environ. 2018, 227, 312–321. [CrossRef] 108. Jo, W.K.; Adinaveen, T.; Vijaya, J.J.; Sagaya Selvam, N.C. References Synthesis of MoS2 nanosheet supported Z-scheme
TiO2/g-C3N4 photocatalysts for the enhanced photocatalytic degradation of organic water pollutants. RSC
Adv. 2016, 6, 10487–10497. 109. Tahir, M.B.; Sagir, M.; Shahzad, K. Removal of acetylsalicylate and methyl-theobromine from aqueous
environment using nano-photocatalyst WO3-TiO2 @g-C3N4 composite. J. Hazard. Mater. 2019, 363, 205–213. [CrossRef] 110. Schubert, J.S.; Popovic, J.; Haselmann, G.M.; Nandan, S.P.; Wang, J.; Giesriegl, A.; Cherevan, A.S.; Eder, D. Immobilization of Co, Mn, Ni and Fe oxide co-catalysts on TiO2 for photocatalytic water splitting reactions. J. Mater. Chem. A 2019, 7, 18568–18579. [CrossRef] 111. Patil, S.B.; Basavarajappa, P.S.; Ganganagappa, N.; Jyothi, M.S.; Raghu, A.V.; Reddy, K.R. Recent advances in
non-metals-doped TiO2 nanostructured photocatalysts for visible-light driven hydrogen production, CO2
reduction and air purification. Int. J. Hydrogen Energy 2019, 44, 13022–13039. [CrossRef] 112. Yang, Q.; Dong, L.; Su, R.; Hu, B.; Wang, Z.; Jin, Y.; Wang, Y.; Besenbacher, F.; Dong, M. Nanostructured
heterogeneous photo-catalysts for hydrogen production and water splitting: A comprehensive insight. Appl. Mater. Today 2019, 17, 159–182. [CrossRef] 113. Bhagya, T.C.; Krishnan, A.; Arunima Rajan, S.; Ameen Sha, M.; Sreelekshmy, B.R.; Jineesh, P.; Shibli, S.M.A. Exploration and evaluation of proton source-assisted photocatalyst for hydrogen generation. Photochem. Photobiol. Sci. 2019, 18, 1716–1726. [CrossRef] 114. Hernández-Majalca,
B.C.;
Meléndez-Zaragoza,
M.J.;
Salinas-Gutiérrez,
J.M.;
López-Ortiz,
A.;
Collins-Martínez, V. Visible-light photo-assisted synthesis of GO-TiO2 composites for the photocatalytic
hydrogen production. Int. J. Hydrogen Energy 2019, 44, 12381–12389. [CrossRef] 115. Wang, D.; Saleh, N.B.; Sun, W.; Park, C.M.; Shen, C.; Aich, N.; Peijnenburg, W.J.G.M.; Zhang, W.; Jin, Y.; Su, C. Next-Generation Multifunctional Carbon–Metal Nanohybrids for Energy and Environmental Applications. Environ. Sci. Technol. 2019, 53, 7265–7287. [CrossRef] [PubMed] 116. Xu, X.; Ray, R.; Gu, Y.; Ploehn, H.J.; Gearheart, L.; Raker, K.; Scrivens, W.A. Electrophoretic analysis and
purification of fluorescent single-walled carbon nanotube fragments. J. Am. Chem. Soc. 2004, 126, 12736–12737. [CrossRef] [PubMed] Materials 2020, 13, 1338 42 of 44 42 of 44 117. Rao, V.N.; Reddy, N.L.; Kumari, M.M.; Cheralathan, K.K.; Ravi, P.; Sathish, M.; Neppolian, B.; Reddy, K.R.;
Shetti, N.P.; Prathap, P.; et al. Sustainable hydrogen production for the greener environment by quantum
dots-based efficient photocatalysts: A review. J. Environ. Manag. 2019, 248, 109246. [CrossRef] 118. Yi, L.; Lan, F.; Li, J.; Zhao, C. Efficient Noble-Metal-Free Co-NG/TiO2 Photocatalyst for H2 Evolution:
Synergistic Effect between Single-Atom Co and N-Doped Graphene for Enhanced Photocatalytic Activity. ACS Sustain. Chem. Eng. 2018, 6, 12766–12775. [CrossRef] 119. References Rabin, N.N.; Ohmagari, H.; Islam, M.S.; Karim, M.R.; Hayami, S. A procession on photocatalyst for solar fuel
production and waste treatment. J. Incl. Phenom. Macrocycl. Chem. 2019, 94, 263–281. [CrossRef] 120. Lv, T.; Wang, H.; Hong, W.; Wang, P.; Jia, L. In situ self-assembly synthesis of sandwich-like TiO2/reduced
graphene oxide/LaFeO3 Z-scheme ternary heterostructure towards enhanced photocatalytic hydrogen
production. Mol. Catal. 2019, 475, 110497. [CrossRef] 121. Iwase, A.; Ng, Y.H.; Ishiguro, Y.; Kudo, A.; Amal, R. Reduced Graphene Oxide as a Solid-State Electron Mediator
in Z-Scheme Photocatalytic Water Splitting under Visible Light. J. Am. Chem. Soc. 2011, 133, 11054–11057. [CrossRef] 122. Samal, A.; Das, D.P. Transfiguring UV light active “metal oxides” to visible light active photocatayst by
reduced graphene oxide hypostatization. Catal. Today 2018, 300, 124–135. [CrossRef] 123. Ida, S.; Wilson, P.; Neppolian, B.; Sathish, M.; Karthik, P.; Ravi, P. Ultrasonically aided selective stabilization
of pyrrolic type nitrogen by one pot nitrogen doped and hydrothermally reduced Graphene oxide/Titania
nanocomposite (N-TiO2/N-RGO) for H2 production. Ultrason. Sonochem. 2019, 57, 62–72. [CrossRef] 124. Olowoyo, J.O.; Kumar, M.; Jain, S.L.; Babalola, J.O.; Vorontsov, A.V.; Kumar, U. Insights into Reinforced
Photocatalytic Activity of the CNT-TiO2 Nanocomposite for CO2 Reduction and Water Splitting. J. Phys. Chem. C 2019, 123, 367–378. [CrossRef] 125. Umer, M.; Tahir, M.; Azam, M.U.; Tahir, B.; Jaffar, M.M.; Alias, H. Montmorillonite dispersed single wall
carbon nanotubes (SWCNTs)/TiO2 heterojunction composite for enhanced dynamic photocatalytic H2
production under visible light. Appl. Clay Sci. 2019, 174, 110–119. [CrossRef] 126. Bellamkonda, S.; Thangavel, N.; Hafeez, H.Y.; Neppolian, B.; Ranga Rao, G. Highly active and stable
multi-walled carbon nanotubes-graphene-TiO2 nanohybrid: An efficient non-noble metal photocatalyst for
water splitting. Catal. Today 2019, 321, 120–127. [CrossRef] p
g
y
127. Haque, F.; Daeneke, T.; Kalantar-zadeh, K.; Ou, J.Z. Two-Dimensional Transition Metal Oxide and
Chalcogenide-Based Photocatalysts. Nano-Micro Lett. 2018, 10, 23. [CrossRef] [PubMed] 128. Camposeco, R.;
Castillo, S.;
Rodriguez-González, V.;
Hinojosa-Reyes, M.;
Medina-Álvares, M.I.;
Mejía-Centeno, I. Promotional effect of Rh nanoparticles on WO3/TiO2 titanate nanotube photocatalysts for
boosted hydrogen production. J. Photochem. Photobiol. A Chem. 2018, 353, 114–121. [CrossRef] 129. Ren, X.; Gao, P.; Kong, X.; Jiang, R.; Yang, P.; Chen, Y.; Chi, Q.; Li, B. NiO/Ni/TiO2 nanocables with Schottky/p-n
heterojunctions and the improved photocatalytic performance in water splitting under visible light. J. Colloid
Interface Sci. 2018, 530, 1–8. [CrossRef] [PubMed] 130. Madhumitha, A.; Preethi, V.; Kanmani, S. Photocatalytic hydrogen production using TiO2 coated iron-oxide
core shell particles. Int. J. 136. Wei, T.; Zhu, Y.-N.; An, X.; Liu, L.-M.; Cao, X.; Liu, H.; Qu, J. Defect Modulation of Z-Scheme TiO2 /Cu2O
Photocatalysts for Durable Water Splitting. ACS Catal. 2019, 9, 8346–8354. [CrossRef] References Hydrogen Energy 2018, 43, 3946–3956. [CrossRef] 131. Xie, M.Y.; Su, K.Y.; Peng, X.Y.; Wu, R.J.; Chavali, M.; Chang, W.C. Hydrogen production by photocatalytic
water-splitting on Pt-doped TiO2–ZnO under visible light. J. Taiwan Inst. Chem. Eng. 2017, 70, 161–167. [CrossRef] 132. Chen, Q.; Tong, R.; Chen, X.; Xue, Y.; Xie, Z.; Kuang, Q.; Zheng, L. Ultrafine ZnO quantum dot-modified TiO2
composite photocatalysts: The role of the quantum size effect in heterojunction-enhanced photocatalytic
hydrogen evolution. Catal. Sci. Technol. 2018, 8, 1296–1303. [CrossRef] y
g
133. Liu, J.; Ke, J.; Li, Y.; Liu, B.; Wang, L.; Xiao, H.; Wang, S. Co3O4 quantum dots/TiO2 nanobelt hybrids for
highly efficient photocatalytic overall water splitting. Appl. Catal. B Environ. 2018, 236, 396–403. [CrossRef]
134. Zhang, Q.; Hai, Z.; Jian, A.; Xu, H.; Xue, C.; Sang, S. Synthesis of p-Co3O4/n-TiO2 nanoparticles for overall 133. Liu, J.; Ke, J.; Li, Y.; Liu, B.; Wang, L.; Xiao, H.; Wang, S. Co3O4 quantum dots/TiO2 nanobelt hybrids for
highly efficient photocatalytic overall water splitting. Appl. Catal. B Environ. 2018, 236, 396–403. [CrossRef] 133. Liu, J.; Ke, J.; Li, Y.; Liu, B.; Wang, L.; Xiao, H.; Wang, S. Co3O4 quantum dots/TiO2 nanobelt hybrids for
highly efficient photocatalytic overall water splitting. Appl. Catal. B Environ. 2018, 236, 396–403. [CrossRef]
134. Zhang, Q.; Hai, Z.; Jian, A.; Xu, H.; Xue, C.; Sang, S. Synthesis of p-Co3O4/n-TiO2 nanoparticles for overall
water splitting under visible light irradiation. Nanomaterials 2016, 6, 138. [CrossRef] 134. Zhang, Q.; Hai, Z.; Jian, A.; Xu, H.; Xue, C.; Sang, S. Synthesis of p-Co3O4/n-TiO2 nanoparticles for overall
water splitting under visible light irradiation. Nanomaterials 2016, 6, 138. [CrossRef] 135. Wang, L.; Zhang, X.; Gao, H.; Hu, J.; Mao, J.; Liang, C.; Zhang, P.; Shao, G. 3D CuO network supported TiO2
nanosheets with applications for energy storage and water splitting. Sci. Adv. Mater. 2016, 8, 1256–1262. [CrossRef] 136. Wei, T.; Zhu, Y.-N.; An, X.; Liu, L.-M.; Cao, X.; Liu, H.; Qu, J. Defect Modulation of Z-Scheme TiO2 /Cu2O
Photocatalysts for Durable Water Splitting. ACS Catal. 2019, 9, 8346–8354. [CrossRef] 43 of 44 43 of 44 Materials 2020, 13, 1338 137. Rao, V.N.; Reddy, N.L.; Kumari, M.M.; Ravi, P.; Sathish, M.; Neppolian, B.; Shankar, M.V. Synthesis of
titania wrapped cadmium sulfide nanorods for photocatalytic hydrogen generation. Mater. Res. Bull. 2018, 103, 122–132. [CrossRef] 138. References Du, J.; Wang, H.; Yang, M.; Li, K.; Zhao, L.; Zhao, G.; Li, S.; Gu, X.; Zhou, Y.; Wang, L.; et al. Pyramid-like CdS
nanoparticles grown on porous TiO2 monolith: An advanced photocatalyst for H2 production. Electrochim. Acta 2017, 250, 99–107. [CrossRef] 139. Peng, R.; Wu, C.M.; Baltrusaitis, J.; Dimitrijevic, N.M.; Rajh, T.; Koodali, R.T. Solar hydrogen generation over
CdS incorporated in Ti-MCM-48 mesoporous materials under visible light illumination. Int. J. Hydrogen
Energy 2016, 41, 4106–4119. [CrossRef] 140. Zhao, D.; Yang, C.F. Recent advances in the TiO2/CdS nanocomposite used for photocatalytic hydrogen
production and quantum-dot-sensitized solar cells. Renew. Sustain. Energy Rev. 2016, 54, 1048–1059. [CrossRef] 141. Qi, L.; Yu, J.; Jaroniec, M. Preparation and enhanced visible-light photocatalytic H2- production activity of
CdS-sensitized Pt/TiO2 nanosheets with exposed (001) facets. Phys. Chem. Chem. Phys. 2011, 13, 8915–8923. [CrossRef] 142. Li, C.; Yuan, J.; Han, B.; Jiang, L.; Shangguan, W. TiO2 nanotubes incorporated with CdS for photocatalytic
hydrogen production from splitting water under visible light irradiation. Int. J. Hydrogen Energy
2010, 35, 7073–7079. [CrossRef] 143. Zhang, Y.J.; Yan, W.; Wu, Y.P.; Wang, Z.H. Synthesis of TiO2 nanotubes coupled with CdS nanoparticles and
production of hydrogen by photocatalytic water decomposition. Mater. Lett. 2008, 62, 3846–3848. [CrossRef] 144. Chandra, M.; Bhunia, K.; Pradhan, D. Controlled Synthesis of CuS/TiO2 Heterostructured Nanocomposites
for Enhanced Photocatalytic Hydrogen Generation through Water Splitting. Inorg. Chem. 2018, 57, 4524–4533. [CrossRef] 145. Dang, H.; Cheng, Z.; Yang, W.; Chen, W.; Huang, W.; Li, B.; Shi, Z.; Qiu, Y.; Dong, X.; Fan, H. Room-temperature
synthesis of CuxS (x = 1 or 2) co-modified TiO2 nanocomposite and its highly efficient photocatalytic H2
production activity. J. Alloys Compd. 2017, 709, 422–430. [CrossRef] 146. Wang, Q.; Huang, J.; Sun, H.; Ng, Y.H.; Zhang, K.Q.; Lai, Y. MoS2 Quantum Dots@TiO2 Nanotube Arrays: An
Extended-Spectrum-Driven Photocatalyst for Solar Hydrogen Evolution. ChemSusChem 2018, 11, 1708–1721. [CrossRef] [PubMed] 147. Ng, B.J.; Putri, L.K.; Tan, L.L.; Pasbakhsh, P.; Chai, S.P. All-solid-state Z-scheme photocatalyst with carbon
nanotubes as an electron mediator for hydrogen evolution under simulated solar light. Chem. Eng. J. 2017, 316, 41–49. [CrossRef] 148. Yang, C.; Qin, J.; Rajendran, S.; Zhang, X.; Liu, R. WS2 and C-TiO2 Nanorods Acting as Effective Charge
Separators on g-C3N4 to Boost Visible-Light Activated Hydrogen Production from Seawater. ChemSusChem
2018, 11, 4077–4085. [CrossRef] [PubMed] 149. Liu, E.; Xu, C.; Jin, C.; Fan, J.; Hu, X. References Carbon quantum dots bridged TiO2 and Cd0.5Zn0.5S film as solid-state
Z-scheme photocatalyst with enhanced H2 evolution activity. J. Taiwan Inst. Chem. Eng. 2019, 97, 316–325. [CrossRef] 150. Zou, Y.; Shi, J.W.; Ma, D.; Fan, Z.; Niu, C.; Wang, L. Fabrication of g-C3N4/Au/C-TiO2 Hollow Structures as
Visible-Light-Driven Z-Scheme Photocatalysts with Enhanced Photocatalytic H2 Evolution. ChemCatChem
2017, 9, 3752–3761. [CrossRef] 151. Yang, G.; Ding, H.; Chen, D.; Feng, J.; Hao, Q.; Zhu, Y. Construction of urchin-like ZnIn2S4-Au-TiO2
heterostructure with enhanced activity for photocatalytic hydrogen evolution. Appl. Catal. B Environ. 2018, 234, 260–267. [CrossRef] 152. Zhang, L.; Huang, Y.; Dai, C.; Liang, Q.; Yang, P.; Yang, H.; Yan, J. Constructing ZnO/ZnCr2O4@TiO2 -NTA
Nanocomposite for Photovoltaic Conversion and Photocatalytic Hydrogen Evolution. J. Electron. Mater. 2019, 48, 1724–1729. [CrossRef] 153. Ke, X.; Zhang, J.; Dai, K.; Liang, C. Construction of flourinated-TiO2 nanosheets with exposed {001}
facets/CdSe-DETA nanojunction for enhancing visible-light-driven photocatalytic H2 evolution. Ceram. Int. 2020, 46, 866–876. [CrossRef] 154. Kim, Y.K.; Lim, S.K.; Park, H.; Hoffmann, M.R.; Kim, S. Trilayer CdS/carbon nanofiber (CNF) mat/Pt-TiO2
composite structures for solar hydrogen production: Effects of CNF mat thickness. Appl. Catal. B Environ. 2016, 196, 216–222. [CrossRef] Materials 2020, 13, 1338 44 of 44 44 of 44 155. Dai, K.; Lv, J.; Zhang, J.; Zhu, G.; Geng, L.; Liang, C. Efficient Visible-Light-Driven Splitting of Water
into Hydrogen over Surface-Fluorinated Anatase TiO2 Nanosheets with Exposed {001} Facets/Layered
CdS-Diethylenetriamine Nanobelts. ACS Sustain. Chem. Eng. 2018, 6, 12817–12826. [CrossRef] 156. Yuan, W.; Zhang, Z.; Cui, X.; Liu, H.; Tai, C.; Song, Y. Fabrication of Hollow Mesoporous CdS@TiO2@Au
Microspheres with High Photocatalytic Activity for Hydrogen Evolution from Water under Visible Light. ACS Sustain. Chem. Eng. 2018, 6, 13766–13777. [CrossRef] 157. Zhao, H.; Wu, M.; Liu, J.; Deng, Z.; Li, Y.; Su, B.L. Synergistic promotion of solar-driven H2 generation by
three-dimensionally ordered macroporous structured TiO2-Au-CdS ternary photocatalyst. Appl. Catal. B
Environ. 2016, 184, 182–190. [CrossRef] 158. Wu, M.; Zhang, J.; Liu, C.; Gong, Y.; Wang, R.; He, B.; Wang, H. Rational Design and Fabrication
of Noble-metal-free NixP Cocatalyst Embedded 3D N-TiO2/g-C3N4 Heterojunctions with Enhanced
Photocatalytic Hydrogen Evolution. ChemCatChem 2018, 10, 3069–3077. [CrossRef] 159. Sampath, S.; Sellappa, K. Visible-light-driven photocatalysts for hydrogen production by water splitting. Energy Sources Part A Recover. Util. Environ. Eff. 2020, 42, 719–729. [CrossRef] 160. Azam, M.U.; Tahir, M.; Umer, M.; Tahir, B.; Shehzad, N.; Siraj, M. References In-situ synthesis of TiO2/La2O2CO3/rGO
composite under acidic/basic treatment with La3+/Ti3+ as mediators for boosting photocatalytic H2 evolution. Int. J. Hydrogen Energy. 2019, 44, 23669–23688. [CrossRef] 161. Zhu, J.; Zhang, M.; Xiong, J.; Yan, Y.; Li, W.; Cheng, G. Electrostatically assembled construction of ternary
TiO2-Cu@C hybrid with enhanced solar-to-hydrogen evolution employing amorphous carbon dots as
electronic mediator. Chem. Eng. J. 2019, 375, 121902. [CrossRef] © 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/). © 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/). © 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/).
|
https://openalex.org/W2963196827
|
https://projecteuclid.org/journals/electronic-communications-in-probability/volume-23/issue-none/Fluctuations-for-block-spin-Ising-models/10.1214/18-ECP161.pdf
|
English
| null |
Fluctuations for block spin Ising models
|
Electronic communications in probability
| 2,018
|
cc-by
| 8,777
|
Electron. Commun. Probab. 23 (2018), no. 53, 1–12.
https://doi.org/10.1214/18-ECP161
ISSN: 1083-589X Electron. Commun. Probab. 23 (2018), no. 53, 1–12. https://doi.org/10.1214/18-ECP161
ISSN: 1083-589X ELECTRONIC
COMMUNICATIONS
in PROBABILITY y
,
y
@
†University of Münster, Germany. E-mail: kristina.schubert@wwu.de Abstract We analyze the high temperature fluctuations of the magnetization of the so-called
Ising block model. This model was recently introduced by Berthet et al. in [2]. We
prove a Central Limit Theorems (CLT) for the magnetization in the high temperature
regime. At the same time we show that this CLT breaks down at a line of critical
temperatures. At this line we prove a non-standard CLT for the magnetization. Keywords: Ising model; Curie-Weiss model; fluctuations; Central Limit Theorem; block model. AMS MSC 2010: 60F05; 82B05; 60G09. Submitted to ECP on June 22, 2018, final version accepted on August 6, 2018. Fluctuations for block spin Ising models Matthias Löwe*
Kristina Schubert† Matthias Löwe* *University of Münster, Germany. E-mail: maloewe@math.uni-muenster.de
†University of Münster, Germany. E-mail: kristina.schubert@wwu.de versity of Münster, Germany. E-mail: maloewe@math.uni-m • If β + α ≤2, then ρN,α,β weakly converges to the Dirac measure in (0, 0). • If β + α > 2 and α = 0 then ρN,α,β weakly converges to the mixture of Dirac
measures 1
4
P
s1,s2∈{−,+} δ(s1m+(β/2),s2m+(β/2)). • If β + α > 2 and α = 0 then ρN,α,β weakly converges to the mixture of Dirac
measures 1
4
P
s1,s2∈{−,+} δ(s1m+(β/2),s2m+(β/2)). 4
P
s1,s2∈{
,+}
( 1
(β/ ), 2
(β/ ))
• If β + α > 2 and α > 0 then ρN,α,β weakly converges to the mixture of Dirac
measures 1
2(δ(m+( α+β
2
),m+( α+β
2
)) + δ(−m+( α+β
2
),−m+( α+β
2
)). 4
1, 2∈{
,+}
(
( / )
( / ))
• If β + α > 2 and α > 0 then ρN,α,β weakly converges to the mixture of Dirac
measures 1
2(δ(m+( α+β
2
),m+( α+β
2
)) + δ(−m+( α+β
2
),−m+( α+β
2
)). {
}
• If β + α > 2 and α > 0 then ρN,α,β weakly converges to the mixture of Dirac
measures 1
2(δ(m+( α+β
2
),m+( α+β
2
)) + δ(−m+( α+β
2
),−m+( α+β
2
)). Theorem 1.1 is the trigger for another question: In the Curie-Weiss model the phase
transition can also be observed on the level of fluctuations of the magnetization: As is
shown in [13], [12] or in [11, Theorems V.9.4 and V.9.5] or [10], for β < 1 the parameter
√
Nm obeys a standard CLT with expectation 0 and variance
1
1−β , while for β = 1 one
has to scale differently: Then N 1/4m converges in distribution to a random variable that
has Lebesgue density proportional to exp(−1
12x4). Our question in this note is, whether
a similar behaviour can be observed for the block spin Ising model and how the limit
distribution depends on the relation between α and β. To answer this question we show Theorem 1.2. For the block spin Ising model assume that 0 ≤α < β and that β + α < 2 . Then,
√
Nm converges in distribution to a 2-dimensional Gaussian random variable with
expectation 0 and covariance matrix Σ = s2
1
r
r
1
with s2 =
8−4β
(2−β)2−α2 and r =
α
2−β . expectation 0 and covariance matrix Σ = s2
1
r
r
1
with s2 =
8−4β
(2−β)2−α2 and r =
α
2−β . Fluctuations for block spin Ising models Indeed, the Hamiltonian is handily rewritten as Indeed, the Hamiltonian is handily rewritten as HN,α,β,S(σ) = −N
2
1
2αm1m2 + β 1
4m2
1 + 1
4βm2
2
. This observation is not only a convenient way to analyze the block spin Ising model, it
also makes m an obvious choice to describe its behaviour and its phase transitions. To
characterize them, recall that with the above notation for α = β one reobtains the Curie-
{
}N This observation is not only a convenient way to analyze the block spin Ising model, it
also makes m an obvious choice to describe its behaviour and its phase transitions. To
characterize them, recall that with the above notation for α = β one reobtains the Curie-
Weiss or mean-field Ising model at inverse temperature β, i.e. the model on {−1, +1}N
given by HCW (σ) =
1
2N
P
i,j σiσj and Gibbs measure µCW
N,β (σ) = e−βHCW (σ)
ZCW
N,β
. Also, recall
([11]) that the Curie-Weiss model undergoes a phase transition at β = 1. This phase
transition can be described by saying that the distribution of the parameter m = 1
N
P
i σi
(also called the magnetization) weakly converges to the Dirac measure in 0, δ0, if β ≤1
while it converges to the mixture 1
2(δm+(β) +δ−m+(β)), if β > 1. Here m+(β) is the largest
solution of the so-called Curie-Weiss equation z = tanh(βz). A similar result was proven
for the block spin Ising model in [2] (the authors also allow for negative values of α). There the authors (implicitly) show This observation is not only a convenient way to analyze the block spin Ising model, it
also makes m an obvious choice to describe its behaviour and its phase transitions. To
characterize them, recall that with the above notation for α = β one reobtains the Curie-
Weiss or mean-field Ising model at inverse temperature β, i.e. the model on {−1, +1}N
given by HCW (σ) =
1
2N
P
i,j σiσj and Gibbs measure µCW
N,β (σ) = e−βHCW (σ)
ZCW
N,β
. Fluctuations for block spin Ising models Also, recall
([11]) th t th
C
i
W i
d l
d
h
t
iti
t β
1
Thi
h This observation is not only a convenient way to analyze the block spin Ising model, it
also makes m an obvious choice to describe its behaviour and its phase transitions. To
characterize them, recall that with the above notation for α = β one reobtains the Curie-
Weiss or mean-field Ising model at inverse temperature β, i.e. the model on {−1, +1}N
given by HCW (σ) =
1
2N
P
i,j σiσj and Gibbs measure µCW
N,β (σ) = e−βHCW (σ)
ZCW
N,β
. Also, recall
([11]) that the Curie-Weiss model undergoes a phase transition at β = 1. This phase
transition can be described by saying that the distribution of the parameter m = 1
N
P
i σi
(also called the magnetization) weakly converges to the Dirac measure in 0, δ0, if β ≤1
while it converges to the mixture 1
2(δm+(β) +δ−m+(β)), if β > 1. Here m+(β) is the largest
solution of the so-called Curie-Weiss equation z = tanh(βz). A similar result was proven
for the block spin Ising model in [2] (the authors also allow for negative values of α). There the authors (implicitly) show N,β
([11]) that the Curie-Weiss model undergoes a phase transition at β = 1. This phase
transition can be described by saying that the distribution of the parameter m = 1
N
P
i σi
(also called the magnetization) weakly converges to the Dirac measure in 0, δ0, if β ≤1
while it converges to the mixture 1
2(δm+(β) +δ−m+(β)), if β > 1. Here m+(β) is the largest
solution of the so-called Curie-Weiss equation z = tanh(βz). A similar result was proven
for the block spin Ising model in [2] (the authors also allow for negative values of α). There the authors (implicitly) show Theorem 1.1. cf. [2, Proposition 1] In the above assume that 0 ≤α < β and denote by
ρN,α,β the distribution of m under the Gibbs measure µN,α,β. Then • If β + α ≤2, then ρN,α,β weakly converges to the Dirac measure in (0, 0). 1
Introduction In a recent paper Berthet, Rigollet and Srivastavaz studied a block version of the
Curie-Weiss-Ising model [2]. This model is inspired by extensive studies of block models
in the recent past, see e.g. [1], [4], [5], [17], [22]. On the other hand, similar models were
considered a bit earlier in the statistical mechanics literature, see [6], [7], [14], [15], [16]. To define our model, we partition the set {1, . . . , N} for N even into a set S ⊂{1, . . . , N}
with |S| = N
2 and its complement Sc. This segmentation induces a partitioning of the
binary hypercube {−1, +1}N, N ∈N, the state space of the Ising block model. For β > 0
and 0 ≤α ≤β the model we will consider is defined by the Hamiltonian HN,α,β,S(σ) := −β
2N
X
i∼j
σiσj −α
2N
X
i̸∼j
σiσj,
σ ∈{−1, +1}N. Here we write i ∼j, if either i, j ∈S or i, j ∈Sc and i ̸∼j otherwise. This Hamiltonian
induces a Gibbs measure µN,α,β(σ) = µN,α,β,S(σ) :=
e−HN,α,β(σ)
P
σ′ e−HN,α,β(σ′) =: e−HN,α,β(σ)
ZN,α,β
. (1.1) (1.1) A closely related version of this model has been investigated in [19]. However, the
couplings in [19] between the blocks have the same strength of interaction as the
couplings within a block. We were informed that a more general version of the model
will be studied in [20]. Similar to the Curie-Weiss model the Ising block model has an
order parameter: the vector of block magnetizations, m := mN := (mN
1 , mN
2 ), where A closely related version of this model has been investigated in [19]. However, the
couplings in [19] between the blocks have the same strength of interaction as the
couplings within a block. We were informed that a more general version of the model
will be studied in [20]. Similar to the Curie-Weiss model the Ising block model has an
order parameter: the vector of block magnetizations, m := mN := (mN
1 , mN
2 ), where m1 := mN
1 := m1(σ) := 2
N
X
i∈S
σi
and
m2 := mN
2 := m2(σ) := 2
N
X
i/∈S
σi. Fluctuations for block spin Ising models Fluctuations for block spin Ising models We will show Theorems 1.2 and 1.4 in Section 3 using a Hubbard-Stratonovich
transformation for an appropriate function of m. Before, in Section 2, however, we will
give an alternative proof of Theorem 1.1 using the theory of large deviations. This is
not only interesting in its own right, but also provides a way to derive limit theorems in
more complicated settings, see e.g [21]. • If β + α ≤2, then ρN,α,β weakly converges to the Dirac measure in (0, 0). Remark 1.3. It is well known that in the standard Curie-Weiss a CLT also holds in
the presence of an external field [12], i.e. with a Hamiltonian of the form HCW (σ) =
1
2N
P
i,j σiσj +h P
i σi with h > 0. We are firmly convinced that a similar result is true for
our model as well. However, we did not try to prove it. Finally, one might ask, whether –
in the spirit of [10] – Stein’s method may be applied to our situation as well. We consider
this a more challenging question, because a multi-dimensional version of Stein’s method
would be needed. We may consider this problem in a different paper. On the other hand, if β + α = 2 the fluctuations are no longer Gaussian Theorem 1.4. For the block spin Ising model assume that 0 ≤α < β and that β + α = 2. Then, N
1
4 m1 converges in distribution to a probability measure ρ on R. The measure
ρ is absolutely continuous with Lebesgue-density g(x) = exp(−1
12x4)/Z, where Z is
a normalizing constant. The difference between m1 and m2 multiplied by
√
N, i.e. ˜m1 −˜m2 :=
√
N(m1 −m2), however, is asymptotically Gaussian with mean 0 and variance
2
2−(β−α). ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ http://www.imstat.org/ecp/ Page 2/12 2
An LDP for the vector of block magnetizations Differing from the line of arguments in [2], Theorem 1.1 can also be shown by proving
a large deviation principle (LDP) for 0 ≤α ≤β. To this end, we will slightly change our
variables and consider the vector v = (v1, v2) with v1 := 1
2m1 and v2 := 1
2m2. We show Theorem 2.1. For every S ⊂{1, . . . , N} with |S| = N
2 the vector v obeys a principle of
large deviations (LDP) under the Gibbs measure µN,α,β := µN,α,β,S, with speed N and
rate function Jv(x) := supy∈R2[Fv(y)−J(y)]−[Fv(x)−J(x)]. Here Fv : R2 →R is defined Fv(x) := 1
2
βx2
1 + βx2
2 + 2αx1x2
(2.1) (2.1) and for x ∈R2 J(x) := sup
t∈R2
⟨t, x⟩−1
2 log cosh(t1) −1
2 log cosh(t2)
. This implies that the convergence in Theorem 1.1 (for 0 ≤α ≤β) is exponentially fast. Proof. We will prove this theorem in two steps, first we show the LDP, then, how one
derives Theorem 1.1 from it. First, note that the case α = 0 is trivial. Then, the system consists of two independent
Curie-Weiss models on N
2 spins at temperature β. The LDP for the magnetization in each
of the systems is known (cf. e.g. [11]) and transferring these LDPs to the vector v (with
independent components) is trivial. We will thus assume that α > 0. Let us consider the moment generating function of the vector v. To this end let
t = (t1, t2) ∈R2. Then the moment generating function of v in t is given by E exp(N⟨t, v⟩) = cosh(t1)
N
2 cosh(t2)
N
2 , where here E denotes the expectation with respect to the a priori measure 1
2(δ−1 + δ+1). This readily yields limN→∞1
N log E exp(N⟨t, v⟩) = 1
2 log cosh(t1) + 1
2 log cosh(t2). As the
right hand side of this expression is finite and differentiable on all of R2, by the Gärtner-
Ellis Theorem [8, Theorem 2.3.6] this computation implies an LDP for v under the
uniform distribution with speed N and rate function where here E denotes the expectation with respect to the a priori measure 1
2(δ−1 + δ+1). This readily yields limN→∞1
N log E exp(N⟨t, v⟩) = 1
2 log cosh(t1) + 1
2 log cosh(t2). 2
An LDP for the vector of block magnetizations As the
right hand side of this expression is finite and differentiable on all of R2, by the Gärtner-
Ellis Theorem [8, Theorem 2.3.6] this computation implies an LDP for v under the
uniform distribution with speed N and rate function J(x) := sup
t∈R2
⟨t, x⟩−1
2 log cosh(t1) −1
2 log cosh(t2)
= 1
2I (2x1) + 1
2I (2x2) for x ∈R2. Here I(x) := 1
2(1 + x) log(1 + x) + 1
2(1 −x) log(1 −x). Now the Hamiltonian
HN,α,β,S(σ) of our model can also be rewritten in terms of v: HN,α,β,S(σ) = −N
2
βv2
1(σ) + βv2
2(σ) + 2αv1(σ)v2(σ)
. This fact, together with the above LDP and the exponential form of the Gibbs measure
and the LDP for integrals of exponential functions (see e.g. [9, Theorem III.17] – a direct This fact, together with the above LDP and the exponential form of the Gibbs measure
and the LDP for integrals of exponential functions (see e.g. [9, Theorem III.17] – a direct ECP 23 (2018), paper 53. Page 3/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ Page 3/12 Fluctuations for block spin Ising models Fluctuations for block spin Ising models Fluctuations for block spin Ising models consequence of Varadhan’s Lemma [8, Theorem 4.3.1] – implies that the distribution of v
under µN,α,β,S satisfies an LDP with speed N and rate function consequence of Varadhan’s Lemma [8, Theorem 4.3.1] – implies that the distribution of v
under µN,α,β,S satisfies an LDP with speed N and rate function Jv(x) := sup
y∈R2[Fv(y) −J(y)] −[Fv(x) −J(x)], where Fv : R2 →R is given by (2.1). A change of variables yields the desired LDP. Step 2: p
If M denotes the set of minima of Jv and Bε(M) := S
y∈M Bε(y) (where Bε(y) are open
balls of radius ε > 0 centered around y) we obtain from the upper bound of the LDP that P(v /∈Bε(M)) ≤exp
−N
2
inf
x∈Bcε(M) Jv(x)
for N large enough. The inf on the right hand side of the inequality is positive. In this
sense, v concentrates in the minima of Jv exponentially fast. 2
An LDP for the vector of block magnetizations By a change of variables,
again, this implies that m concentrates exponentially fast in the (global) minima of Jm
defined by Jm(x) := sup
y∈R2[Fm(y) −˜J(y)] −[Fm(x) −˜J(x)], Jm(x) := sup
y∈R2[Fm(y) −˜J(y)] −[Fm(x) −˜J(x)], ˜J(x) := 1
2I(x1) + 1
2I(x2)
and
Fm(x) := 1
2
β 1
4x2
1 + β 1
4x2
2 + 1
2αx1x2
. minima of Jm are the maxima of Fm(x) −˜J(x). These satisfy ∇(Fm −˜J)(x) = 0, i.e. The minima of Jm are the maxima of Fm(x) −˜J(x). These satisfy ∇(Fm −˜J)(x) = 0, i.e. The minima of Jm are the maxima of Fm(x) −˜J(x). These satisfy ∇(Fm −˜J)(x) = 0, i.e. 1
2βx1 + 1
2αx2 = artanh(x1)
and
1
2βx2 + 1
2αx1 = artanh(x2). (2.2) (2.2) Note that the vector (0, 0) is always a solution to this system of equations and hence a
critical point of Fm −˜J. We start with the high temperature regime, i.e. we consider β + α < 2. By an easy
calculation we find that the Hesse matrix of Fm(x, y) −˜J(x, y) is given by 1
2
1
2β −
1
1−x2
1
2α
1
2α
1
2β −
1
1−y2
! . Hence, the Hesse matrix in the point (0, 0) is negative definite, i.e. (0, 0) is a local
maximum of Fm(x) −˜J(x), if 0 ≤α ≤β < 2 and
1 −1
2β
2 −1
4α2 > 0. This is true, if
α + β < 2. Hence, the Hesse matrix in the point (0, 0) is negative definite, i.e. (0, 0) is a local
maximum of Fm(x) −˜J(x), if 0 ≤α ≤β < 2 and
1 −1
2β
2 −1
4α2 > 0. This is true, if
α + β < 2. Next, we will see that, in this case, the point (0, 0) is the only solution to the system
of equations in (2.2), and hence the global maximum of Fm(x) −˜J(x). To this end, we
rewrite the equations in (2.2) as x2 = 2
α
artanh(x1) −1
2βx1
and
x1 = 2
α
artanh(x2) −1
2βx2
. Hence, for |x| < 1 and f(x) := 2
α
artanh(x) −1
2βx
we have
x1 = f(x2),
x2 = f(x1)
resp. x1 = f 2(x1),
x2 = f 2(x2). 2
An LDP for the vector of block magnetizations x2 = 2
α
artanh(x1) −1
2βx1
and
x1 = 2
α
artanh(x2) −1
2βx2
. nd f(x) := 2
α
artanh(x) −1
2βx
we have Hence, for |x| < 1 and f(x) := 2
α
artanh(x) −1
2βx
we have x1 = f(x2),
x2 = f(x1)
resp. x1 = f 2(x1),
x2 = f 2(x2). This means we are looking for the fixed points of f 2. We note that for 0 ≤β ≤2, we have
for all |x| < 1 This means we are looking for the fixed points of f 2. We note that for 0 ≤β ≤2, we have
for all |x| < 1 for all |x| < 1 f ′(x) = 2
α
1
1 −x2 −1
2β
> 0 ECP 23 (2018), paper 53. Page 4/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. ECP 23 (2018), paper 53. Page 4/12 Page 4/12 Fluctuations for block spin Ising models and hence f is invertible. The fixed points of f 2 are thus the same as the fixed points
of (f −1)2 resp. of (f −1). We see that f −1 is a strong contraction for α + β < 2 from
(f −1)′ =
1
f ′ and for all y ∈(−1, 1) 1
f ′(y) = α
2
1
1
1−y2 −1
2β
≤α
2
1
1 −1
2β
≤1 −ε, for ε > 0 small enough. Thus, by Banach’s fixed point theorem, there is a single fixed
point, which has to be equal to zero. Next consider the critical line β + α = 2. Here the arguments are almost the same. The only difference is, that now
1
f ′(y)
y=0 = 1, while
1
f ′(y) < 1 for all other y. Hence f −1
is a weak contraction for α + β = 2. However, the magnetizations m1 and m2 live on the
compact interval [−1, 1], such that we can again conclude that f −1 has the unique fixed
point 0. Now we consider α + β > 2. In this case (0, 0) is still a solution to (2.2). However, in
this case, it is either a saddle point or a local minimum of Fm −˜J, because it is not a
maximum. Indeed, choose x = y, i.e. 2
An LDP for the vector of block magnetizations Fm(x, x) −˜J(x, x) = 1
2
β + α
2
x2 −I(x). From the one dimensional Curie-Weiss model we know that for α + β > 2 the maximum
at attained away from zero. Hence, (0, 0) is not a maximum of Fm −˜J. Recalling that
α > 0, we see directly from the definition of Fm and ˜J that a point (x, y) can only be a
maximum, if x and y have the same sign. We will see that f and thus f 2 has exactly one
positive fixed point m∗and one negative fixed point −m∗. This again is shown using a
fixed point argument for f −1. Note that now
1
f ′(0) > 1. However, the function y 7→
1
f ′(y)
is always non-negative on [0, 1], it is decreasing, and depends continuously on y and by
the intermediate value theorem there is y0 such that
1
f ′(y) ≤1 for all y ∈[y0, 1]. Thus,
f −1 restricted to [y0, 1] is a weakly contracting self-map and therefore has a unique fixed
point. But this fixed point of f −1 is the fixed point of f and is easily checked to satisfy tanh
α + β
2
m∗
= m∗. tanh
α + β
2
m∗
= m∗. This proves the claim. Remark 2.2. In the spirit of [19], one can also allow for other sizes of S, i.e. for 0 < γ < 1
we can consider sets S ⊂{1, . . . , N} with |S| = γN. Here, we assume, for simplicity, γN
to be an integer. In this case, the Hamiltonian HN,α,β,S is the same as in (1.1) and the
magnetizations are m1(σ) =
1
γN
P
i∈S σi, and m2(σ) :=
1
(1−γ)N
P
i/∈S σi. The Hamiltonian
can then be rewritten as HN α β S(σ) = −N
2
2γ(1 −γ)αm1m2 + βγ2m2
1 + (1 −γ)2βm2
2
. Remark 2.2. In the spirit of [19], one can also allow for other sizes of S, i.e. for 0 < γ < 1
we can consider sets S ⊂{1, . . . , N} with |S| = γN. Here, we assume, for simplicity, γN
to be an integer. 2
An LDP for the vector of block magnetizations In this case, the Hamiltonian HN,α,β,S is the same as in (1.1) and the
magnetizations are m1(σ) =
1
γN
P
i∈S σi, and m2(σ) :=
1
(1−γ)N
P
i/∈S σi. The Hamiltonian
can then be rewritten as HN,α,β,S(σ) = −N
2
2γ(1 −γ)αm1m2 + βγ2m2
1 + (1 −γ)2βm2
2
. We believe that a result analogue to Theorem 1.1 can be shown by generalizing the
large deviation techniques in the proof of Theorem 2.1. In the same spirit as in Theorem
1.2 and Theorem 1.4, one can also show a Central Limit Theorem for this generalized
setting. The technical problems are, however, more demanding. We will return to these
questions in a later publication. ECP 23 (2018), paper 53. ECP 23 (2018), paper 53.
Page 5/12
http://www.imstat.org/ecp/ 3
Proof of Theorem 1.2 and 1.4 The proofs of Theorems 1.2 and 1.4 rely on the same idea. We will first prove limit
theorems for two other parameters, that are closely related to m1 and m2. To this end ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ http://www.imstat.org/ecp/ Page 5/12 Page 5/12 Fluctuations for block spin Ising models Fluctuations for block spin Ising models Fluctuations for block spin Ising models we introduce the random variables we introduce the random variables we introduce the random variables w1 := w1(σ) := 1
N
N
X
i=1
σi
and
w2 := w2(σ) := 1
N
X
i∈S
σi −
X
i/∈S
σi
! and the corresponding standardized versions and the corresponding standardized versions ˜w1 :=
√
Nw1
and
˜w2 :=
√
Nw2. ˜w1 :=
√
Nw1
and
˜w2 :=
√
Nw2. Note that m1 = w1 + w2 and m2 = w1 −w2 and thus limit theorems for w = (w1, w2) will
imply limit theorems for m and vice versa. Again, note that the Hamiltonian HN,α,β,S can also be rewritten in terms of the
variables w1 and w2 resp. in terms of ˜w1 and ˜w2 as HN,α,β,S(σ) = −N
2
2α1
4m1m2 + β 1
4m2
1 + β 1
4m2
2
= −1
4((α + β) ˜
w1
2 + (β −α) ˜
w2
2). HN,α,β,S(σ) = −N
2
2α1
4m1m2 + β 1
4m2
1 + β 1
4m2
2
= −1
4((α + β) ˜
w1
2 + (β −α) ˜
w2
2). Next we will show a Central Limit Theorem for the vector ˜w := ( ˜w1, ˜w2) in the high
temperature region 0 ≤α < β < 2 and α + β < 2. Lemma 3.1. Assume that 0 ≤α < β and β + α < 2. Then, as N →∞, under the Gibbs
measure µα,β,S the vector ˜w converges to a 2-dimensional Gaussian distribution with
1
0
! Lemma 3.1. Assume that 0 ≤α < β and β + α < 2. Then, as N →∞, under the Gibbs
measure µα,β,S the vector ˜w converges to a 2-dimensional Gaussian distribution with
expectation 0 and covariance matrix Σ =
1
1−α+β
2
0
0
1
β
! . measure µα,β,S the vector w converges to a 2-dimensional Gaussian distribution with
expectation 0 and covariance matrix Σ =
1
1−α+β
2
0
0
1
1−β−α
2
! . 3
Proof of Theorem 1.2 and 1.4 expectation 0 and covariance matrix Σ =
1
1−α+β
2
0
0
1
1−β−α
2
! . Proof. Our principal strategy consists of computing a suitable Hubbard-Stratonovich
transform of our measure of interest (as e.g. in [18]) and expanding it. To this end,
let N(0, C) denote a two-dimensional Gaussian distribution with expectation 0 and
covariance matrix C given by C =
2
β+α
0
0
2
β−α
! . We will now compute the density of
χN,α,β := µN,α,β( ˜w)−1 ∗N(0, C): Let A be a Borel subset of R2 and let ϱN,β(σ) denote
the density of µN,α,β. Then Proof. Our principal strategy consists of computing a suitable Hubbard-Stratonovich
transform of our measure of interest (as e.g. in [18]) and expanding it. To this end,
let N(0, C) denote a two-dimensional Gaussian distribution with expectation 0 and covariance matrix C given by C =
2
β+α
0
0
2
β−α
! . We will now c covariance matrix C given by C =
β+α
0
2
β−α
! . We will now compute the density of
χN,α,β := µN,α,β( ˜w)−1 ∗N(0, C): Let A be a Borel subset of R2 and let ϱN,β(σ) denote
the density of µN,α,β. Then ! χN,α,β := µN,α,β( ˜w)−1 ∗N(0, C): Let A be a Borel subset of R2 and let ϱN,β(σ) denote
the density of µN,α,β. 3
Proof of Theorem 1.2 and 1.4 Then χN,α,β(A) = ( ˜w)−1 ∗N(0, C)(A) =
X
σ∈{−1,1}N
N(0, C)(A −˜w)µN,α,β(σ) χN,α,β(A) = ( ˜w)−1 ∗N(0, C)(A) =
X
σ∈{−1,1}N
N(0, C)(A −˜w)µN,α,β(σ)
=K1
X
σ∈{−1,1}N
Z
A−
√
˜
w
exp
−1
2
α + β
2
x2 + β −α
2
y2
ϱN,β(σ)dxdy
=K1
X
σ∈{−1,1}N
Z
A
exp
−1
2
α + β
2
(x −˜
w1)2 + β −α
2
(y −˜
w2)2
ϱN,β(σ)dxdy
=K2
X
σ∈{−1,1}N
Z
A
exp
−1
2
α + β
2
(x −˜
w1)2 + β −α
2
(y −˜
w2)2
× exp
1
4
(α + β) ˜
w1
2 + (β −α) ˜
w2
2
dxdy
=K2
X
σ∈{−1,1}N
Z
A
exp
−α + β
4
x2 −β −α
4
y2
exp
α + β
2
x ˜
w1 + β −α
2
y ˜
w2
dxdy
=K3
Z
A
exp
−α + β
4
x2 −β −α
4
y2
exp
N
2 log cosh
1
√
N
α + β
2
x + β −α
2
y
+ N
2 log cosh
1
√
N
α + β
2
x −β −α
2
y
dxdy =K1
X
σ∈{−1,1}N
Z
A−
√
˜
w
exp
−1
2
α + β
2
x2 + β −α
2
y2
ϱN,β(σ)dxdy =K1
X
σ∈{−1,1}N
Z
A
exp
−1
2
α + β
2
(x −˜
w1)2 + β −α
2
(y −˜
w2)2
ϱN,β(σ)dxdy ECP 23 (2018), paper 53. Page 6/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. Page 6/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. ECP 23 (2018), paper 53. Page 6/12 Fluctuations for block spin Ising models Fluctuations for block spin Ising models Here, we used K1 :=
1
2π
√
det C , K2 :=
1
2π
√
det CZN,α,β and
K3 := K22N. Denote by
Φ(x, y) :=ΦN,α,β,S(x, y)
:=1
4(α + β)x2 + 1
4(β −α)y2 −N
2 log cosh
1
√
N
α + β
2
x + β −α
2
y
−N
2 log cosh
1
√
N
α + β
2
x −β −α
2
y
. Now recall the second order Taylor expansion log cosh(x) = 1
2x2 + O(x4). 3
Proof of Theorem 1.2 and 1.4 Thus Φ(x, y) =1
4(α + β)x2 + 1
4(β −α)y2 −1
4
(α + β)2
4
x2 + β2 −α2
2
xy + (β −α)2
4
y2
−1
4
(α + β)2
4
x2 −β2 −α2
2
xy + (β −α)2
4
y2
+ O(N −1),
=x2
2
α + β
2
−
α + β
2
2! + y2
2
β −α
2
−
β −α
2
2! + O(N −1), where the constant in the O(N −1)-term depends on x and y. However, the convergence
is uniform on compact subsets of R2. Thus χN,α,β(A) = K3
Z
A
e−Φ(x,y)dx dy
=K3
Z
A
exp
"
−x2
2
α + β
2
−
α + β
2
2! −y2
2
β −α
2
−
β −α
2
2! + O(N −1)
#
dx dy and the convergence in the O(N −1)-term is uniform on compact subsets of R2. To turn this into a weak convergence statement, we need to control integrals over
unbounded sets as well, in particular, we need to treat the case A = R2 to see that K3
converges to
1
2π
√
detΣ′ for Σ′ :=
α+β
2
−
α+β
2
2−1
0
0
β−α
2
−
β−α
2
2−1
. (3.1) (3.1) Hence, for any measurable set A ⊂R2 we write Hence, for any measurable set A ⊂R2 we write Hence, for any measurable set A ⊂R2 we write Z
A
Ψ(x, y)dx dy
=
Z
A∩B(0,R)
Ψ(x, y)dx dy +
Z
A∩B(0,R)c∩B(0,r
√
N)
Ψ(x, y)dx dy +
Z
A∩B(0,r
√
N)c Ψ(x, y)dx dy,
(3.2) Z
A
=
Z
A∩B(0,R)
Ψ(x, y)dx dy +
Z
A∩B(0,R)c∩B(0,r
√
N)
Ψ(x, y)dx dy +
Z
A∩B(0,r
√
N)c Ψ(x, y)dx dy,
(3.2) (3.2) where we set where we set Ψ(x, y) := e−Φ(x,y). Ψ(x, y) := e−Φ(x,y). Here for any l > 0 we denote by B(0, l) the ball in R2 with center in 0 and radius l. Further, we consider numbers R > 0 and r > 0 and we will send R to ∞and consider
r sufficiently small. We will refer to the summands on the right hand of (3.2) as inner
region, intermediate region and outer region, respectively. The goal is to see that the
inner region contributes all mass to the integral as R →∞. ECP 23 (2018), paper 53. As already marked above for fixed R > 0 As already marked above for fixed R > 0 As already marked above for fixed R > 0 lim
N→∞
Z
A∩B(0,R)
Ψ(x, y)dx dy
=
Z
A∩B(0,R)
exp
"
−1
2x2
α + β
2
−
α + β
2
2! −1
2y2
β −α
2
−
β −α
2
2!#
dx dy. N→∞
Z
A∩B(0,R)
=
Z
A∩B(0,R)
exp
"
−1
2x2
α + β
2
−
α + β
2
2! −1
2y2
β −α
2
−
β −α
2
2!#
dx dy. Next, we treat the outer region. Let us rewrite the exponent in this case as Φ(x, y) = N ˜Φ
x
√
N
,
y
√
N
, Φ(x, y) = N ˜Φ
x
√
N
,
y
√
N
, where ˜Φ(x, y) :=1
4(α + β)x2 + 1
4(β −α)y2
−1
2 log cosh
α + β
2
x + β −α
2
y
−1
2 log cosh
α + β
2
x −β −α
2
y
. Analyzing ˜Φ we see that it becomes minimal only if ∇˜Φ = 0 and ∇˜Φ(x, y) =
1
2c1x −c1
4 tanh( c1x+c2y
2
) −c1
4 tanh( c1x−c2y
2
)
1
2c2y −c2
4 tanh( c1x+c2y
2
) + c2
4 tanh( c1x−c2y
2
)
, where we abbreviate c1 := α + β and c2 := β −α. This means, we aim to solve where we abbreviate c1 := α + β and c2 := β −α. This means, we aim to solve where we abbreviate c1 := α + β and c2 := β −α. This means, we aim to solve x = 1
2 tanh
c1x + c2y
2
+ 1
2 tanh
c1x −c2y
2
y = 1
2 tanh
c1x + c2y
2
−1
2 tanh
c1x −c2y
2
. This is done in the spirit of the arguments in Section 2. ECP 23 (2018), paper 53. 3
Proof of Theorem 1.2 and 1.4 http://www.imstat.org/ecp/ http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. Page 7/12 Page 7/12 Page 7/12 Fluctuations for block spin Ising models As already marked above for fixed R > 0 Indeed, denoting by G(x, y) :=
1
2 tanh( c1x+c2y
2
) + 1
2 tanh( c1x−c2y
2
)
1
2 tanh( c1x+c2y
2
) −1
2 tanh( c1x−c2y
2
)
, we see that its Jacobian is given by we see that its Jacobian is given by JG(x, y) =
c1
4
1
cosh2( c1x+c2y
2
) +
1
cosh2( c1x−c2y
2
)
c2
4
1
cosh2( c1x+c2y
2
) −
1
cosh2( c1x−c2y
2
)
c1
4
1
cosh2( c1x+c2y
2
) −
1
cosh2( c1x−c2y
2
)
c2
4
1
cosh2( c1x+c2y
2
) +
1
cosh2( c1x−c2y
2
)
. This means that ||JG(x, y)||1 ≤max
c1
2 , c2
2
= c1
2 < 1, where ∥· ∥1 denotes the maximum
absolute column sum of a matrix. Thus G is a (strict) contraction with fixed point (0, 0)
and hence ˜Φ is minimal in (0, 0). Therefore, for every r > 0. inf
(x,y)/∈B(0,r)
˜Φ(x, y) > 0. This implies that This implies that lim
N→∞
Z
A∩B(0,r
√
N)c Ψ(x, y)dx dy = lim
N→∞
Z
A∩B(0,r
√
N)c e
−N ˜Φ
x
√
N ,
y
√
N
= 0 for any r > 0. Therefore, the outer region is asymptotically negligible. for any r > 0. Therefore, the outer region is asymptotically negligible. Let us turn to the intermediate region. Here we take again a Taylor expansion of the
log cosh on an interval [−z0, z0], z0 > 0, around the origin to first order with a Lagrange ECP 23 (2018), paper 53. Page 8/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ Page 8/12 Fluctuations for block spin Ising models Fluctuations for block spin Ising models bound on the remainder: log cosh(z) = z2
2 + Cz4 with a constant C that depends on z0. As already marked above for fixed R > 0 This yields for
x
√
N ,
y
√
N ∈B(0, r) N ˜Φ
x
√
N
,
y
√
N
= x2
2
α + β
2
+ y2
2
β −α
2
−N
4
(α + β)x
2
√
N
+ (β −α)y
2
√
N
2
−N
4
(α + β)x
2
√
N
−(β −α)y
2
√
N
2
−CrN
"(α + β)x
2
√
N
+ (β −α)y
2
√
N
4
+
(α + β)x
2
√
N
−(β −α)y
2
√
N
4#
≥x2
2
α + β
2
−
α + β
2
2! + y2
2
β −α
2
−
β −α
2
2! −2CrN
|x| + |y|
√
N
4
, where we used α+β
2
≤1. However, on Ar
R := A ∩B(0, R)c ∩B(0, r
√
N) we can estimate
the last line by 2CrN
|x| + |y|
√
N
4
= 2Cr (|x| + |y|)2
|x| + |y|
√
N
2
≤8Cr(x2 + y2)r2 =: ˜
Crr2(x2 + y2) for ˜Cr uniformly on B(0, r
√
N). Note that ˜Crr2 depends continuously on r and converges
to 0 as r →0. In particular, if r is small enough we have that
α+β
2
−
α+β
2
2
−˜Crr2 > 0
2 as well as
β−α
2
−
β−α
2
2
−˜Crr2 > 0. But for this choice of r and ˜Cr we arrive at as well as
β−α
2
−
β−α
2
2
−˜Crr2 > 0. But for this choice of r and ˜Cr we arrive at as well as
β−α
2
−
β−α
2
2
−˜Crr2 > 0. But for this choice of r and ˜Cr we arrive at Z
Ar
R
Ψ(x, y) dx dy
≤
Z
Ar
R
exp
"
−x2
2
α + β
2
−
α + β
2
2
−Crr2
! −y2
2
β −α
2
−
β −α
2
2
−Crr2
!#
dxdy R
≤
Z
Ar
R
exp
"
−x2
2
α + β
2
−
α + β
2
2
−Crr2
! −y2
2
β −α
2
−
β −α
2
2
−Crr2
!#
dxdy and the right hand side is an integrable function. Thus for R →∞the right hand side as
well as the left hand side converges to 0. As already marked above for fixed R > 0 Putting the estimates together, we have seen that χN,α,β converges weakly to the
2-dimensional Gaussian distribution with expectation 0 and covariance matrix Σ′, where
Σ′ is given in (3.1). This weak convergence is equivalent to the convergence of the
characteristic functions. Computing the characteristic functions of the Gaussian distribution involved in the
above proof, we have therefore shown that the characteristic function of ˜w in the point
t = (t1, t2) ∈R2 satisfies lim
N→∞E(eit ˜
w)e−1
2 (
2
α+β )t2
1−1
2 (
2
β−α )t2
2 = e
−1
2 t2
1[ α+β
2
−
α+β
2
2]−1−1
2 t2
2[ β−α
2
−
β−α
2
2]−1
. This implies that lim
N→
E(eit ˜
w) = e
1
2 (
2
α+β )t2
1+ 1
2 (
2
β−α )t2
2e
−1
2 t2
1[ α+β
2
−
α+β
2
2]−1−1
2 t2
2[ β−α
2
−
β−α
2
2]−1 lim
N→∞E(eit ˜
w) = e
1
2 (
2
α+β )t2
1+ 1
2 (
2
β−α )t2
2e
−1
2 t2
1[ α+β
2
−
α+β
2
2]−1−1
2 t2
2[ β−α
2
−
β−α
2
2]−1
= e
−1
2 t2
1
1
1−α+β
2
−1
2 t2
2
1
1−β−α
2
. lim
N→∞E(eit ˜
w) = e
1
2 (
2
α+β )t2
1+ 1
2 (
2
β−α )t2
2e
−1
2 t2
1[ α+β
2
−
α+β
2
2]−1−1
2 t2
2[ β−α
2
−
β−α
2
2]−1 = e
−1
2 t2
1
1
1−α+β
2
−1
2 t2
2
1
1−β−α
2
. Turning this into a weak convergence statement again, we obtain Turning this into a weak convergence statement again, we obtain ( ˜
w1, ˜
w2)
N→∞
−−−−→N(0, Σ),
Σ =
1
1−α+β
2
0
0
1
1−β−α
2
! in distribution. Fluctuations for block spin Ising models Proof of Theorem 1.2. The proof of Theorem 1.2 is straightforward from the above
lemma. As observed we have that m1 = w1 + w2 and m2 = w1 −w2, thus
√
Nm1 =
√
N(w1 + w2) = ˜
w1 + ˜
w2 as well as
√
Nm2 =
√
N(w1 −w2) = ˜
w1 −˜
w2. Thus Lemma 3.1
gives that m1 and m2 are asymptotically normal with mean 0 and variance lim
N→∞V(
√
Nm1) =
1
1 −β+α
2
+
1
1 −β−α
2
=
2
2 −β −α +
2
2 −β + α =
4(2 −β)
(2 −β)2 −α2 . Moreover, the same considerations together with Lemma 3.1 show that their covariance
is given by Moreover, the same considerations together with Lemma 3.1 show that their covariance
is given by lim
N→∞Cov(
√
Nm1,
√
Nm2) = V ˜w1 −V ˜w2 =
4α
(2 −β)2 −α2 =
4(2 −β)
(2 −β)2 −α2
α
2 −β as proposed. as proposed. On the other hand, the proof Lemma 3.1 also inspires the proof of Theorem 1.4. Indeed, redoing the computations there shows that for α + β = 2 the quadratic term in
the first component of χN,α,β cancels. To this end we have to rescale ˜w1 to make the
second term in the Taylor expansion of log cosh appear (as a matter of fact this is very y
p
g
pp
(
y
similar, to what happens in the Curie-Weiss model at its critical temperature β = 1). Proof of Theorem 1.4. As motivated above we will now consider the vector ˆw = ( ˆw1, ˆw2)
consisting of the components ˆw1 := N 1/4w1
and
ˆw2 :=
√
Nw2 = ˜w2. This time we will convolute the distribution of ˆw under the Gibbs measure µN,α,β,S with a
two-dimensional Gaussian distribution N(0, ˆC), where ˆC =
1
√
N
0
0
2
β−α
! (note that this
is well defined since β > α). ECP 23 (2018), paper 53.
Page 10/12
http://www.imstat.org/ecp/ in distribution. ECP 23 (2018), paper 53. Page 9/12
http://www.imstat.org/ecp/ ECP 23 (2018), paper 53. ECP 23 (2018), paper 53. Page 9/12 Fluctuations for block spin Ising models Fluctuations for block spin Ising models Fluctuations for block spin Ising models Computing the density of ˆχN,α,β := µN,α,β( ˆw)−1 ∗N(0, ˆC)
as in the proof of Lemma 3.1 we obtain building on the fact that now α + β = 2 ˆχN,α,β(A)
= ˆK1
X
σ∈{−1,1}N
Z
A
exp
−1
2(
√
N(x −ˆ
w1)2 + β −α
2
(y −ˆ
w2)2
ϱN,α,β(σ)dxdy
= ˆK2
X
σ∈{−1,1}N
Z
A
exp
−1
2(
√
N(x −ˆ
w1)2 + β −α
2
(y −ˆ
w2)2
× exp
1
4(2
√
N ˆ
w1
2 + (β −α) ˜
w2
2)
dxdy
= ˆK2
X
σ∈{−1,1}N
Z
A
exp
−
√
N
2 x2 −1
4(β −α)y2
! exp
√
Nx ˆ
w1 + 1
2(β −α)y ˜
w2
dxdy
= ˆK3
Z
A
exp
−
√
N
2 x2 −1
4(β −α)y2
! exp
N
2 log cosh
x
N 1/4 + β −α
2
√
N
y
+N
2 log cosh
x
N 1/4 −β −α
2
√
N
y
dxdy with the normalizing constants ˆK1, ˆK2, and ˆK3 chosen similarly to K1, K2, and K3 in
the proof of Lemma 3.1. Now we expand the log cosh to fourth order: log cosh(z) =
z2
2 −1
12z4 + O(z6). We thus see that the x2 terms in the exponent cancel and so do the ECP 23 (2018), paper 53. Page 10/12 Fluctuations for block spin Ising models s (fortunately). For fixed x and y only the x4 is of vanishing order. The y2 terms
ted as in the proof of Lemma 3.1. We thus see that xy-terms (fortunately). For fixed x and y only the x4 is of vanishing order. The y2 terms
are treated as in the proof of Lemma 3.1. We thus see that −
√
N
2 x2 −1
4(β −α)y2 + N
2 log cosh
x
N 1/4 + β −α
2
√
N
y
+ N
2 log cosh
x
N 1/4 −β −α
2
√
N
y
= −1
12x4 −1
2y2
β −α
2
−
β −α
2
2! + O(N −1
2 ) with a O(N −1
2 ) term that depends on x and y. To conclude the convergence of ˆχN,α,β(A)
we now proceed as in the proof of Lemma 3.1. Here we will only sketch the differences,
because many steps are very similar. Fluctuations for block spin Ising models The exact steps are left to the reader. The main
differences to the above proof of Lemma 3.1 is that the inner region is again B(0, R),
while the intermediate region now is the rectangle [−, N
1
4 r] × [−r
√
N, r
√
N] to take into
account that the Taylor expansion gives another order for x than for y. Correspondingly
in the intermediate region the integrable function that dominates with a O(N −1
2 ) term that depends on x and y. To conclude the convergence of ˆχN,α,β(A)
we now proceed as in the proof of Lemma 3.1. Here we will only sketch the differences,
because many steps are very similar. The exact steps are left to the reader. The main
differences to the above proof of Lemma 3.1 is that the inner region is again B(0, R),
while the intermediate region now is the rectangle [−, N
1
4 r] × [−r
√
N, r
√
N] to take into
account that the Taylor expansion gives another order for x than for y. Correspondingly
in the intermediate region the integrable function that dominates exp
"
−
√
N
2 x2 −1
4(β −α)y2
#
× exp
N
2 log cosh
x
N 1/4 + β −α
2
√
N
y
+ N
2 log cosh
x
N 1/4 −β −α
2
√
N
y
is given by exp(−d1x4 −d2y2) for suitable constants d1, d2 > 0. With these changes we
see that ˆχN,α,β(A) converges to a 2-dimensional distribution with density proportional to exp
−1
12x4 −1
2y2
β −α
2
−
β −α
2
2!! with respect to the 2-dimensional Lebesgue measure. However, from here we see that
ˆw1 converges in distribution to a random variable with density proportional to e−1
12 x4
since the Gaussian measure we convoluted the first coordinate of ˆw with converges to 0
in probability. Moreover, the same computation as in Lemma 3.1 shows that ˆw2 = ˜w2
converges to a normal distribution with mean 0 and variance
2
2−(β−α). (β
)
However, the latter convergence implies that N
1
4 w2 converges to 0 in probability. Thus N
1
4 m1 = N
1
4 w1 + N
1
4 w2 also converges in distribution to a random variable with
density proportional to e−1
12 x4 (see e.g. [3, Theorem 3.1]). [1] Arash A. Amini and Elizaveta Levina, On semidefinite relaxations for the block model, Ann.
Statist. 46 (2018), no. 1, 149–179. MR-3766949 [2] Quentin Berthet, Philippe Rigollet, and Piyush Srivastavaz, Exact recovery in the ising
blockmodel, Preprint, arXiv:1612.03880v1 (2016), 1–29. Fluctuations for block spin Ising models [6] Francesca Collet, Macroscopic limit of a bipartite Curie-Weiss model: a dynamical approach,
J. Stat. Phys. 157 (2014), no. 6, 1301–1319. MR-3277768 [7] Pierluigi Contucci, Ignacio Gallo, and Giulia Menconi, Phase transitions in social sciences:
Two-population mean field theory, International Journal of Modern Physics B 22 (2008),
no. 14, 2199–2212. [8] Amir Dembo and Ofer Zeitouni, Large deviations techniques and applications, Stochastic
Modelling and Applied Probability, vol. 38, Springer-Verlag, Berlin, 2010, Corrected reprint
of the second (1998) edition. MR-2571413 [9] Frank den Hollander, Large deviations, Fields Institute Monographs, vol. 14, American
Mathematical Society, Providence, RI, 2000. MR-1739680 [10] Peter Eichelsbacher and Matthias Löwe, Stein’s method for dependent random variables oc-
curring in statistical mechanics, Electron. J. Probab. 15 (2010), no. 30, 962–988. MR-2659754 [11] Richard S. Ellis, Entropy, large deviations, and statistical mechanics, Classics in Mathematics,
Springer-Verlag, Berlin, 2006, Reprint of the 1985 original. MR-2189669 [12] Richard S. Ellis and Charles M. Newman, Limit theorems for sums of dependent random
variables occurring in statistical mechanics, Z. Wahrsch. Verw. Gebiete 44 (1978), no. 2,
117–139. MR-0503333 [13] Richard S. Ellis and Charles M. Newman, The statistics of Curie-Weiss models, J. Statist. Phys. 19 (1978), no. 2, 149–161. MR-0503332 [14] Micaela Fedele and Francesco Unguendoli, Rigorous results on the bipartite mean-field
model, J. Phys. A 45 (2012), no. 38, 385001, 18. MR-2970551 [15] Ignacio Gallo, Adriano Barra, and Pierluigi Contucci, Parameter evaluation of a simple mean-
field model of social interaction, Math. Models Methods Appl. Sci. 19 (2009), no. suppl.,
1427–1439. MR-2554157 [16] Ignacio Gallo and Pierluigi Contucci, Bipartite mean field spin systems. Existence and solution,
Math. Phys. Electron. J. 14 (2008), Paper 1, 21. MR-2407199 [17] Chao Gao, Zongming Ma, Anderson Y. Zhang, and Harrison H. Zhou, Achieving optimal
misclassification proportion in stochastic block models, J. Mach. Learn. Res. 18 (2017), Paper
No. 60, 45. MR-3687603 [18] Barbara Gentz and Matthias Löwe, The fluctuations of the overlap in the Hopfield model with
finitely many patterns at the critical temperature, Probab. Theory Related Fields 115 (1999),
no. 3, 357–381. MR-1725405 [19] Werner
Kirsch
and
Gabor
Toth,
Two
groups
in
a
Curie-Weiss
model,
preprint,
arXiv:1712.08477 (2017). [20] Werner Kirsch and Gabor Toth, Two groups in a Curie-Weiss model with heterogeneous
coupling, preprint (2018). [21] Matthias Löwe, Kristina Schubert, and Franck Vermet, Block spin Ising models on random
graphs, preprint, in preparation (2018). References [1] Arash A. Amini and Elizaveta Levina, On semidefinite relaxations for the block model, Ann. Statist. 46 (2018), no. 1, 149–179. MR-3766949 [2] Quentin Berthet, Philippe Rigollet, and Piyush Srivastavaz, Exact recovery in the ising
blockmodel, Preprint, arXiv:1612.03880v1 (2016), 1–29. [3] Patrick Billingsley, Convergence of probability measures, second ed., Wiley Series in Proba-
bility and Statistics: Probability and Statistics, John Wiley & Sons, Inc., New York, 1999, A
Wiley-Interscience Publication. MR-1700749 [4] Guy Bresler, Efficiently learning Ising models on arbitrary graphs [extended abstract],
STOC’15—Proceedings of the 2015 ACM Symposium on Theory of Computing, ACM, New
York, 2015, pp. 771–782. MR-3388257 [5] Guy Bresler, Elchanan Mossel, and Allan Sly, Reconstruction of Markov random fields from
samples: some observations and algorithms, SIAM J. Comput. 42 (2013), no. 2, 563–578. MR-3037003 ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ Page 11/12 Fluctuations for block spin Ising models [22] Elchanan Mossel, Joe Neeman, and Allan Sly, Belief propagation, robust reconstruction
and optimal recovery of block models, Ann. Appl. Probab. 26 (2016), no. 4, 2211–2256. MR-3543895 Acknowledgments. We are grateful to an anonymous referee for many valuable re-
marks. Acknowledgments. We are grateful to an anonymous referee for many valuable re-
marks. ECP 23 (2018), paper 53. http://www.imstat.org/ecp/ Page 12/12 Page 12/12
|
https://openalex.org/W2942921297
|
https://hal.univ-lorraine.fr/hal-03228694/file/NR-g-PHB%20VERSION%20FINALISEE.pdf
|
English
| null |
Influence of the synthesis parameters on the properties of natural rubber grafted poly-3-hydroxybutyrate
|
IOP conference series. Materials science and engineering
| 2,019
|
cc-by
| 8,528
|
To cite this version: Asmaa Zainal Abidin, Noor Hana Hanif Abu Bakar, Denis Roizard, Anne Jonquieres, Carole Arnal-
Herault, et al.. Influence of the synthesis parameters on the properties of natural rubber grafted
poly-3-hydroxybutyrate (acte de congrès - 10 pages). 13th Joint Conference in Chemistry, Sep 2018,
Semarang, Indonesia. pp.012024, 10.1088/1757-899X/509/1/012024. hal-03228694 Influence of the synthesis parameters on the properties
of natural rubber grafted poly-3-hydroxybutyrate (acte
de congrès - 10 pages) Asmaa Zainal Abidin, Noor Hana Hanif Abu Bakar, Denis Roizard, Anne
Jonquieres, Carole Arnal-Herault, Mohamad Abu Bakar, Rosniza Hamzah Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03228694
https://hal.univ-lorraine.fr/hal-03228694v1
Submitted on 18 May 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1.0 Introduction The development of materials based on renewable resources is currently being considered
as one of the most promising ways for overcoming the environmental concerns related to major
synthetic polymers. Polymers derived from biomass such as poly(3-hydroxybutyrate) (PHB) have
received considerable attention nowadays. PHB is a natural occurring polyester produced by the
microorganism, known as Bacillus megaterium and was discovered by Lemoigne in 1926 [1]. PHB
is a semi-crystalline thermoplastic which is biocompostable, biocompatible as well as renewable
in nature [2]. Because of these properties, PHB has been applied in vast applications especially in
the biomedical field. PHB not only acts as a vehicle to transport nutrient, drug and bioactive
molecule to the tissue or cell, but it is also employed in tissue scaffolding for bone and nerve
regeneration, cardiovascular as well as cartilage support respectively [3, 4]. Apart from this, PHB
is also used in the field of water treatment. Heitmann and coworkers [5] demonstrated the
degradation of methylene blue using PHB as a support to disperse nanostructured niobium
oxyhydroxide. While PHB has shown excellent properties for certain applications, it is a very brittle plastic
and prone to thermal degradation at temperatures above the melting point [6]. To a certain extent,
this actually limits its application. In an effort to extend its use, various methods have been devised
to improve the properties of PHB. Studies have shown that PHB can be blended with other
polymers such as ethylene propylene rubber (EPR) [7], poly(methyl methacrylate) (PMMA) [8],
polyethylene oxide (PEO) [9] and poly-caprolactone (PCL) [10]. However, as expected, most of
the works reported that the polymers did not mix well, and a miscible system rarely occurred. As
an example, Graco and Martuscelli [11] studied the blending of PHB with ethylene propylene
rubber (EPR). The authors concluded that no interactions occurred between PHB and EPR after
two distinct Tg values were observed. Hence, interests in PHB based blends have gradually shifted
to reactive systems which allow better mixing and simple preparation method. Chemical modification of PHB via grafting with NR is an alternative method to incorporate
desirable properties without sacrificing its biodegradable nature. NR is a good candidate as it has
high elasticity and good rebound resilience [12]. Thus, NR will act as a soft component in the
grafted copolymers to achieve better properties or processability which can be employed in
biomedical application. A few works have been done on PHB grafting. INFLUENCE OF VARIOUS SYNTHESIS PARAMETERS ON THE PROPERTIES OF
NATURAL RUBBER GRAFTED POLY-3-HYDROXYBUTYRATE 1Nanoscience Research Laboratory, School of Chemical Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia 1Nanoscience Research Laboratory, School of Chemical Sciences, Universiti Sains Malaysia,
11800 Penang, Malaysia 2Laboratory of Reactions and Process Engineering, Université de Lorraine, UMR CNRS, LRGP,
7274, 1 rue Grandville, 54000 Nancy, France 3Laboratoire de Chimie Physique Macromoléculaire, Université de Lorraine, CNRS, LCPM,
ENSIC, 1 rue Grandville, BP 20451, F-54 000 Nancy, France 3Laboratoire de Chimie Physique Macromoléculaire, Université de Lorraine, CNRS, LCPM,
ENSIC, 1 rue Grandville, BP 20451, F-54 000 Nancy, France 4Center of Excellence Geopolymer and Green Technology (CEGeoGTech), Faculty of
Engineering Technology (FETech), Universiti Malaysia Perlis (UniMAP), Level 1 Block S2,
UniCITI Alam Campus, Sungai Chucuh, Padang Besar, 02100, Perlis, Malaysia
Email: hana_hanif@usm.my Natural rubber (NR) graft poly-3-hydroxybutyrate (PHB) with a ratio of 60:40 was synthesized in
chlorobenzene solvent. Two types of initiators namely azobisisobutyronitrile (AIBN) and benzoyl
peroxide (BPO) were utilized to initiate the free radical grafting of the two polymers. The influence
of the various types of initiator loadings was also investigated. The position of the PHB grafting
on the NR was then determined using 1H, 13C, DEPT 90 and DEPT 145 nuclear magnetic resonance
(NMR) analyses and Fourier transformed infrared spectroscopy (FTIR). The thermal stability and
crystallization behavior of NR-g-PHB was studied using thermogravimetric analysis (TGA) as
well as differential scanning calorimetry (DSC) respectively. The increase in initiator loading
improved the grafting. Moreover, single glass transition temperature (Tg) was observed for NR-g-
PHB which indicated that no phase separation occurred for the PHB grafts and the thermal stability
of the grafted copolymers was improved compared to that of pristine NR and PHB alone. 1.0 Introduction 1.0 Introduction As an example, Jiang and Hu [13] investigated the graft polymerization of isoprene onto PHB using radiation technique. A
maximum grafting degree of 18% was obtained by varying the solvent, isoprene concentration and
irradiation time. Subsequently, Chen et al. [14] employed maleic anhydride (MA) as the grafting
monomer to be grafted onto PHB chains. Authors discovered that introducing MA onto PHB
chains could disturb the regularity of the PHB chains, control the morphological structures as well
as improve its properties. Other works also reported the grafting of PHB onto cellulose via reactive
extrusion using dicumyl peroxide as radical initiator [6]. They showed that the thermal stability of
PHB was improved after grafting reaction due to the formation of new bonds between PHB and
cellulose. Nevertheless, most previous works on PHB grafting used high temperatures around 175
-180 oC for the grafting reaction [6, 15, 16]. For instance, PHB grafted maleic anhydride was
prepared at 175 oC [15]. According to the authors, as a result of the use of this high temperature,
thermal degradation of PHB occurred almost exclusively by random chain-scission. Therefore, in
the present study, medium temperature was employed to graft NR with PHB to minimize the
occurrence of PHB degradation. This study describes the grafting of NR with PHB in solution
using different types of initiators and various parameters that affect the grafting process. Apart
from that, several characterization techniques were utilized in this study. The possible structure of
the grafted copolymers was further scrutinized using 1D and 2D NMR to confirm the grafting
position, since previous literature on NR-g-PHB copolymers did not investigate this in detail [13]. 2.1 Preparation of dry rubber (DR) DR was obtained by coagulation of NRL in a glass bottle containing acetone at room temperature. The acetone was removed after coagulation and the coagulates were washed again using acetone
several times. Finally, the coagulates were dried in a vacuum oven at 40 °C until constant weight
was reached. 2.0 Materials Natural rubber latex (NRL, (C5H8)n) with 60 % dry rubber content (DRC) was obtained from
Malaysian Rubber Board, Kuala Lumpur. Poly(3-hydroxybutyrate) (PHB, 433,000 g/mol) was
purchased from BIOCYCLE (Brazil) and purified before use [17]. Chlorobenzene (C6H5Cl, 99.7
%) and acetone (C3H6O) were purchased from Sigma Aldrich (USA). Azobisisobutyronitrile
(AIBN, C8H12N4) and benzoyl peroxide (BPO, C14H10O4) were manufactured by Fluka (UK). Besides that, deuterated chloroform (99.8%, CDCl3) for NMR analysis was purchased from Merck
(USA) and argon (99% purity) gas was purchased from Linde (Malaysia). 2.2 Preparation of NR-g- poly (3-hydroxybutyrate) copolymers DR and PHB with a 60:40 wt/wt ratio were dissolved in chlorobenzene at 105 °C overnight. The
initiator, AIBN, was added into the cooled polymer mixture at 30 °C followed by 15 min stirring. The polymer mixture was cast in a glass mold, purged with argon gas, and covered with a glass
plate. Afterwards, the NR-g-PHB was kept in the oven for 2 days at a certain casting temperature
to maximize the grafting and cross-linking reactions. The glass plate cover was then removed to
achieve solvent evaporation at casting temperature. Different casting temperatures and initiator
loadings were investigated as in Table 1. These steps were repeated with another initiator (BPO)
allowing working at slightly higher temperature. Table 1: Parameters for the study of NR-g-PHB synthesis. NR-g-PHB
Type of initiator
Casting Temperature
(oC)
Initiator
loading (% mol)
60:40
AIBN
90
7
BPO
90
7
60:40
BPO
105
3
BPO
105
5
BPO
105
7
BPO
105
10 rameters for the study of NR-g-PHB synthesis. Table 1: Parameters for the study of NR-g-PHB synthesis. 2.3 Characterization 2.3 Characterization 2.3 Characterization The structural characterization of the grafting reaction was carried out by using NMR
spectroscopy. The spectra were obtained using Bruker BioSpin ADVANCE at 500 MHz and 300
MHz for 1H NMR and 13C NMR, respectively. Complementary DEPT 90 and DEPT 135 analyses were performed at 25 oC in a flow of air. The samples were dissolved in deuterated chloroform
(CDCl3) and tetramethylsilane (TMS) was used as an internal standard. In addition, the possible
structural changes of the samples were confirmed by Fourier Transformed Infrared (FTIR)
spectroscopy. The samples were scanned from 400-4000 cm-1 using a Frontier Universal ATR
Perkin Elmer FT-NIR Spectrometer. The thermal properties of the samples were studied using a
Mettler Toledo Differential Scanning Calorimetry. As much as 10 mg of the samples were weighed
in an aluminum pan and covered with an aluminum lid. The sample was heated from -100 to 190
oC and held for 2 min at a heating rate of 20 oC min-1 under nitrogen environment. Next, the
samples were quenched to -100 oC at a scanning rate of 20 oC min-1 and held for another 2 min. Finally, the samples were again heated to 190 oC at a heating rate of 20 oC min-1. Meanwhile, 5.00
mg of the samples were heated from 30 oC to 900 oC at a heating rate of 20 oC min-1 under nitrogen
flow to examine thermal stability using Perkin Elmer STA 6000 simultaneous thermal analyzer. 3.1 Effect of initiator Two initiators, AIBN and BPO were studied using the same initiator concentration of 7 %
mol at 90 oC to investigate their effectiveness in grafting NR with PHB. When AIBN initiator was
employed, the NR-g-PHB copolymer formed a lot of small separated pieces on the glass mold as
compared to the BPO initiator, which gave a smoother surface as shown in Figure 1 (a-b). The
breakages in the NR-g-PHB membrane could be due to the insufficient occurrence of grafting
and/or crosslinking between NR and PHB, which can be proven by the rapid dissolution of the
grafted polymer in chloroform solvent. AIBN is known as a good thermal initiator for moderate
temperatures (e.g. 60°C). At 90°C, it was not an effective initiator mainly because at this higher
temperature, its decomposition rate was too high. Therefore, an initiator (BPO) with a moderate
decomposition rate at the grafting temperature was then chosen to ensure that the initiating phenyl
radicals would be produced during the whole grafting process. BPO contains a weak sigma bond
(O-O bond), which could rapidly undergo bond homolysis at the grafting temperature to generate
a pair of radicals, which were then fragmentated to provide phenyl radicals as depicted in Figure
2 (b). These phenyl radicals are capable in abstracting a hydrogen atom from a carbon-hydrogen
bond in either NR or PHB [19]. In addition, BPO could also be used at temperatures 90-110 oC
due to the fact that its half-life in chlorobenzene is much longer in comparison to AIBN. In this work, the choice of the grafting temperature range (90-105 °C) resulted from a compromise. It had
to be high enough for improving NR grafting and, at the same time, it had to be limited for avoiding
PHB degradation. Therefore, it would provide advantages compared to previous works reporting
grafting and crosslinking for NR usually at much higher temperatures (170-180 oC) [20]. Hence,
for the next part of this study, BPO was selected as the initiator in the preparation of NR-g-PHB
copolymers in the temperature range 90-105 °C. (a) (b)
Figure 1: Images of membranes obtained for different initiator (a) AIBN and (b) BPO using 7 %
mol at 90 oC. (b) (a) (b) (a) Figure 1: Images of membranes obtained for different initiator (a) AIBN and (b) BPO using 7 %
mol at 90 oC. 3.1 Effect of initiator H3C
C
N
N
C
CH3
CH3
CH3
CN
CN
H3C
C
CH3
CN
2
+
N2
C
O
O
C
O
O
C
O
O
2
(b)
∆
∆
(a) H3C
C
N
N
C
CH3
CH3
CH3
CN
CN
(a) (a) C
O
O
C
O
O
C
O
O
2
(b)
∆ (b) C
O
O
2
2
+
CO2
Figure 2: Radical formation from (a) AIBN and (b) BPO. ∆ 2 Figure 2: Radical formation from (a) AIBN and (b) BPO. The effect of initiator loadings on the preparation of NR-g-PHB was investigated and the
membrane images are illustrated in Figure 3. The experiments were performed using various BPO
initiator concentrations ranging from 3 to 10 % mol. The same weight ratio 60:40 for NR and PHB
was used for the synthesis of the NR-g-PHB copolymers with reaction temperature of 105 °C. When the initiator loading was increased, the membrane breakage was greatly reduced. For the
highest initiator loadings, homogeneous membranes were obtained. This improvement can be
explained as due to the increasing amounts of free radicals that were produced from the
dissociation of BPO, resulting in more active sites on the NR and PHB backbones, thus facilitating
grafting. However, for the maximum initiator loading of 10 % mol., the membrane retracted from
the mold border (Fig 3d). Therefore, from these results, the optimum BPO amount was taken to be
7 % mol (Fig 3c). (a)
(b) (b) (a) (b) (a) (a) (c) (d)
Figure 3: Images of NR-g-PHB membranes obtained for (a) 3, (b) 5, (c) 7 and (d) 10 % mol of
BPO loadings at 105 °C. (d) (c) (d) (c) Figure 3: Images of NR-g-PHB membranes obtained for (a) 3, (b) 5, (c) 7 and (d) 10 % mol of
BPO loadings at 105 °C. 3.2.1 NMR Spectroscopy The confirmation of the chemical structure of the pristine NR, PHB and NR-g-PHB was
further obtained using 1H and 13C NMR spectrum as presented in Figures 4, 5 and 6, respectively. In this work, the NR, PHB and NR-g-PHB structures were labeled to identify the carbon position
and its respective proton(s). For facilitating the assignments of the different peaks for the grafted
copolymers, labelling by numbering was chosen for the main chain (NR) and labelling by lettering
was selected for the grafts (PHB). For the 1H NMR spectrum of NR in Figure 4 (a), the unsaturated
methyne proton 3 of the double bonds, the methyl protons 5 and allylic methylene protons 1 and
4 resonated at δH 5.04-5.06 ppm, δH 1.61 ppm and δH 1.97 ppm, respectively. On the other hand,
five 13C NMR signals of NR resonated at δC 23.42 ppm, δC 26.40 ppm, δC 29.90 ppm, δC 125.04
ppm as well as δC 135.21 ppm as observed in Figure 4 (b). These carbon signals are attributed to
the methyl carbon 5, allylic methylene carbons 4 and 1, methyne carbon 3 and the other carbon of
the double bonds corresponding to carbon 2. These chemical shifts in the 1H and 13C NMR of NR
were in good agreement with previous works [21-26]. (a)
Figure 4: NMR spectrum for (a) 1H and (b) 13C of NR. (b)
1H chemical shifts of NR (ppm)
5 (δH 1.61)
1 and 4 (δH 1.97)
3 (δH 5.04-5.06)
5
1, 4
3
5
4
1
3
2
13C chemical shifts of NR
(ppm)
5 (δC 23.42)
4 (δC 26.40)
1 (δC 29.90)
3 (δC 125.04)
2 (δC 135.21)
Solvent
(NR) (a)
1H chemical shifts of NR (ppm)
5 (δH 1.61)
1 and 4 (δH 1.97)
3 (δH 5.04-5.06)
5
1, 4
3
13C chemical shifts of NR
(ppm)
(NR) (a)
1H chemical shifts of NR (ppm)
5 (δH 1.61)
1 and 4 (δH 1.97)
3 (δH 5.04-5.06)
5
1, 4
3
(NR) (a) 1H chemical shifts of NR (ppm)
5 (δH 1.61)
1 and 4 (δH 1.97)
3 (δH 5.04-5.06)
5
1, 4
3
(NR) 1H chemical shifts of NR (ppm) (NR) Figure 4: NMR spectrum for (a) 1H and (b) 13C of NR. 3.2.1 NMR Spectroscopy (b)
5
4
1
3
2
13C chemical shifts of NR
(ppm)
5 (δC 23.42)
4 (δC 26.40)
1 (δC 29.90)
3 (δC 125.04)
2 (δC 135.21)
Solvent (b) Figure 4: NMR spectrum for (a) 1H and (b) 13C of NR. As can be seen in Figure 5 (a), the 1H NMR spectrum of PHB showed a doublet at the δH
1.20-1.21 ppm which is attributed to the methyl protons D. These protons were highly shielded
and absorbed at the most upfield region. Besides that, the methylene protons B showed a mirror
quartet signal that resonated in the range of δH 2.38-2.56 ppm. Meanwhile and as expected, the
multiplet signal in the range δH 5.17-5.21 ppm corresponded to the methyne proton A. This signal
resonated at the most downfield region because these methyne protons were deshielded by an
electronegative oxygen atom due to inductive effect of the ester group. In Figure 5 (b), PHB
exhibited four carbon peaks at δC 19.76 ppm, δC 40.81 ppm, δC 67.62 ppm and δC 169.12 ppm that
were assigned to methyl carbon D, methylene carbon B, methyne carbon A and carbon C of the
carbonyl of the ester group, respectively. These NMR spectra confirmed the purity of PHB, and
the assignments of the different peaks were consistent with previous works [27-32]. (a)
(PHB)
D
B
A
1H chemical shifts of PHB (ppm)
D (δH 1.20-1.21)
B (δH 2.38-2.56)
A (δH 5.17-5.21) (a) (a) (PHB)
D
B
A
1H chemical shifts of PHB (ppm)
D (δH 1.20-1.21)
B (δH 2.38-2.56)
A (δH 5.17-5.21) (PHB)
D
B
A
D (δH 1.20-1.21)
B (δH 2.38-2.56)
A (δH 5.17-5.21) (b)
Figure 5: NMR spectrum for (a) 1H and (b) 13C of PHB. 13C chemical shifts of PHB (ppm)
D (δC 19.76)
B (δC 40.81)
A (δC 67.62)
C (δC 169.12)
D
B
A
C
Solvent (b) Figure 5: NMR spectrum for (a) 1H and (b) 13C of PHB. Figure 5: NMR spectrum for (a) 1H and (b) 13C of PHB. The grafting of PHB onto NR in the presence of BPO at high temperature can proceed in
two different ways (Figure 6). In type I grafting, an allylic H is abstracted from the NR chain by
the phenyl radical resulting from BPO decomposition, followed by coupling of the resulting allylic
radical with a PHB chain bearing a radical. 3.2.1 NMR Spectroscopy The formation of such allylic radical is very easy
because this allylic radical is strongly stabilized by resonance involving the adjacent double bond. In this first type of grafting, the NR double bonds remain unchanged. In type II grafting, first, there
is an addition of the phenyl radical onto the NR double bond. Then, the corresponding radical is
coupled with a PHB chain bearing a radical. The formation of a radical onto PHB mainly occurs
through H abstraction by the phenyl radical. The most favored site for this abstraction is on the
ternary carbon atom to generate a stabilized tertiary radical. After coupling, this ternary carbon
atom becomes a quaternary carbon, which does not bear any hydrogen atom. (a) (b)
Figure 6: Mechanisms for PHB grafting onto NR. (a) Type I grafting, an allylic H abstraction
followed by radical coupling and (b) Type II grafting, an addition of phenyl radical onto
NR double bond followed by radical coupling. (b) Figure 6: Mechanisms for PHB grafting onto NR. (a) Type I grafting, an allylic H abstraction Figure 6: Mechanisms for PHB grafting onto NR. (a) Type I grafting, an allylic H abstraction
followed by radical coupling and (b) Type II grafting, an addition of phenyl radical onto
NR double bond followed by radical coupling. Figure 6: Mechanisms for PHB grafting onto NR. (a) Type I grafting, an allylic H abstraction
followed by radical coupling and (b) Type II grafting, an addition of phenyl radical onto
NR double bond followed by radical coupling. As illustrated in Figure 7 (a) and (b), 1H and 13C NMR spectra of NR-g-PHB showed all
the signals for the NR and PHB ungrafted monomer units because of the low grafting rate. In
particular, the methyne protons of the NR double bonds are responsible for a peak at δH 5.04-5.05
ppm in the 1H NMR spectrum (Fig 7 (a)). These double bonds were still numerous after grafting,
because it only disappears by type II grafting after the addition of phenyl radical to the double
bonds. Meanwhile, in type I grafting, the double bonds remained in the corresponding grafted
monomer units after the allylic H abstraction. These double bonds were also responsible for two
peaks in the 13C NMR spectrum at δC 124.2 ppm (carbons 3) and δC 134.5 ppm (carbons 2) (Fig 7
(b)). 3.2.1 NMR Spectroscopy As expected from the low grafting rate, new NMR signals related to the grafted monomer
units were scarce and of weak intensity. The broad tiny peak in the range of δH 7.33-7.42 ppm in
the 1H NMR spectrum was characteristic for the proton of the phenyl rings attached the NR chains
after type II grafting. The very weak intensity of this broad peak shows that type II grafting was
minor. In the 13C NMR spectrum, as expected, the new corresponding peaks were hardly detectable
at δC 127.12-128.42 ppm due to the much lower 13C abundance. The new methyne protons related to the grafting sites were not detected in the 1H NMR
spectrum, most likely because this peak was overlapped by other peaks (1,4). However, in the 13C
NMR spectrum, two tiny new peaks were characteristic for these grafted sites. The first one at δC
31.19 ppm corresponded to the carbons 4’ of the methyne group attached to PHB during type I
grafting. This assignment is in good agreement with the chemical shift calculated by ChemDraw
Ultra simulation (δC 32.8 ppm) for this methyne carbon. It is important to note that the chemical
shift (δC 40 ppm) calculated for the methyne carbon 3” resulting from type II grafting occurs at a
significantly higher chemical shift due to the attached phenyl ring. The intensity of the peak related
to this methyne carbon 3” was too low to be detected in the 13C NMR spectrum, as expected from
the hardly detectable phenyl carbons attached to the NR chains during type II grafting. The other new peak at δC 71.90 ppm corresponded to the quaternary carbon A’ of the
grafted PHB monomer units. As expected, the intensity of this new peak related to carbon A’ is
much lower than that of the peak for the related carbon 4’, because carbon A’ are quaternary carbon
not bearing any hydrogen atom. Here again, the quaternary carbon A” resulting from type II
grafting, which would appear at a slightly higher chemical shift, were too scarce to be detected on the 13C NMR spectrum. Therefore, the 1H and 13C NMR spectra of the grafted copolymer both
show that type I grafting prevailed and that the grafting rate was low, as expected because all the
grafted copolymers remained soluble in chloroform. the 13C NMR spectrum. 3.2.1 NMR Spectroscopy Therefore, the 1H and 13C NMR spectra of the grafted copolymer both
show that type I grafting prevailed and that the grafting rate was low, as expected because all the
grafted copolymers remained soluble in chloroform. (a)
(Type I NR-g-PHB)
(Type II NR-g-PHB) a) (a) (Type I NR-g-PHB)
(Type II NR-g-PHB) (Type I NR-g-PHB) (Type II NR-g-PHB) D
5
1,4
Acetone
(acetone)
e’
B
3
A
Solvent
Phenyl
1H chemical shifts of NR-g-PHB (ppm)
Related to NR monomer units:
5 (δH 1.61), 1,4 (δH 1.97)
3 (δH 5.04-5.05)
Related to PHB monomer units:
D (δH 1.20-1.21) B (δH 2.38-2.56)
A (δH 5.16-5.22)
New peaks after grafting:
Phenyl (δH 7.33-7.42) D
5
1,4
Acetone
(acetone)
e’
B
3
A
Solvent
Phenyl
1H chemical shifts of NR-g-PHB (ppm)
Related to NR monomer units:
5 (δH 1.61), 1,4 (δH 1.97)
3 (δH 5.04-5.05)
Related to PHB monomer units:
D (δH 1.20-1.21) B (δH 2.38-2.56)
A (δH 5.16-5.22)
New peaks after grafting:
Phenyl (δH 7.33-7.42) 1H chemical shifts of NR-g-PHB (ppm) D
1H chemical shifts of NR-g-PHB (ppm)
Related to NR monomer units:
5 (δH 1.61), 1,4 (δH 1.97)
3 (δH 5.04-5.05)
Related to PHB monomer units:
D (δH 1.20-1.21) B (δH 2.38-2.56)
A (δH 5.16-5.22)
New peaks after grafting:
Phenyl (δH 7.33-7.42) Related to NR monomer units:
5 (δH 1.61), 1,4 (δH 1.97)
3 (δH 5.04-5.05)
Related to PHB monomer units:
D (δH 1.20-1.21) B (δH 2.38-2.56)
A (δH 5.16-5.22)
New peaks after grafting:
Phenyl (δH 7.33-7.42) (b) Figure7: Enlarged NMR spectrum for (a) 1H and (b) 13C of NR-g-PHB. D
4 5
1
4’
B
A
A’
Phenyl
C
3
2
Solvent
13C chemical shifts of NR-g-PHB (ppm)
Related to NR monomer units:
5 (δC 22.42), 4 (δC 25.38), 1 (δC 29.89),
3 (δC 124.2), 2 (δC 134.5)
Related to PHB monomer units:
D (δC 18.75), B (δC 39.78)
A (δC 66.61), C (δC 168.15)
New peaks after grafting:
4’ (δC 31.19)
A’ (δC 71.90)
Phenyl (δC 127.12-128.42) Figure7: Enlarged NMR spectrum for (a) 1H and (b) 13C of NR-g-PHB. To further confirm assignment of the new peaks resulting from grafting, all carbon signals
were characterized using DEPT 90 and 135 experiments. As shown in Figure 8 (a), four methyne
signals were observed in the DEPT 90. 3.2.2 FTIR Spectroscopy Figure 12 show the FTIR spectra of pristine NR and PHB. PHB showed several peaks as
presented in Figure 12 (a). The most important characteristic peak in the PHB spectra is the
carbonyl stretching vibration (C=O) which occurred at 1720 cm-1. The peak at 1099 cm-1
corresponded to C-O-C stretching vibration and the peaks at 2932 and 2974 cm-1 are assigned to
sp3 –CH bond respectively. In addition, the various modes of CH3 deformation give rise to peaks
at 1453 cm-1 (asymmetric deformation) and 1379 cm-1 (symmetric deformation). All the data here
closely resembled data obtained by previous studies [30, 35]. In addition, Figure 12 (b) showed
the FTIR spectrum of NR. The NR spectrum confirmed the presence of the C=C bond at 1661 cm-
1 (stretching vibration of medium intensity) and (=C-H) out-of-plane bending which causes the
peak at 833 cm-1. Other peaks occur at 2961 cm-1 which refers to the asymmetric vibration of CH3,
at 2920 cm-1 which is attributed to the asymmetric vibration of CH2 and at 2852 cm-1 which
corresponds to the symmetric vibrations of CH3 and CH2. On the other hand, the peaks at 1375 cm-
1 and 1446 cm-1 represents the CH in-plane deformation related to the NR double bond and CH2
bending, respectively. Subsequently, Figure 12 (c) showed a typical NR-g-PHB spectrum prepared using 7 % mol
of BPO. As can be seen here, both characteristic peaks of PHB and NR were observed in the NR-
g-PHB spectra. As an example, the peak for asymmetric vibration of -CH3 of NR was found at
2869 cm-1, meanwhile the peak for C=O of PHB was observed at 1720 cm-1. However, the peak at
1661 cm-1 related to the stretching vibration of the NR double bonds did not clearly appear in the
NR-g-PHB spectrum. However, from the NMR data, it was obvious that these double bonds were
still present in the grafted copolymer and this weak FTIR peak overlapped with the strong C=O
peak of PHB as illustrated in Figure 12 (c). Interestingly, the peak at 827 cm-1 characteristic for
the (=C-H) out-of-plane bending related to the NR double bonds was still clearly observed in the
spectrum of the grafted copolymer. 3.2.1 NMR Spectroscopy These signals resonated at δC 66.61 ppm, δC 31.19 ppm, δC
124.2 ppm and δC 127.12-128.42 ppm, which were assigned to the methyne carbon A, 4’, 3 and
phenyl rings attached to the NR main chain after type I and II grafting, respectively. The
assignment of the new peak related to the methyne carbon 4’ obtained after type I grafting was
thus confirmed by the DEPT 90 analysis. In contrast, three peaks for the methylene carbon (4, 1
and B) of NR-g-PHB showed negative signals in the DEPT 135 spectrum shown in Figure 8 (b). Meanwhile, in the positive region, the two methyl carbon signals (D and 5) of PHB and NR
resonated at δC 18.75 ppm and δC 22.42 ppm. As expected, similar methyne carbon signals as in
the DEPT 90 also appeared in the positive region of DEPT 135. As can be seen here, a new signal
at δC 71.90 ppm corresponding to carbons A’ did not appear in the DEPT 90 and 135, which proves that this signal corresponded to quaternary carbon atoms not bearing any hydrogen atom in good
agreement with the proposed structure in Figure 6. agreement with the proposed structure in Figure 6. Figure 8: DEPT (a) 90 and (b) 135 spectrum of NR-g-PHB. G1
Phenyl
A
4
4’
B
1
4’
(a)
(b)
A
3
Phenyl
3
D
5
’ (a) G1
Phenyl
4’
A
3 Figure 8: DEPT (a) 90 and (b) 135 spectrum of NR-g-PHB. A
4
4’
B
1
(b)
Phenyl
3
D
5
’ (b) Figure 8: DEPT (a) 90 and (b) 135 spectrum of NR-g-PHB
A
4
4’
B
1
(b)
Phenyl
3
D
5
’ Figure 8: DEPT (a) 90 and (b) 135 spectrum of NR-g-PHB. 3.2.2 FTIR Spectroscopy Its reduced intensity compared to that of pure NR was related
to presence of 40 wt% of PHB in the grafted copolymer and also to the reaction of some of the NR
double bonds during NR grafting. These findings corroborate with the NMR results from the
previous section. Furthermore, the change in the crystalline phase of PHB could further support that
modification occurred. As can be seen in Figure 12 (a), three prominent peaks attributed to the crystallinity of PHB occurred at 980 cm-1, 1230 cm-1 as well as 1720 cm-1 [6]. Noticeably, the peak
intensity attributed to crystalline PHB at 980 cm-1 and 1230 cm-1 for NR-g-PHB was reduced when
compared to PHB alone. In addition, an intense and broad shoulder at 1720 cm-1 attributed to the
amorphous region of PHB was observed in Figure 13. The entanglement of the grafted NR might
have obstructed the lamellar structure of the crystalline phase of PHB, resulting in a more
amorphous PHB. Wei et al. [6], discovered a similar observation when grafting PHB onto cellulose
causing a reduction in the intensity of the crystalline peak and a broad shoulder attributed to
amorphous PHB. Thus, it could be deduced from FTIR results, that NR had successfully modified
PHB. Figure 12: FTIR spectra of (a) pristine PHB, (b) NR and (c) typical of 7 % mol of BPO
initiator on NR grafting by PHB at 105 oC. Figure 12: FTIR spectra of (a) pristine PHB, (b) NR and (c) typical of 7 % mol of BPO
initiator on NR grafting by PHB at 105 oC. Figure 13: Expanded region of FTIR spectra for PHB and NR-g-PHB with various initiator
loading. Figure 13: Expanded region of FTIR spectra for PHB and NR-g-PHB with various initiator
loading. 3.4 Thermogravimetry Analysis (TG-DTG) TG and DTG thermograms of pristine NR, PHB and NR-g-PHB prepared using different
% mol of BPO initiator are presented in Figure 14 (a) and (b). The Tonset values as well as maximum
degradation temperature (Tmax) are summarized in Table 2. As can be seen, NR and PHB exhibited
a single stage thermal degradation profile, meanwhile a three stage thermal degradation profile
was found for NR-g-PHB prepared using 3, 5 and 10 % mol of BPO. However, a two stage thermal
degradation profile was observed when 7 % mol BPO was used. NR and PHB showed that the
Tonset1 occurred at 363 oC and 286 oC with Tmax1 values at 380 oC and 292 oC respectively. Meanwhile, for the NR-g-PHB, the first stage of degradation temperature (Tonset1) occurred
at around 155-198 oC with Tmax1 at 209-232 oC. This stage is attributed to the short chain length of
PHB which is produced from the chain scission reaction. This finding was similar to Lee and
coworkers [17]. They discovered that the melt reaction of PHB/ENR starts with a random thermal
scission of long chain PHB molecules to shorter chains bearing carboxyl ends. The second
degradation stage which has a Tonset2 at 272-285 oC and Tmax2 of 297-312 oC was attributed to the
grafted portion of PHB chains. The value of Tonset2 for the NR-g-PHB prepared using 3, 5, 7 and
10 % mol of BPO was slightly lower when compared to Tonset1 of neat PHB. This phenomenon is due to the heat transfer effect from the shorter PHB chains (first stage) which triggered the Tonset
at the second stage to occur earlier. Finally, the last stage has a Tonset3 in the temperature range of
363-380 oC with a Tmax3 between 412-427 oC. This degradation stage can be attributed to cross-
linked and grafted NR. The Tmax3 values of this stage is higher than pristine NR and PHB as shown
clearly in the derivative thermogravimetric graph. This indicated that the NR-g-PHB had improved
thermal stability compared to pristine NR and PHB. Furthermore, the amount of char residue of
NR and PHB was not found whilst it was about 1-5 % residual for the NR-g-PHB. In addition,
grafting with 7 % mol of initiator showed a large residual at around 5.13 %. This result
demonstrated the ability of NR-g-PHB to resist heat higher when compared to NR and PHB alone. 3.5 Differential Scanning Calorimetry (DSC) Figure 15 (a) and (b) showed the DSC heating curves of NR and PHB as well as NR-g-
PHB samples from -100 oC to 190 oC. The corresponding thermal data, including the glass
transition temperature (Tg), melting temperature (Tm), crystallization temperature (Tc) and the
calculated polymer’s crystallinity (χc) are listed in Table 3. Generally, the DSC curve in Figure 15
(a) showed only a single Tg at 8.6 oC for PHB, while the Tm peak of the purified PHB occurred at
172.1 oC. Similar result was obtained in a previous report [28]. In contrast, the measured Tg of NR
is -60.9 oC with the absence of Tm and Tc peaks. A single and distinct Tg was observed in all NR-g-PHB samples. The Tg for 3, 5, 7 and 10
% mol of BPO were 0.6 oC, -1 oC, 2.9 oC and -0.5 oC respectively. Herein, the appearance of single
a Tg indicated no phase separation and confirmed that grafting reaction occurred. In addition, the
7 % mol of BPO loadings showed a higher Tg probably due to the presence of the bulky grafted
PHB on the NR molecule which caused restriction on the chain mobility. Besides that, two Tm peaks were observed in all grafted samples as shown in Figure 15 (b). The Tm of all NR-g-PHB samples was slightly shifted to lower temperature as compared to pristine
PHB. For example, the Tm peaks for PHB was 172 oC, while for NR-g-PHB prepared using 3, 5,
7 and 10 % mol, Tm values were 143-154 oC, 141.3-153.4 oC, 141.2-155.1 oC as well as 144.7-
158.0 oC correspondingly. This occurrence may be due to the presence of ungrafted and shorter
PHB chains in the NR-g-PHB. Furthermore, the double melting behavior might be explained by
the dispersion of NR particles in PHB matrix that inhibited the crystallization of PHB, which lead
to the formation of imperfect crystals. Meanwhile, Xu et al. [36] revealed the formation of the
double Tm peaks was attributed to the melting of the crystals formed during the non-isothermal
crystallization for the first Tm peak, whereas the second Tm peak corresponded to the melting of
the crystals formed through recrystallization and reorganization of the crystals of the first Tm peak
during the subsequent DSC heating scans. Subsequently, pristine PHB has no crystallization peak temperature (Tc). 3.4 Thermogravimetry Analysis (TG-DTG) (a) 100
200
300
400
500
600
700
800
900
-20
0
20
40
60
80
100
120
Weight loss (%)
Temperature (
oC)
Natural Rubber
PHB
3 %
5 %
7 %
10 %
(a) 100
200
300
400
500
600
700
800
900
-20
0
20
40
60
80
100
120
Weight loss (%)
Temperature (
oC)
Natural Rubber
PHB
3 %
5 %
7 %
10 %
(a) 100
200
300
400
500
600
700
800
900
-20
0
20
40
60
80
100
120
Weight loss (%)
Temperature (
oC)
Natural Rubber
PHB
3 %
5 %
7 %
10 %
(a) 100
200
300
400
500
600
700
800
900
-20
0
20
40
60
80
100
120
Weight loss (%)
Temperature (
oC)
Natural Rubber
PHB
3 %
5 %
7 %
10 % Temperature (
oC) 100
200
300
400
500
600
700
800
900
-100
-80
-60
-40
-20
0
20
Derivative Weight loss (%)
Temperature (
oC)
NR
PHB
3 %
5 %
7 %
10 %
Figure 14: (a) TG and (b) DTG curves obtained for (a) NR and PHB as well as (b) NR-g-PHB
prepared using different mol % of BPO. (b) 100
200
300
400
500
600
700
800
900
-100
-80
-60
-40
-20
0
20
Derivative Weight loss (%)
T
(
oC)
NR
PHB
3 %
5 %
7 %
10 %
(b) (b) Figure 14: (a) TG and (b) DTG curves obtained for (a) NR and PHB as well as (b) NR-g-PHB
prepared using different mol % of BPO. Table 2: Thermal data of pristine NR, PHB and NR-g-PHB with different loading of BPO
initiator. Thermal data of pristine NR, PHB and NR-g-PHB with different loading of BPO
initiator Table 2: Thermal data of pristine NR, PHB and NR-g-PHB with different loading o
initiator. Table 2: Thermal data of pristine NR, PHB and NR-g-PHB with different loading of BPO
initiator. Different loading
of BPO
Tonset (TG curve)
Tmax (DTG curve)
Final Residual
T1
T2
T3
Tmax1
Tmax2
Tmax3
NR
363
-
-
380
-
-
0
PHB
286
-
-
292
-
-
0
3
195
272
371
209
297
425
1.76
5
198
279
364
215
290
425
3.28
7
-
283
363
-
310
412
5.13
10
155
285
380
232
312
427
1.88 3.5 Differential Scanning Calorimetry (DSC) 4.0 Conclusion In summary, this work describes a grafting study of PHB onto NR. The use of BPO enabled PHB
to be grafted onto NR in a simple process at relatively mild temperature for limiting PHB thermal
degradation. The properties of the NR-g-PHB materials could be tuned by varying reaction
conditions. Herein, 105 oC for reaction temperature with 7 % mol of BPO are the optimum
conditions for grafting NR with PHB. The grafting sites between PHB and NR were analyzed
using complementary NMR techniques and the reaction mechanism was confirmed by the
appearance of new 1H and 13C NMR peaks. Besides that, DEPT 90 and 135 also showed consistent
results with the proposed structure for the grafted copolymer. Additionally, FTIR show a consistent
trend with NMR analyses. Meanwhile, a single glass transition temperature was observed from
DSC, which indicated no phase separation occurred for the grafted samples. In addition, due to the
new bonds formed via grafting the thermal stability of NR-g-PHB was improved as compared to
pristine PHB and NR. 3.5 Differential Scanning Calorimetry (DSC) However, in the
case of NR-g-PHB prepared using 3, 5, 7 and 10 % mol, Tc occurs to around 77.3 oC, 87.5 oC, 90.3
oC and 85.9 oC respectively. It was obvious that Tc of the NR-g-PHB was always high, indicating
that the presence of NR played a role in accelerating the crystallization of PHB due to the
nucleation effect in the graft polymer. Similar phenomenon was reported elsewhere [36]. The values for degree of crystallinity were lower after grafting in comparison with the PHB 55 %. This
trend is due to the existence of amorphous NR which hindered the crystal structure of PHB [6]. This was in good agreement with the increased crystallization temperatures (Tc) of all grafted
copolymers as compared to bulk PHB. This was in good agreement with the increased crystallization temperatures (Tc) of all grafted
copolymers as compared to bulk PHB. (a) (a) a) (a) (b) Figure 15: DSC thermogram for NR, PHB and NR-g-PHB using various initiators 3, 5, 7 and
10 % mol. (b) Figure 15: DSC thermogram for NR, PHB and NR-g-PHB using various initiators 3, 5, 7 and
10 % mol. Table 3: Thermal transitions of NR, PHB and various NR-g-PHB. Table 3: Thermal transitions of NR, PHB and various NR-g-PHB. BPO
Loadings
2nd heating scan
Tg1
(oC)
Tm1
(oC)
Tm2
(oC)
TC
(oC)
χc (%)
NR
-60.9
-
-
-
-
3
0.6
143.0
154.0
77.3
25.0
5
-1.0
141.3
153.4
87.5
32.0
7
2.9
141.2
155.1
90.3
39.7
10
-0.5
144.7
158.0
85.9
40.6
PHB
8.6
172.1
-
-
55.0 Table 3: Thermal transitions of NR, PHB and various NR-g-PHB. Acknowledgement The authors would like to acknowledge (i) the financial support from research grant scheme (RU
1001/PKIMIA/8011071) and also ministry of higher education for scholarship. 5.0 References [1] R.A.J. Verlinden, D.J. Hill, M.A. Kenward, C.D. Williams and I. Radecka, Bacterial
Synthesis of biodegradable polyhydroxyalkanoates, Journal of Applied Microbiology, 102
(2007), 1437–1449. [2] P. Ma, D. G. Hristova-Bogaerds, P. J. Lemstra, Y. Zhang, S. Wang, Toughening of
PHBV/PBS and PHB/PBS blends via in situ compatibilization using dicumyl peroxide as a
free-radical grafting initiator, Macromolecular Materials Engineering, 297 (2012), 402–410 [2] P. Ma, D. G. Hristova-Bogaerds, P. J. Lemstra, Y. Zhang, S. Wang, Toughening of
PHBV/PBS and PHB/PBS blends via in situ compatibilization using dicumyl peroxide as a
free-radical grafting initiator, Macromolecular Materials Engineering, 297 (2012), 402–410
[3] M. I. Artsis, A. P. Bonartsev, A. L. Iordanskii, G. A. Bonartseva, G. E. Zaikov, [2] P. Ma, D. G. Hristova-Bogaerds, P. J. Lemstra, Y. Zhang, S. Wang, Toughening of
PHBV/PBS and PHB/PBS blends via in situ compatibilization using dicumyl peroxide as a
free-radical grafting initiator, Macromolecular Materials Engineering, 297 (2012), 402–410 [3] M. I. Artsis, A. P. Bonartsev, A. L. Iordanskii, G. A. Bonartseva, G. E. Zaikov,
Biodegradation and medical application of microbial poly (3-Hydroxybutyrate), Molecular
Crystallization Liquid Crystallization, 555 (2012), 232–262. Biodegradation and medical application of microbial poly (3-Hydroxybutyrate), Molecular
Crystallization Liquid Crystallization, 555 (2012), 232–262. [4] I. Manavitehrani, A. Fathi, H. Badr, S. Daly, A. N. Shirazi and F. Dehghani, Biomedical
applications of biodegradable polyesters, Polymers, 8 (2016), 1-32. [4] I. Manavitehrani, A. Fathi, H. Badr, S. Daly, A. N. Shirazi and F. Dehghani, Biomedical
applications of biodegradable polyesters, Polymers, 8 (2016), 1-32. [5] Ana P. Heitmanna, Patrícia S.O. Patrício, Italo R. Coura, Emerson F. Pedroso, Patterson P. [5] Ana P. Heitmanna, Patrícia S.O. Patrício, Italo R. Coura, Emerson F. Pedroso, Patterson P. Souza, Herman S. Mansur, Alexandra Mansur, Luiz C.A. Oliveira, Nanostructured Niobium
Oxyhydroxide Dispersed Poly (3-hydroxybutyrate) (PHB) films: Highly Efficient
Photocatalysts for Degradation Methylene Blue Dye, Applied Catalysis B: Environmental, 189
(2016), 141–150. Souza, Herman S. Mansur, Alexandra Mansur, Luiz C.A. Oliveira, Nanostructured Souza, Herman S. Mansur, Alexandra Mansur, Luiz C.A. Oliveira, Nanostructured Niobium
Oxyhydroxide Dispersed Poly (3-hydroxybutyrate) (PHB) films: Highly Efficient
Photocatalysts for Degradation Methylene Blue Dye, Applied Catalysis B: Environmental, 189
(2016), 141–150. [6] L. Wei, A. G. McDonald, N. M. Stark, Grafting of Bacterial Polyhydroxybutyrate (PHB)
onto Cellulose via In-Situ Reactive Extrusion with Dicumyl Peroxide, Biomacromolecules,
Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00049. [7] A.A. Ol’khov, L.S. Shibryaeva, Y. V. Tertyshnaya, A.N. Kovaleva, E.L. Kucherenko, A.L. Zhul’kina, and A.L. 5.0 References Iordanskii, Resistance to Thermal Oxidation of Ethylene Propylene
Rubber and Polyhydroxybutyrate Blends, International Polymer Science and Technology, 44
(2017), 6-10. [8] M. Avella, M. E. Errico, B. I. M. Malinconico, L. F. L. Paolillo, Preparation of Poly(β‐
hydroxybutyrate)/Poly(methyl methacrylate) Blends by Reactive Blending and Their
Characterization, Macromolecule Chemical Physic, 199 (1998), 1901-1907. [9] H. J. Chiu, J. W. You, Lamellar Morphology of Poly (3-hydroxybutyrate)/Poly (ethylene
oxide) Blends as Studied via Small Angle X-ray Scattering, Journal of Polymer Research, 10
(2003), 79–85. [10] D. Lovera, L. Ma´rquez, V. Balsamo, A. Taddei, C. Castelli, A. J. Mu¨ller, Crystallization,
Morphology, and Enzymatic Degradation of Polyhydroxybutyrate/Polycaprolactone
(PHB/PCL) Blends, Macromolecule Chemical Physic, 208 (2007), 924–937. [11] P. Greco, E. Martuscelli, Crystallization and Thermal Behaviour of poly(D-3-hyd
roxybutyrate)-Based Blends, Polymer, 30 (1989), 1475-1483. [12] J. T. Sakdapipanich, P. Rojrutha, Molecular Structure of Natural Rubber and Its
Characteristics Based on Recent Evidence, 13 (2012)214-238. [13] T. Jiang, P. Hu, Radiation Induced Graft Polymerization of Isoprene onto
Polyhydroxybutyrate, Polymer Journal, 33 (2001), 647-653. [13] T. Jiang, P. Hu, Radiation Induced Graft Polymerization of Isoprene onto
Polyhydroxybutyrate, Polymer Journal, 33 (2001), 647-653. [14] C. Chen, S. Peng, B. Fei, Y. Zhuang, L. Dong, Z. Feng, S. Chen, H. Xia, Synthesis and
Characterization of Maleated Poly(3-hydroxybutyrate), Journal of Applied Polymer Science,
88 (2003), 659 – 668. [14] C. Chen, S. Peng, B. Fei, Y. Zhuang, L. Dong, Z. Feng, S. Chen, H. Xia, Synthesis and
Characterization of Maleated Poly(3-hydroxybutyrate), Journal of Applied Polymer Science,
88 (2003), 659 – 668. [15] S. G. Hong, Y. C. Lin, C. H. Lin, Improvement of the Thermal Stability of [15] S. G. Hong, Y. C. Lin, C. H. Lin, Improvement of the Thermal Stability of Polyhydroxybutyrates by Grafting with Maleic Anhydride by Different Methods: Polyhydroxybutyrates by Grafting with Maleic Anhydride by Different Methods: ifferential Scanning Calorimetry, Thermogravimetric Analysis, and Gel Permeatio Differential Scanning Calorimetry, Thermogravimetric Analysis, and Gel Permeation
Chromatography, Journal of Applied Polymer Science, 110 (2008), 2718–2726. Differential Scanning Calorimetry, Thermogravimetric Analysis, and Gel Permeation
Chromatography, Journal of Applied Polymer Science, 110 (2008), 2718–2726. g
y
g
y
Chromatography, Journal of Applied Polymer Science, 110 (2008), 2718–2726. [16] P. Ma, X. Cai, X. Lou, W. Dong, M. Chen, P. J. Lemstra, Styrene-Assisted Melt Free-
Radical Grafting of Maleic Anhydride onto Poly(b-hydroxybutyrate), Polymer Degradation
and Stability, 100 (2014), 93-100. [16] P. Ma, X. Cai, X. Lou, W. Dong, M. Chen, P. J. [24] T. Saito, W. Klinklai, S. Kawahara, Characterization of Epoxidized Natural Rubber by 2D 5.0 References Lemstra, Styrene-Assisted Melt Free-
Radical Grafting of Maleic Anhydride onto Poly(b-hydroxybutyrate), Polymer Degradation
and Stability, 100 (2014), 93-100. [17] H. K. Lee, J. Ismail, H. W. Kammer, M. A. Bakar, Melt Reaction in Blends of Poly(3-
hydroxybutyrate) (PHB) and Epoxidized Natural Rubber (ENR-50), Journal of Applied
Polymer Science, 95 (2005), 113–129. [18] B. N. Misra, R. Dogra, I. Kaur, D. Sood, Grafting onto Starch. II.Graft Copolymerization of
Vinyl Acetate onto Starch by Radical Initiator, Journal Polymer Science: Polymer Chemistry
Edition, 18 (1980), 341-344. [19] S. Moolsin, N. Saksayamkul, A. N. Wichien, Natural Rubber Grafted Poly (methy
methacrylate) as Compatibilizer in 50/50 Natural Rubber/Nitrile Rubber Blend, Journal of
Elastomers and Plastics, 49 (2016), 422-439. [20] K. Formela, M. Marc, S. Wang, M. R. Saeb, Interrelationship between Total Volatile
Organic Compounds Emissions, Structure and Properties of Natural Rubber/
Polycaprolactone Bio-Blends Cross-linked with Peroxides, Polymer Testing, 60
(2017), 405-412. [21] F. Dghima, M. Bouaziz, I. Mezghani, M. Boukhris, M. Neffati, Laticifers identification and
Natural Rubber Characterization from The Latex of Periploca Angustifolia Labill. (Apocynaceae), Flora, 217 (2015), 90–98. [22] S. Kawaharaa, O. Chaikumpollerta, S. Sakuraib, Y. Yamamotoa, K. Akabori, Crosslinking
Junctions of Vulcanized Natural Rubber Analyzed by Solid-State NMR Spectroscopy
Equipped with Field-Gradient-Magic Angle Spinning Probe, Polymer, 50 (2009), 1626-
1631. [23] P. C. D. Oliveira, A. M. D. Oliveira, A. Garcia, J. C. D. Souza Barboza, C. A. D. Carvalho
Zavaglia, A. M. D. Santos, Modification of Natural Rubber: A Study by 1H NMR to Assess
The Degree of Graftization of Poly-DMAEMA or Poly-MMA onto Rubber Particles Under
Latex Form in The Presence of a Redox Couple Initiator, European Polymer Journal, 41
(2005), 1883–1892. [24] T. Saito, W. Klinklai, S. Kawahara, Characterization of Epoxidized Natural Rubber by 2D NMR Spectroscopy, Polymer, 48 (2007), 750-757. NMR Spectroscopy, Polymer, 48 (2007), 750-757. NMR Spectroscopy, Polymer, 48 (2007), 750-757. [25] S. Kawahara, J. Ukawa, J. Sakai, Y. Yamamoto, Y. Isono, High- Resolution Latex-State
13C-NMR spectroscopy for natural rubber vulcanizates, Rubber Chemistry And
Technology, 80 (2007), 751-761. [26] N. Choothong, K. Kosugi, Y. Yamamoto, S. Kawahara, Characterization of Brominated
Natural Rubber by Solution-state 2D NMR Spectroscopy, Reactive and functional
Polymers, 113 (2017) 6-12. [27] M. A. Hassan, E. K. Bakhiet, S. G. Ali, H. R. Hussien, Production and Characterization of
Polyhydroxybutyrate (PHB) Produced by Bacillus sp. Isolated from Egypt, Journal of
Applied Pharmaceutical Science, 6 (2016), 46-51. [28] V. U. Irorere, S. Bagherias, M. Blevins, I. Kwiecie, A. Stamboulis, I. Radecka, Electrospun
Fibres of Polyhydroxybutyrate Synthesized by Ralstonia eutropha from Different Carbon
Sources, International Journal of Polymer Science, 8 (2014), 1-11. [29] A. Brinda Devi, C. Valli Nachiyar, T. Kaviyarasi, Antony V. Samrot, Characterization of
Polyhydroxybutyrate Synthesized by Bacillus Cereus, International Journal Pharmaceutical
Science, 7 (2015) 140-144. [30] W.L.Tan, N.N. Yaakob, A. Zainal Abidin, M. Abu Bakar and N.H.H. Abu Bakar, Metal
Chloride Induced Formation of Porous Polyhydroxybutyrate (PHB) Films: Morphology,
Thermal Properties and Crystallinity, IOP Conf. Series: Materials Science and Engineering,
133 (2016), 1-12. [31] J. A. López, J. M. Naranjo, J. C. Higuita, M. A. Cubitto, C. A. Cardona, M. A. Villar,
Biosynthesis of PHB from a New Isolated Bacillus Megaterium Strain: Outlook on Future
Developments with Endospore Forming Bacteria, Biotechnology and Bioprocess
Engineering, 17 (2012), 250-258. [32] Y. Doi, M. Kunioka, Y. Nakamura, K. Soga, Proton and Carbon NMR Analysis of Poly(β-
hydroxybutyrate) Isolated from Bacillus Megaterium, Macromolecules, 19 (1986), 1271-
1276. [33] C. R. Arza, P. Jannasch, P. Johansson, P. Magnusson, A. Werker, F. H. J. Maurer, Effect of
Additives on The Melt Rheology and Thermal Degradation of Poly[(R)-3-Hydroxybutyric
acid], Journal of Applied Polymer Science, 132 (2015), 1-6. [34] E. Bugnicourt, P. Cinelli, A. Lazzeri, V. Alvarez, Polyhydroxyalkanoate (PHA): Review of Synthesis, Characteristics, Processing and Potential Applications in Packaging, eXPRESS
Polymer Letters, 8 (2014), 791–808. Synthesis, Characteristics, Processing and Potential Applications in Packaging, eXPRESS
Polymer Letters, 8 (2014), 791–808. Polymer Letters, 8 (2014), 791–808. [35] S. Rolere, S. Liengprayoon, L. Vaysse , J. Sainte-Beuve, F. Bonfils, Investigating Natural
Rubber Composition with Fourier Transform Infrared (FT-IR) Spectroscopy: A Rapid and
Non-Destructive Method to Determine Both Protein and Lipid Contents Simultaneously,
Polymer Testing, 43 (2015), 83-93. [36] C. Xu, Z. NMR Spectroscopy, Polymer, 48 (2007), 750-757. Qiu, Non-isothermal Melt Crystallization and Subsequent Melting Behavior of
Biodegradable Poly (hydroxybutyrate)/ Multiwalled Carbon Nanotubes Nanocomposites,
Journal of Polymer Science: Part B: Polymer Physics, 47(2009), 2238–2246. [35] S. Rolere, S. Liengprayoon, L. Vaysse , J. Sainte-Beuve, F. Bonfils, Investigating Natural
Rubber Composition with Fourier Transform Infrared (FT-IR) Spectroscopy: A Rapid and
Non-Destructive Method to Determine Both Protein and Lipid Contents Simultaneously,
Polymer Testing, 43 (2015), 83-93.
|
https://openalex.org/W2558873071
|
https://ojs.lib.uwo.ca/index.php/fpq/article/download/3047/2312
|
English
| null |
From Epistemic Responsibility to Ecological Thinking: The Importance of Advocacy for Epistemic Community
|
Feminist philosophy quarterly
| 2,016
|
cc-by
| 6,655
|
Recommended Citation
Maloney, Catherine. 2016. "From Epistemic Responsibility to Ecological Thinking: The Importance of Advocacy for Epistemic
Community."Feminist Philosophy Quarterly2, (2). Article 7. doi:10.5206/fpq/2016.2.7. Feminist Philosophy Quarterly
Volume 2
Issue 2 Fall 2016
Article 7
2016
From Epistemic Responsibility to
Ecological Thinking: The Importance of
Advocacy for Epistemic Community
Catherine Maloney
York University, cathy.maloney@utoronto.ca
Recommended Citation
Maloney, Catherine. 2016. "From Epistemic Responsibility to Ecological Thinking: The Importance of Advocacy for Epistemic
Community."Feminist Philosophy Quarterly2, (2). Article 7. doi:10.5206/fpq/2016.2.7. Feminist Philosophy Quarterly Volume 2
Issue 2 Fall 2016 Volume 2
Issue 2 Fall 2016 Article 7 From Epistemic Responsibility to
Ecological Thinking: The Importance of
Advocacy for Epistemic Community Catherine Maloney
York University, cathy.maloney@utoronto.ca Catherine Maloney York University, cathy.maloney@utoronto.ca Maloney: From Epistemic Responsibility to Ecological Thinking From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney Lorraine Code’s 1987 book Epistemic Responsibility connects the
epistemological realm with the ethical realm and develops the idea that knowing
well is possible when knowers are engaged in and can draw on the resources of an
epistemic community. Her work has been influential upon ensuing work in feminist
epistemology and social epistemology more generally, having helped to open the
way for discussions that challenge the notion of value- and context-independent
epistemology. Revisiting and building on the notion of epistemic responsibility in her
2006 book Ecological Thinking: The Politics of Epistemic Location, Code suggests that
while her earlier work was sound in its basic assumptions, it relied on “an
excessively benign conception of community, imagined without contest to provide
space for and uniform access to debate” (2006, vii). By contrast her 2006 work
provides a robust account of the social imaginary—both instituted and instituting—
and its connection to epistemic responsibility, and introduces advocacy as a crucial
mode of knowing across difference and destabilizing epistemologies of mastery. This
paper will highlight some of the common threads in the two works, as well as where
they diverge. I will approach this mini-genealogy by focussing on three concepts:
epistemic responsibility, which is central and common across both works; cognitive
interdependence which is common to both works, but undergoes a major
transformation in Ecological Thinking; and advocacy, which is entirely absent from
the discussion in Epistemic Responsibility. Concurrent to developing this mini-
genealogy of Code’s thought, I will consider how her work intersects with specific
aspects of the work of two other thinkers—Miranda Fricker’s hermeneutic injustice
and Mikhail Bakhtin’s creative understanding—as a means of bringing out the
nuances of Code’s argument and delving deeper into the troublesome issues of
knowing across difference, reflexive thinking, and destabilizing hegemonic ways of
knowing, all of which are integral to knowing well. I will conclude that advocacy as it
emerges in Ecological Thinking must include a dialogical process with the other that
leads both to and from greater self-understanding if it is to do the work of
destabilizing dominant modes of knowing; and further, I will conclude that advocacy
is both necessary for, and can only happen within, epistemic community. The idea that knowers are responsible for “what and how they know” (Code
2006, ix) shapes the landscape of both books. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney In Epistemic Responsibility, Code
introduces the idea that there is a moral element to knowing well beginning with
examples suggested from legal and political situations where not being informed of Published by Scholarship@Western, 2016 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 local laws or political situations is not enough to relieve a person from her burden of
duty to act correctly—for example, the legal obligation to drive on the correct side
of the road (1987, 1). From here Code builds to the more significant claim that
“knowers, or would-be knowers, come to bear as much of the onus of credibility as
‘the known’” (1987, 8–9). The shift she is effecting here is away from the
predominant epistemological tradition’s focus on “products [or] end-states of
cognition” (Code 1987, 8) and towards the act of knowing or “cognitive activity”. As
is now both well known and well understood, the import of this shift is in
acknowledging the particularity and locatedness of knowers and the consequent
impact this has on the construction of knowledge. What is known is no longer a
static object “waiting only to be read” (Haraway 1991, 198) and understood in the
same way by every passing knower (who is himself interchangeable with every other
generic knower), but rather is the result of a dynamic between the specific knower
and the known. It is a process of making meaning that is located in specificity but
still constrained by reality. This epistemological approach begun in Epistemic
Responsibility continues right through Ecological Thinking. As Christine Koggel writes
in her 2008 essay on Ecological Thinking, Code is able to “reveal the spaces between
realism and relativism” (178) and in this way allows access to previously discounted
knowledges without losing the ability to make judgments about the quality of what
is known. The shift in thinking that Code affected in her 1987 book remains central
to her work, and the work of many others, today. Where the two works begin to diverge is in how community or the cognitive
interdependence of knowers is construed. The inherently social nature of
knowledge and the resultant impossibility of opting out of epistemic community
(Code 1987, 188) remains in Ecological Thinking as a core tenet that opposes the
regulative principle that good knowing is autonomous knowing. http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney However, whereas
Epistemic Responsibility conceives the structure of epistemic community as a
combined form of life/contract/practice model in which one knowing subject has
more or less the same access to the resources of the community, Ecological Thinking
gives much greater weight to the role of testimony and considers its epistemological
weight within the context of communities of knowers who are governed by
particular social imaginaries. In opposition to the image of an autonomous knower
(2006, 171) Code makes the strong claim that “testimony makes knowledgeable
living possible” (2006, 173). The idea that each person’s knowledge base is
communally built from information that we have learned from others (e.g., the
location of the North and South Poles) is a shift away from the possibility of
autonomous knowing, but locating testimony and knowing within a social imaginary
is a radical reconfiguring of the epistemic landscape. In Epistemic Responsibility, the
knowing subject builds her understanding by experiencing the world and interacting
with others, and then processing that interaction within the creative synthesis of her http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 Maloney: From Epistemic Responsibility to Ecological Thinking Maloney: From Epistemic Responsibility to Ecological Thinking own imagination. By contrast, in Ecological Thinking, the knowing subject is
embedded in a relational framework of knowing that situates and shapes the limits
of understanding in a way that was not at issue with the more straightforward
cognitive interdependence of the earlier work—that is, in Ecological Thinking,
understanding happens within an ecosystem of meaning making. own imagination. By contrast, in Ecological Thinking, the knowing subject is
embedded in a relational framework of knowing that situates and shapes the limits
of understanding in a way that was not at issue with the more straightforward
cognitive interdependence of the earlier work—that is, in Ecological Thinking,
understanding happens within an ecosystem of meaning making. At the end of Code’s 1991 book What Can She Know, she outlines the
framework and potential benefits of an ecological model of knowing. She writes that an ecological model can shift epistemological inquiry away from autonomy-
obsession toward an analysis explicitly cognizant of the fact that every
cognitive act takes place at a point of intersection of innumerable relations,
events, circumstances, and histories that make the knower and the known
what they are, at that time. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney (269) This suggestion of the benefits of an ecological model of knowing announced at the
end of the 1991 book is fully developed in the 2006 book and is a marked shift from
the framework of Epistemic Responsibility. The knower is now bound to and by the
limits of her community’s imaginary, complete with all its sociopolitical implications. The addition of a sociopolitical dimension to what was formerly simply an ethico-
epistemological discussion adds a layer of significant complexity to the possibility of
knowing across difference. Having brought in the sociopolitical dimension of cognitive activity,
Ecological Thinking diverges even further from Epistemic Responsibility with the
introduction of the need for advocacy as an element of responsible knowing. Recognising the imbalance of power between various epistemic communities and
the impact that this imbalance has on the acceptance of testimony, advocacy
becomes a necessary tool. Code writes that advocacy practices work to get at truths operating imperceptibly, implicitly,
below the surface of the assumed self-transparency of evidence. They can be
strategically effective in claiming discursive space for ‘subjugated
knowledges,’ putting such knowledge into circulation where it can claim
acknowledgement, working to ensure informed, emancipatory moral-
political effects. (2006, 176) Advocacy becomes a means of “developing an instituting social imaginary” (2006,
170) which is capable of destabilising the instituted imaginary and making space for
subjugated ways of knowing. Responsible knowing takes on more aspects under this
view of advocacy practices than it did in Epistemic Responsibility. In addition to the
increased complexity of the epistemic terrain in the latter work, a responsible Published by Scholarship@Western, 2016 Published by Scholarship@Western, 2016 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 knower now has a responsibility beyond her own imperative to know well. Now a
responsible knower has the additional duty of “putting such knowledge into
circulation” (Code 2006, 176). This may mean acting as an advocate under
appropriate circumstances, but more importantly it means being attentive to
difficult, uncomfortable, and potentially even seemingly incoherent testimony. It
means listening to and taking seriously the testimony of others, even when that
testimony seems at first hearing to have no merit—perhaps because it is so far
outside the hearer’s own imaginary. This may be the testimony of first-hand
experiencers or of second-hand advocates. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney Advocacy becomes an
irreplaceable element of responsible epistemic community. But how is advocacy possible at all when knowing across difference in the
first place is so difficult? That is, how does the advocate come to understand? Further, how does advocacy function to destabilize an instituted imaginary when the
testimony of the original experiencer was not able even to enter the ring with the
dominant or centralizing imaginary, let alone unsettle it? In chapter six of Ecological
Thinking, Code brings in the concept of “imaginative empathy” (231). In contrast to
the imagination’s creative synthesis in Epistemic Responsibility, Code is here
excruciatingly aware of the pitfalls and difficulty entailed in understanding across
difference. She writes that “‘We’ may indeed be imaginative creatures, but
prototypes and hegemonic imaginaries block responsible imaginings at least as
frequently as they enable them” (2006, 229). While Code concludes the chapter with 1 During the presentation of a version of this paper at CSWIP 2015, Naomi Scheman
suggested that there may in fact be an additional problem to consider in relation to
advocacy. That is, that those advocated for may not benefit from having their
experiences made comprehensible within the dominant social imaginary. This
reminded me of a woman’s testimony as recounted in the Irish documentary The
Love That Dare Not Speak Its Name, by Bill Hughes, which screened on the Irish
television station RTE in 2000. In one particular scene a woman recounted her
experience of living as a lesbian before and after her experience became more
widely understood in her country. Previously she had been able to “fly under the
radar” and, for example, easily check in to B&Bs and other establishments with her
female lover without problem, because the social imaginary at the time she was
speaking of did not include the possibility of two women being anything more than
platonic friends. After a shift in public understanding, however, her relationship
became visible, and she no longer had the freedom she had previously enjoyed. While I would certainly not want to trivialise the difficult and often violent eras that
becoming visible can entail for individuals in newly visible groups (consider the
recent and tragic events in Orlando), nor would I want to suggest that remaining
invisible is ultimately a desirable alternative (at least as it plays out in this example—
consider that same-sex marriage is now legal in Ireland). From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney Code is quick to acknowledge that advocacy faces at least two legitimate
obstacles: the first is a reputation that is at odds with responsible knowing, when for
instance it is used to obfuscate the truth (think for example of the tobacco lobby);
and the second is the very real worry that advocacy is “tantamount to paternalism”
(2006, 179). While acknowledging that both are dangers, Code states that neither is
inevitable (2006, 178). The response to the first point is fairly straightforward. That
is, yes, advocacy has been and sometimes is not epistemically responsible, but this is
of course not the type of advocacy under discussion. It would be quite easy to
discern between a self-interested “advocacy” that aims only to promote its own
ends and one that aims to uncover truths that are uncomfortable and disruptive to
dominant ways of knowing. The former is monologic and the latter dialogic. Thinking
through a frame that is both epistemically responsible and, now, ecologically
sensitive, advocacy in this latter, dialogic form—in opposition to the autonomy
ideal—would pay close attention to testimony while mapping the “interrelations,
consonances, and contrasts” (Code 2006, 51) involved. The second concern Code raises regarding paternalism is tougher, but still
not insurmountable. In contrast to a paternalistic practice that would keep the
subject of its actions reliant on or beholden to a master, advocacy that is
epistemically responsible should ultimately yield autonomy; an “autonomy
remodelled” as Code writes (2006, 195). That is, while advocacy can seem to strip
those who are spoken for of their autonomy, by using whatever dialogical power the
advocate has (i.e., her greater credibility) to push against “intransigent imaginaries”
(Code 2006, 178), advocacy can in fact bring formerly silenced voices forward for the
first time. Code writes that “when advocacy is effective, those advocated for may
come to be well placed to claim the autonomy of acknowledged knowledgeability”
(2006, 180). So for example, drawing on one of Code’s examples, when the Harvard
Women’s Health Watch wrote an article corroborating the experiences of women
with Syndrome X, the credibility of the testimony of people with this illness http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 Maloney: From Epistemic Responsibility to Ecological Thinking increased (2006, 193).1 The social-political complexity that Code’s articulation of
autonomy, testimony and advocacy in Ecological Thinking adds to the concept of
epistemic community in her earlier work is transformational. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney 7 the suggestion that her argument is in fact inconclusive and further that her
“epistemic stance . . . enjoins skepticism about the possibility of understanding
across differences” (2006, 233), she goes some way in addressing both the first and
second of the questions above. Starting with the first question, the advocate comes
to understand across difference by engaging in at least three practices: by shifting
the fulcrum of understanding, by remaining in the in-between space of commonality
and particularity, and by remaining committed to contextual and relational modes
of understanding. Starting with the first practice, imaginative empathy of the kind
that Code wants to endorse “is less about knowing than about believing” (2006,
231). This means that understanding across difference, which opens the possibility
of advocacy, requires open or willing listening of the kind that Jay Lampert, writing
about Gadamer’s hermeneutics, refers to when he says that to engage dialogically
an interpreter must “freely invite aliens into one’s home” (1997, 359). A person who
wants to understand across difference cannot do so without opening herself to new
possibilities. This involves risk to the knower’s own ways of perceiving and sense of
self, but without this open belief of the other, knowledge remains only a monologic
repetition of the knower’s own understanding. The second practice is what Code
calls “the productive (thus not aporetic) tension” (2006, 228) of thinking both in
particulars and in commonalities. Without inhabiting this in-between space, a
knower falls into one of two extremes: universal thinking that is unaccountable for
its social-political location or “radical particularity” that becomes incommensurable
to the point of unknowability (Code, 228). Finally, imaginative empathy requires a
commitment to contextual and relational thinking. Part of understanding
ecologically means understanding the system in which an embodied subject lives
and operates: “she or he has to be understood . . . as situated within the habitus or
ethos of a society” (2006, 232). It is neither desirable nor possible to understand a
subject stripped of context. Understanding across difference requires all three of
these practices in order to have a chance of succeeding. The second question about advocacy’s ability to destabilize an instituted
imaginary relies in large part on the effectiveness of listening practices and
willingness “to engage with the affective dimensions of lives situated outside the
norms of epistemic sameness” (Code 2006, 233). From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney In either case however, the
idea that advocacy can be problematic in this way is certainly worth pursuing
further. For the purpose of this paper, please consider that advocacy practices refer
to situations in which it is either clearly beneficial for the person or groups in
question, as I believe it is in the example of Syndrome X, or at least arguably
beneficial. 1 During the presentation of a version of this paper at CSWIP 2015, Naomi Scheman
suggested that there may in fact be an additional problem to consider in relation to
advocacy. That is, that those advocated for may not benefit from having their
experiences made comprehensible within the dominant social imaginary. This
reminded me of a woman’s testimony as recounted in the Irish documentary The
Love That Dare Not Speak Its Name, by Bill Hughes, which screened on the Irish
television station RTE in 2000. In one particular scene a woman recounted her
experience of living as a lesbian before and after her experience became more
widely understood in her country. Previously she had been able to “fly under the
radar” and, for example, easily check in to B&Bs and other establishments with her
female lover without problem, because the social imaginary at the time she was
speaking of did not include the possibility of two women being anything more than
platonic friends. After a shift in public understanding, however, her relationship
became visible, and she no longer had the freedom she had previously enjoyed. While I would certainly not want to trivialise the difficult and often violent eras that
becoming visible can entail for individuals in newly visible groups (consider the
recent and tragic events in Orlando), nor would I want to suggest that remaining
invisible is ultimately a desirable alternative (at least as it plays out in this example—
consider that same-sex marriage is now legal in Ireland). In either case however, the
idea that advocacy can be problematic in this way is certainly worth pursuing
further. For the purpose of this paper, please consider that advocacy practices refer
to situations in which it is either clearly beneficial for the person or groups in
question, as I believe it is in the example of Syndrome X, or at least arguably
beneficial. Published by Scholarship@Western, 2016 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney A knower who is committed to
understanding across difference in this way is open to the extra-cognitive elements
of the other’s story and by so being allows that understanding to be transformative. Certain types of narratives can leave a knower “pulled up short by the text”
(Gadamer, 268) and, as Jennifer Geddes writes, help “us unlearn what we have
presumed to know or to be able to imagine” (Geddes, quoted in Code 2006, 234). The role of the advocate in this connection is two-fold: first, she must engage in the
practices that make imaginative empathy possible in the first place, and second, she
must devise means of provoking particularly tenacious beliefs in the wider social The second question about advocacy’s ability to destabilize an instituted
imaginary relies in large part on the effectiveness of listening practices and
willingness “to engage with the affective dimensions of lives situated outside the
norms of epistemic sameness” (Code 2006, 233). A knower who is committed to
understanding across difference in this way is open to the extra-cognitive elements
of the other’s story and by so being allows that understanding to be transformative. http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 Maloney: From Epistemic Responsibility to Ecological Thinking Maloney: From Epistemic Responsibility to Ecological Thinking imaginary. This is neither a simple nor unproblematic task, but one can see potential
for a way forward. One way in which Code’s work is particularly salient to me is in its connection
to my own paid work at the Centre for International Experience at the University of
Toronto. One of my core projects at U of T is developing programming to promote
intercultural learning among University of Toronto students. What this means in
practice is helping students to develop (or improve) epistemically responsible
practices in their thinking about their own cultural positionality and then, only in the
context of self-reflexive practices, to consider the cultural positionality of others. The majority of the students I have worked with so far are undergraduates, although
I am increasingly working with graduate students and sometimes staff and faculty. These students come from different years and programs of study and widely
different life experiences. They are both domestic and international students; some
are going out on exchanges or other mobility programs, and others are participating
in internationalization at home opportunities. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney (3) In relation to the evolution of Code’s work, it further solidifies the
importance, and necessity, of advocacy for responsible epistemic community. importance, and necessity, of advocacy for responsible epistemic community. Miranda Fricker’s work explicitly works through the idea that an experiencer
may not fully understand her own experience due to social power structures. Fricker’s “epistemic injustice” functions as what Code has called a “conceptual
apparatus” (2008, 32) for thinking through the link between the social imaginary and
harms done to individuals in their capacity as knowers. “Hermeneutic injustice”—
one of the “two forms of epistemic injustice” which Fricker identifies as
“distinctively epistemic in kind” (2007, 1)—in particular connects gaps in the
“collective hermeneutic resource” (2007, 168) to harm, and Fricker further points
out that inequity in social power tends to “skew shared hermeneutical resources” so
that the powerful have access to shared conceptions of their experiences whereas
the less powerful “are more likely to find themselves having some social experiences
through a glass darkly” (2007, 148). This leaves the less socially powerful vulnerable
to abuses of power and, significantly, seemingly incoherent in their expressions of
meaning. The explicit link between social positionality and vulnerability to epistemic
harm that Fricker develops is particularly compelling and useful. One of Fricker’s
examples of this is the story of Carmita Wood, a woman who experienced sexual
harassment in her workplace at a time when that concept was not part of the
Western social imaginary. As a result of her situation Wood developed health
complications, left her job, and was not able to receive any kind of compensation as
she could not articulate the connection between her workplace environment and
her poor health. In her memoir, Susan Brownmiller writes, “When the claims
investigator asked why she had left her job after eight years, Wood was at a loss to
describe the hateful episodes. She was ashamed and embarrassed. Under
prodding—the blank form needed to be filled in—she answered that her reasons
had been personal. Her claim for unemployment benefits was denied” (1999, 280–
281) While I find this a striking example of a case of hermeneutic injustice at work, I
have never quite been comfortable with Fricker’s claim that Wood herself did not
have a grip on her own experience. José Medina makes a criticism of this aspect of
Fricker’s argument. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney Many of these students are very
sophisticated in their thinking about understanding across differences, and I have
learned quite a lot from them; for others, the idea of knowledge as socially and
politically constructed is a big shift in thinking. An analogically rich metaphor like
Code’s ecological thinking is useful in helping students to think through the
relational nature of what they know; however, the implications of relational or
interdependent knowing are, unsurprisingly, not always clear. I mentioned in my
introduction that I wanted to put Code’s work in conversation with both Miranda
Fricker and Mikhail Bakhtin’s work, as a means of bringing out the nuances of her
argument and thinking through what I consider some troublesome bits of knowing
across difference. To do this, I will consider the claim found in all three thinkers that
an individual does not always understand her own experience. While it is common
across all three, this claim does however arise differently in each of their work: In
Code’s work, not fully understanding one’s own experience is an implicit
concomitant of understanding within the framework of a social imaginary. In
Fricker’s work it is an explicit component of her unpacking of hermeneutic injustice. Within Bakhtin’s work it is explicit in his claim that outsideness is necessary for self-
understanding. In one way this claim is an unproblematic statement of fact, but
taken another way it can seem to lead to the kind of paternalism that Code was
concerned to avoid in her discussion of advocacy. Thinking through this claim does a
few different things: (1) It complicates the idea of advocacy in an interesting way. Advocacy may not only be about helping others to see what they are missing;
rather, advocacy—and the dialogical process it necessitates—may also do work to
help the experiencer understand herself more fully. (2) It helps to unpack the way
social imaginaries both allow for understanding and limit it. For the students I work
with, this can be helpful in showing more clearly the implications of relational Published by Scholarship@Western, 2016 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 knowing. (3) In relation to the evolution of Code’s work, it further solidifies the
importance, and necessity, of advocacy for responsible epistemic community. knowing. http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney He points out that caution should be exercised in tying “too
closely people’s hermeneutical capacities to the repertoire of available terms and
coined concepts, for oppressed subjects often find ways of expressing their suffering
well before such articulations are available” (2012, 208–209). Indeed Wood did
know that something was wrong, but she couldn’t coherently express it either to
herself or to others. What I find interesting and particularly useful for the current
investigation is the way that Wood did finally make sense of her experience. It was
in dialogue with others: http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 Maloney: From Epistemic Responsibility to Ecological Thinking Maloney: From Epistemic Responsibility to Ecological Thinking “Lin’s students had been talking in her seminar about the unwanted sexual
advances they’d encountered on their summer jobs,” Sauvigne relates. “And
then Carmita Wood comes in and tells Lin her story. We realized that to a
person, every one of us—the women on staff, Carmita, the students—had
had an experience like this at some point, you know? And none of us had
ever told anyone before. It was one of those click, aha! moments, a profound
revelation.” . . . “We decided we had to hold a speak-out in order to break the
silence about this.” The “this” they were going to break the silence about had no name. “Eight of us were sitting in an office of Human Affairs,” Sauvigne remembers,
“brainstorming about what we were going to write on the posters for the
speak-out. We were referring to it as ‘sexual intimidation’, ‘sexual coercion,’
‘sexual exploitation on the job.’ None of those names seemed quite right. We wanted something that embraced a whole range of subtle and unsubtle
persistent behaviours. Somebody came up with ‘harassment.’ Sexual
harassment! Instantly we agreed. That’s what it was.” (Brownmiller 1999,
281) The others with whom Carmita Wood was in dialogue were primed to hear and work
through her experiences as they could draw on similar moments from their own
lives. It was only thinking through their experiences in community that they were
able to achieve the transformative moment of understanding. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney Considering this
example through the lens of Code’s imaginative empathy, all three elements were in
place: This group had no problem believing Wood’s experience in the absence of
knowing what it was; they could draw on the particularity of their own experience
but still see the commonality of the experience of the group; and they were
sensitive to the situated or contextual nature in which these experiences were
occurring. Thinking back to Code’s work on advocacy and the model of ecological
thinking generally, I think my concern with Fricker’s claim was not so much that I
couldn’t imagine that someone might not fully understand their own experience in
this case, but was more a worry about Fricker’s “reflexive hearer” slipping into a
paternalistic mode. What emerges when looking at this case through the lens of
Code’s concept of advocacy is a kind of mutual advocacy, enabled by dialogically
working through the issues in community. In their search for understanding of
Wood’s situation and their own, these women acted as advocates of sorts, both for
themselves and each other; they helped each other to form a coherent narrative
around their experiences that could be understood within the larger social
imaginary. Once this internal coherence or self-understanding was achieved they
could then embark upon advocacy in the usual sense. Armed with a name that Published by Scholarship@Western, 2016 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 seemed to fit their experiences and emboldened to draw in others with similar
experiences, their formerly silenced voices were now audible. seemed to fit their experiences and emboldened to draw in others with similar
experiences, their formerly silenced voices were now audible. Mikhail Bakhtin adds an extra dimension to the discussion of advocacy,
suggesting that understanding can really only occur in community. That is, others
are necessary for self-understanding and also for understanding across difference. In
his late essay “Response to a Question from the Novy Mir Editorial Staff,” Bakhtin
articulates a concept he calls “creative understanding.” Creative understanding is
both situated and requires “outsideness”: There exists a very strong, but one-sided and thus untrustworthy, idea that in
order better to understand a foreign culture, one must enter into it,
forgetting one’s own, and view the world through the eyes of this foreign
culture. . . . http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney If this were the only aspect of understanding it would merely be
duplication and would not entail anything new or enriching. Creative
understanding does not renounce itself, its own place in time, its own
culture; and it forgets nothing. In order to understand it is immensely
important for the person who understands to be located outside the object
of his or her creative understanding—in time, in space, in culture. For one
cannot even really see one’s own exterior and comprehend it as a whole, and
no mirrors or photographs can help; our real exterior can be seen and
understood only by other people, because they are located outside us in
space and because they are others. (1986, 6–7) Eschewing the notion that it is possible to see through the eyes of another, Bakhtin
indicates a commitment to situated knowing. He recognises that unsituated knowing
leads only to “duplication,” or repeating the located knowledge of the one who
claims to see universally. It seems curious then that at the same time as he
articulates a located concept of understanding he claims that in order to understand
ourselves we must gain an exterior view of ourselves. This exterior view turns out
not to involve leaving one’s own epistemic location, but engaging in a dialogic
encounter with others: In the realm of culture, outsideness is a most powerful factor in
understanding. It is only in the eyes of another culture that foreign culture
reveals itself fully and profoundly. . . . A meaning only reveals its depths once
it has encountered and come into contact with another, foreign meaning:
they engage in a kind of dialogue, which surmounts the closedness and one-
sidedness of these particular meanings, these cultures. We raise new
questions for a foreign culture, ones that it did not raise itself; we seek
answers to our own questions in it; and the foreign culture responds to us by http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 10 Maloney: From Epistemic Responsibility to Ecological Thinking Maloney: From Epistemic Responsibility to Ecological Thinking Maloney: From Epistemic Responsibility to Ecological Thinking revealing to us its new aspects and new semantic depths. Without one’s own
questions one cannot creatively understand anything other or foreign. . . . Such a dialogic encounter of two cultures does not result in merging or
mixing. Each retains its own unity and open totality, but they are mutually
enriched. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney (1986, 7) revealing to us its new aspects and new semantic depths. Without one’s own
questions one cannot creatively understand anything other or foreign. . . . Such a dialogic encounter of two cultures does not result in merging or
mixing. Each retains its own unity and open totality, but they are mutually
enriched. (1986, 7) The idea that meaning can only reveal itself through dialogue removes any hint of
paternalism in the suggestion that one may not understand one’s own experience
fully. Outsideness is necessary for all social understanding—whether that is self-
understanding or understanding another—and achieving outsideness happens
through engaging dialogically. Reflexive thinking can also only happen in community
as one’s own questions in dialogue with the questions of the other force reflexive
thinking in a way that thinking something through on one’s own simply cannot
achieve. Taking this passage together with Fricker’s concept of hermeneutic injustice
in mind and Code’s concept of advocacy as epistemically responsible and responsive
to testimony, some really important points arise here which are relevant to the
Carmita Wood situation. Far from making the paternalistic claim that the socially
subjugated need others to speak for them, Bakhtin’s understanding of
outsideness—which is clearly a located, socially positioned, outsideness—can help
to clarify the process of coming to understand (whether that is one’s own
experiences or someone else’s). Fricker’s “reflexive hearer” must be in conversation
with Wood, bringing her own understandings to the conversation and considering
where the disconnects and tensions in the story are. Through this dialogic-creative
process, the participants can potentially broaden both their own understanding and
that of the wider social imaginary. This is very similar to the “critical-creative
activity” (2006, 195) which Code’s robust account of the decentring “instituting
epistemic-moral-political imaginary” (2006, 35) accomplishes. She writes that ecology (metaphorically) draws the conclusions of situated inquiries
together, maps their interrelations, consonances, and contrasts, their
impoverishing or mutually sustaining consequences, from a commitment to
generating a creatively interrogative, instituting social imaginary to
denaturalise the instituted imaginary of mastery that represents itself as “the
[only] natural way” of being and knowing. (2006, 51) Ecological thinking as articulated here maintains the situated nature of knowledge,
maps the variety of complementary and contrasting situated knowledges, and
invokes the creativity and reflexivity required for decentering an instituted social
imaginary. From Epistemic Responsibility to Ecological Thinking:
The Importance of Advocacy for Epistemic Community
Cathy Maloney While the experiencer, in this case Carmita Wood, may not fully Published by Scholarship@Western, 2016 Published by Scholarship@Western, 2016 11 11 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 Feminist Philosophy Quarterly, Vol. 2 [2016], Iss. 2, Art. 7 understand her experience, she is a necessary part of the process of coming to
understand. The advocate also would not “have a grip” on the situation without the
dialogical-ecological encounter. Reading Code, Fricker, and Bakhtin together helps
me to see this more clearly and reaffirms the necessary role of advocacy within
epistemic community. understand her experience, she is a necessary part of the process of coming to
understand. The advocate also would not “have a grip” on the situation without the
dialogical-ecological encounter. Reading Code, Fricker, and Bakhtin together helps
me to see this more clearly and reaffirms the necessary role of advocacy within
epistemic community. Published by Scholarship@Western, 2016 References Bakhtin, Mikhail. 1986. “Response to a Question from the Novy Mir Editorial Staff.”
In Speech Genres and Other Late Essays, translated by Vern W. McGee and
edited by Caryl Emerson and Michael Holquist. 1–9. Austin: University of
Texas Press. Brownmiller, Susan. 1999. In Our Time: Memoir of a Revolution. New York: Dial
Press. Code, Lorraine. 2008. “Advocacy, Negotiation, and the Politics of Unknowing.” The
Southern Journal of Philosophy 46:32–51. ———. 2006. Ecological Thinking: The Politics of Epistemic Location. New York:
Oxford University Press. ———. 1991. What Can She Know: Feminist Theory and the Construction of
Knowledge. Ithaca, NY: Cornell University Press. ———. 1987. Epistemic Responsibility. Hanover, NH: University Press of New England
for Brown University Press. Fricker, Miranda. 2007. Epistemic Injustice: Power and the Ethics of Knowing. New
York: Oxford University Press. Gadamer, Hans-Georg. 2004. Truth and Method, 2nd rev. ed., translated by Joel
Weinsheimer and Donald G. Marshall. New York: The Continuum Publishing
Company. Haraway, Donna. 1991. “Situated Knowledges: The Science Question in Feminism
and the Privilege of Partial Perspective.” In Simians, Cyborgs and Women:
The Reinvention of Nature. 183–201. London: Free Association Books. and the Privilege of Partial Perspective. In Simians, Cyborgs and Women:
The Reinvention of Nature. 183–201. London: Free Association Books. Koggel, Christine. M. 2008. “Ecological Thinking and Epistemic Location: The Local
and the Global.” Hypatia 23 (1): 177–186. L
J
1997 “G d
d C
C l
l H
i
” Phil
hi
l Koggel, Christine. M. 2008. “Ecological Thinking and Epistemic Location: The Local
and the Global.” Hypatia 23 (1): 177–186. Lampert, Jay. 1997. “Gadamer and Cross-Cultural Hermeneutics.” Philosophical
Forum 28 (4-1): 351–368. Medina, José. 2012. “Hermeneutical Injustice and Polyphonic Contextualism: Social
Silences and Shared Hermeneutical Responsibilities.” Social Epistemology 26
(2): 201–220. http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 12 http://ir.lib.uwo.ca/fpq/vol2/iss2/7
DOI: 10.5206/fpq/2016.2.7 12 Maloney: From Epistemic Responsibility to Ecological Thinking CATHY MALONEY is a PhD candidate in the Department of Philosophy at York
University and the Manager, Intercultural Initiatives and Learning Strategy, at the
Centre for International Experience, University of Toronto. Her dissertation
research focusses on dialogical models for understanding across difference. Published by Scholarship@Western, 2016 13 13
|
https://openalex.org/W4233550199
|
https://journals.plos.org/plosone/article/file?type=printable&id=10.1371/journal.pone.0009077
|
English
| null |
Correction: Responses to Environmental Enrichment Differ with Sex and Genotype in a Transgenic Mouse Model of Huntington's Disease
|
PloS one
| 2,010
|
cc-by
| 11,810
|
Abstract doi:10.1371/journal.pone.0009077
Editor: Xiao-Jiang Li, Emory University School of Medicine, United States of America
Received November 10, 2009; Accepted January 17, 2010; Published February 12, 2010
Copyright: 2010 Wood et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to AJM from CHDI Foundation (www.highqfoundation.org). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: niw20@cam.ac.uk Copyright: 2010 Wood 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. Copyright: 2010 Wood et al. This is an open-access article distributed under the terms of the Creative Commons Attributi
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to AJM from CHDI Foundation (www.highqfoundation.org). The funders had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: niw20@cam.ac.uk Competing Interests: The authors have declared that no competing interests exist. * E-mail: niw20@cam.ac.uk Responses to Environmental Enrichment Differ with Sex
and Genotype in a Transgenic Mouse Model of
Huntington’s Disease Nigel I. Wood*, Valentina Carta, Stefan Milde, Elizabeth A. Skillings, Catherine J. McAllister, Y.L. Mabel
Ang, Alasdair Duguid, Nadeev Wijesuriya, Samira Mohd Afzal, Joe X. Fernandes, T.W. Leong, Jennifer
Morton Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 Abstract Background: Environmental enrichment (EE) in laboratory animals improves neurological function and motor/cognitive
performance, and is proposed as a strategy for treating neurodegenerative diseases. EE has been investigated in the R6/2
mouse model of Huntington’s disease (HD), where increased social interaction, sensory stimulation, exploration, and
physical activity improved survival. We have also shown previously that HD patients and R6/2 mice have disrupted circadian
rhythms, treatment of which may improve cognition, general health, and survival. Methodology/Principal Findings: We examined the effects of EE on the behavioral phenotype and circadian activity of R6/2
mice. Our mice are typically housed in an ‘‘enriched’’ environment, so the EE that the mice received was in addition to these
enhanced housing conditions. Mice were either kept in their home cages or exposed daily to the EE (a large playground box
containing running wheels and other toys). The ‘‘home cage’’ and ‘‘playground’’ groups were subdivided into ‘‘handling’’
(stimulated throughout the experimental period) and ‘‘no-handling’’ groups. All mice were assessed for survival, body
weight, and cognitive performance in the Morris water maze (MWM). Mice in the playground groups were more active
throughout the enrichment period than home cage mice. Furthermore, R6/2 mice in the EE/no-handling groups had better
survival than those in the home cage/no-handling groups. Sex differences were seen in response to EE. Handling was
detrimental to R6/2 female mice, but EE increased the body weight of male R6/2 and WT mice in the handling group. EE
combined with handling significantly improved MWM performance in female, but not male, R6/2 mice. Conclusions/Significance: We show that even when mice are living in an enriched home cage, further EE had beneficial
effects. However, the improvements in cognition and survival vary with sex and genotype. These results indicate that EE
may improve the quality of life of HD patients, but we suggest that EE as a therapy should be tailored to individuals. Citation: Wood NI, Carta V, Milde S, Skillings EA, McAllister CJ, et al. (2010) Responses to Environmental Enrichment Differ with Sex and Genotype in a Transgenic
Mouse Model of Huntington’s Disease. PLoS ONE 5(2): e9077. doi:10.1371/journal.pone.0009077 Citation: Wood NI, Carta V, Milde S, Skillings EA, McAllister CJ, et al. (2010) Responses to Environmental Enrichment Differ with Sex and Genotype in a Transgenic
Mouse Model of Huntington’s Disease. PLoS ONE 5(2): e9077. Introduction To convert the CAG repeat numbers
determined by GeneMapper technique to the CAG repeat number
determined by sequencing technique (which more closely represents
the true CAG repeat number) the following formula needs to be
applied: SEQ CAG no. (true CAG no.)= 1.0427 * GM CAG no. +
1.1695; personal communication, Dr J. Li, Laragen). To explain the beneficial effects of EE on motor and
cognitive symptoms and survival of HD transgenic mice,
several
potential
mechanisms
have
been
suggested. For
example, EE may cause an increase in striatal and hippocampal
levels of brain-derived neurotrophic factor (BDNF; [8]), levels
of which are known to be reduced in the striatum and
hippocampus of HD mouse models [9] and in the post mortem
brains of HD patients [10]. EE has also been found to
specifically increase neurogenesis in the dentate gyrus of the
hippocampus of a mouse model of HD [11]. Given that such
changes in hippocampal function are possible, and
that
hippocampus-dependent learning is impaired in R6/2 mice
[12], it is logical to expect that enrichment might have a
beneficial effect on hippocampal learning and memory tasks. However, while evidence for improved spatial learning in
response to EE has been found for the R6/1 model [13,14], no
such study has been conducted in R6/2 mice. For this reason,
the first aim of the current study was to investigate the effects of
EE on cognitive performance in the MWM task in R6/2 mice. Mice were kept in home cages comprising single sex, single
genotype groups of 10. Our mice live as standard in an enhanced
environment with increased amounts of bedding and nesting
materials, and additional hydrated food (see below). Clean cages
were provided twice weekly, with grade 8/10-corncob bedding,
finely shredded paper for nesting, and a red plastic nest box. The
mice were maintained on a 12 hour light: 12 hour dark cycle, at a
temperature of 21–23uC and a humidity of 55610%. The mice
had ad libitum access to water (using water bottles with elongated
spouts) and standard dry laboratory food (RM3(E) rodent pellets,
Special Diet Services, Witham, UK). In addition, once a day, a
mash was prepared by soaking 100 g dry food in 230 ml water
until the pellets were soft and fully expanded. The mash was
placed on the cage floor, improving access to food and water for
the R6/2 transgenic mice. Introduction of HD. Indeed, it has been suggested that physical, social and
cognitive stimulation has beneficial effects in HD patients [3,4]. HD is a genetic neurodegenerative disorder that is caused by an
expanded CAG repeat in the coding region of the HD gene [1]. The disease is characterised by progressive striatal atrophy and the
loss of neurons in frontal and temporal cortex, although by the end
stages of the disease degeneration is also present in many
subcortical regions. Patients with HD develop progressive motor,
cognitive and psychological symptoms that invariably lead to
death within 17–20 years after the onset of first symptoms. To study the mechanisms by which lifestyle elements influence
the progression of HD, aspects of an enhanced lifestyle can be
mimicked in laboratory animals by EE. There were two aims of
the current study. EE has been shown to have beneficial effects on
the progression of motor symptoms and survival in the R6/1 and
R6/2 mouse models of HD [5,6,7]. In these studies, EE was
provided through multiple different forms of enhanced home
cages, all of which had beneficial effects. An improved feeding
regime, accompanied by regular behavioural testing, significantly
enhanced the general well-being and life expectancy of R6/2 mice
[5]. An enriched home cage delayed onset of motor symptoms,
decreased severity of the clasping phenotype, and reduced the loss
of peristriatal cerebral volume in R6/1 mice [6]. Even a low level
of enrichment with food pellets and a cardboard tube placed in the There is no effective treatment available yet for HD. However,
it has been shown that an active lifestyle (involving enhanced
social, physical and mental components) protects against dementia
and Alzheimer’s disease in human patients (reviewed in [2]). It is
thus possible that increased environmental stimulation of patients
could be used to improve the symptoms and slow the progression PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 1 February 2010 | Volume 5 | Issue 2 | e9077 Enrichment in R6/2 Mice home cage slowed the decline in rotarod performance in R6/2
mice [7], although a greater level of enrichment induced a marked
improvement in rotarod tests, and delayed the loss of peristriatal
cerebral volume in R6/2 brain [7]. the CAG repeat number measured by GeneMapper differs from that
measured by sequencing. Introduction This feeding regime has been shown
previously to be beneficial [5]. g
p
The second aim of the current study was to investigate the
effects of sleep deprivation during circadian day. Disturbance of
the sleep-wake cycle is commonly observed in HD patients and is
mirrored by a progressive disintegration of circadian patterns of
activity and a disruption of circadian clock gene expression in the
suprachiasmatic nucleus (SCN) of R6/2 mice [15]. The treatment
of R6/2 mice with the sedative drug Alprazolam, a therapy
intended to restore their circadian rhythms, slowed cognitive
decline, and improved clock-gene related functions [16]. Modu-
lation of the sleep-wake cycle with Modafinil as well as Alprazolam
also had beneficial effects on cognitive function and improved
apathy [17]. This raises the possibility of an effective behavioural
therapy involving sleep regulation to manage the symptoms of HD
patients. Given that drug-induced wakefulness had beneficial
effects on the cognitive performance of R6/2 mice [17], a period
of continuous activity induced by enrichment might also impose
sleep without the need for sedative drugs and have similar
beneficial effects. The constraints of our animal facility mean that,
in the current study, enrichment had to take place during
circadian day. However, enrichment during circadian day might
result in sleep deprivation and thus lead to deleterious effects. To
test this possibility, we included groups in which we enforced
wakefulness on mice, by physically handling these mice to
keep
them
awake
throughout
the
6-hour
daily
period
of
experimentation. Environmental Enrichment The mice were tested in four different groups with variations in
environmental enrichment conditions: N Home cage/no handling
N Home cage/handling
N Playground/no handling
N Playground/handling N Home cage/no handling
N Home cage/handling
N Playground/no handling
N Playground/handling N Home cage/no handling N Home cage/handling N Playground/no handling N Playground/handling Each group comprised four cages, containing male WT (n = 10),
female WT (n = 10), male R6/2 (n = 10) and female R6/2 (n = 10)
mice. The timeline for testing and treatments for the whole
experiment is shown in Figure 1. Mice in the home cage/no handling groups were confined to
their home cages without additional enrichment throughout the
whole experiment. EE was provided to the other groups of mice
from 10 to 16 weeks of age for 6 hours each day (13:15h to
19:15h). Enrichment was
administered as either
access to
playgrounds
or
through
gentle
handling. The
playgrounds
consisted of large PerspexTM boxes (60630645 cm) containing
ropes, ladders, running wheels and toys (Movie S1). The toys and
their configuration were changed daily to maintain the element of
novelty, maximising the stimulating nature of the environment. After each session, playground cages and toys were cleaned using
1% acetic acid. Handling involved gentle manipulation of the mice
if they spent longer than 60 seconds immobile throughout the
period of enrichment (Movie S2). Ethics Statement All components of this study were carried out in accordance with
the UK Animals (Scientific Procedures) Act, 1986, and with the
approval of the University of Cambridge Licence Review Committee. Behavior and Handling Data Collection Records were kept throughout the daily experimental period
(1315h to 1915h) of the handling given and also of the behavioural
activity of the mice. For the groups receiving handling, individual
records were kept of every occasion on which each mouse required
handling. The behaviour of each mouse in every cage was
recorded by a trained observer every 15 minutes. Three or 4
observers were randomly assigned to observe the mice during a
specific 2-hour time slot within the daily experimental period (6
hours). Due to the overt phenotype of the R6/2 mice, observers
could not be blind to the genotype of the mice. The observed
behaviours were classified as ‘‘active’’ (score 1) or ‘‘inactive’’ (score February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org Morris Water Maze Task Performance in the probe trials was evaluated by measuring the
time spent in virtual quadrants and zones in the water maze,
proximity to the platform location and swim speed. For statistical
analysis of percent time spent in quadrants and zones of the water
maze, a five-way repeated measures ANOVA was used (factors:
sex, genotype, handling/no handling, home cage/playground and
quadrant or zone number). For statistical analysis of proximity and
swim speed we used a four-way repeated measures ANOVA
(factors: sex, genotype, handling/no handling, home cage/
playground). Group comparisons were made using Sidak- adjusted
multiple comparisons. Behavioural scores reflecting activity of the
mice during the experimental period were analysed using 4-way
repeated measures ANOVA as above. The number of handling
events was analysed using three-way repeated measures ANOVA
(factors: sex, genotype, playground/home cage) with subsequent
Sidak-adjusted multiple comparisons. The body weight data for
WT and R6/2 mice were analysed in separate three-way repeated
measures ANOVA, (factors: sex, playground/home cage and
handling/no handling). Survival data were analysed using a log
rank test. Spatial learning was tested using the protocol as described in
Wood et al. [20]. Briefly, a circular white plastic pool 120 cm in
diameter and 50 cm in height was filled to a depth of 30 cm with
water and maintained at 23uC. A small quantity of non-toxic white
paint was added to render the water opaque. Four positions
around the edge of the tank were arbitrarily designated as N, S, E,
and W, providing four alternative start positions and dividing the
tank into four quadrants: NE, SE, SW and NW. A circular clear
Perspex platform of 10 cm diameter was placed at the midpoint of
one of the four quadrants and submerged 0.5 cm below the water
surface. Extramaze cues were minimised by placing screens
around the tank. Various visible cues were added to the screens
to aid spatial discrimination. Mice were tracked in the maze using
the HVS tracker system (HVS Image 2020, Hampton, UK). During training, mice received four trials per day with an inter-
trial interval of 10–15 minutes. Each mouse was placed in the pool
facing the wall at one of four pseudorandomly chosen starting
positions (N, S, E, W), and allowed to swim until it located and
climbed onto the submerged platform. Any mouse that failed to
locate the platform within 60 seconds was placed on the platform
by hand. Body Weight and Survival Analysis Body weight of all mice was recorded twice weekly from the
start of treatment (age 9 weeks) throughout the experiment (6
weeks) and after that until death (for R6/2 mice) or 27 weeks of
age (for WT mice). Note that we have only presented R6/2
weights up to 19 weeks, since beyond this age mice started to reach
the end-stage of the phenotype, and were killed for humane
reasons. We have shown in previous studies that mice that lose the
most body weight tend to die first, and that this distorts weight
data [19]. Therefore, statistical analysis was conducted on R6/2
weights between the ages of 9–19 weeks while data from 9–27
weeks of age was used for the analysis of WT weights. Age of death was recorded for all R6/2 mice. Mice were killed if
they were moribund, or lacked a righting reflex, or failed to rouse
for their mash, or did not respond to gentle stimulation. Morris Water Maze Task All mice remained on the platform for 15 seconds, after
which they were briefly dried with paper tissues before being
returned to a cage containing clean shredded paper bedding, and
warmed by a heating lamp. On completion of all four trials, the
mice were dried thoroughly and returned to their home cages. A
probe trial consisted of a single 60 second trial with the platform
removed. After 60 seconds the mice were removed to a drying
cage as before. Statistical analyses were performed using SPSS Statistics 17.0
(SPSS Inc., Chicago, USA) or Prism 4 (GraphPad Software Inc.,
San Diego, USA). 0) and these scores were used for quantitative analysis of
behaviour. 0) and these scores were used for quantitative analysis of
behaviour. Baseline data were obtained from the mice at 9 weeks of age in
the MWM (see Figure 1) before they were assigned to their
experimental groups. Mice were tested under a standard protocol
(6 days training, with 4 trials a day, to a single platform position,
followed on day 7 by a single probe trial). After a two week period
of EE, mice received a single probe trial (retention), followed by
five days of training (4 trials per day) to the same platform position
as in the baseline test and a single probe trial (re-acquisition). After
a further two weeks of enrichment, mice underwent a third session
in the MWM. This took the same form as the second session
(single probe trial, then five days of training, then a further single
probe trial). During the second and third MWM sessions, mice
continued to be exposed to their respective enrichment condition. Training or testing in the MWM took place in the morning while
enrichment was provided in the second half of the light period
(13:15–19:15 hrs) as described above. After the end of the last
MWM session at 16 weeks of age, mice were returned to their
home cages. Mice Mice were taken from a colony of R6/2 transgenic mice [18] that
is established in the Centre for Brain Repair, University of
Cambridge, and maintained by backcrossing onto CBA x C57BL6
F1 female mice. Genotyping and CAG repeat length measurement
were carried out by Laragen (Los Angeles, CA, USA). The number
of CAG repeats of R6/2 mice used in this study (N= 80) was
24960.6 (mean 6 SEM) as determined by GeneMapper (note that PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 2 Enrichment in R6/2 Mice Figure 1. Experimental timeline for enrichment and testing. Baseline data in the MWM were obtained at 9 weeks of age. Further MWM
training and testing were performed at 12 and 15 weeks of age. Environmental enrichment took place between 10 to 16 weeks of age. doi:10.1371/journal.pone.0009077.g001 Figure 1. Experimental timeline for enrichment and testing. Baseline data in the MWM were obtained at 9 weeks of age. Further MWM
training and testing were performed at 12 and 15 weeks of age. Environmental enrichment took place between 10 to 16 weeks of age. doi:10.1371/journal.pone.0009077.g001 Morris Water Maze WT mice in all enrichment groups improved their
performance during the course of the experiment by swimming
closer to the original platform position (F(4,135) = 61.459, p,0.001;
Figure 4A). R6/2 mice, in contrast, showed no change in
proximity throughout the experiment, suggesting that the R6/2
mice were impaired in learning the task (Figure 4B). This
combined analysis revealed no overall effect of enrichment in
playgrounds on MWM performance in either WT (Figure 4C) or
R6/2 mice (Figure 4D), suggesting that EE had no effect on the
rate of learning of these mice. Throughout the experiment, WT
mice performed better than R6/2 mice, irrespective of sex or
enrichment (F(1,138) = 224.657, p,0.001; Figure 1E). ( ,
)
We measured swim speed in the MWM, as an index of motor
performance. Neither WT nor R6/2 mice showed any changes in
swim speed over time (Figure 5A, B). An analysis of main effects
showed that playground mice were faster swimmers than home
cage mice (F(1,141) = 8.100, p = 0.005). This effect was evident in
R6/2 mice from the first probe trial after the start of enrichment
and onwards (F(1,141) = 11.947, p,0.001; Figure 5D) but was not
seen in WT mice (Figure 5C). However, it was also found that, as
expected, R6/2 mice were consistently slower swimmers than WT
mice (F(1,141) = 105.580, p,0.001; Figure 5E). During each probe
trial, R6/2 mice spent more time floating than WT mice
(F(1,141) = 45.510, p,0.001; Figure 5F). There was, however, a
significant decrease in time spent floating by R6/2 mice over the
course of the experiment (p,0.001), suggesting that floating was
not a strategy adopted by R6/2 mice because their phenotype
made them too weak to swim. gy
g
In an analysis of the percent time spent in the target zone there
was a main effect of genotype (F(1,136) = 34.933, p,0.001). No
main effects were found for sex or handling (not shown) and so
data from both sexes and handling conditions were combined for
presentation in Figure 3. There was, however, a main effect of
playground exposure (F(2,405) = 5.738, p = 0.003). Post hoc analysis
revealed that this effect was present in WT (p = 0.014) but not R6/
2 mice (p = 0.309), suggesting a beneficial effect of enrichment on
the zone preference of WT mice. Morris Water Maze With the exception of the first
probe trial, WT mice spent significantly more time in the target
zone than R6/2 mice (Figure 3A). In the probe trials after training
in the first MWM session and after the retention interval before
the second training session, neither WT nor R6/2 mice showed a
preference for the target zone, irrespective of their enrichment
condition (Figure 3B, C). The preference for the outer zone of the
MWM observed in these trials in all groups of mice reflects the
initial response to the MWM that was also present in the first
training trial of the first MWM session (Figure S1B). During
training in the second MWM session, WT playground mice
developed a preference for the target zone while WT home cage
mice did not differentiate between the outer and the target zones
(Figure 3D). The same difference between home cage and
playground WT mice was found after the retention interval before
the third MWM session (Figure 3E). After training in the third
MWM session, both WT groups showed significant preference for
the target zone (Figure 3F ).Throughout all training sessions, R6/2
mice never showed a preference for the target zone (Figure 3A-F). Overall, data from the analyses of percent times spent in the target
quadrant and zone during probe trials suggests that WT mice
successfully learned the platform position while R6/2 mice
consistently failed to do so. While enrichment by playground
exposure did not affect the MWM performance of R6/2 mice,
WT playground mice showed a faster learning of the correct
MWM zone than WT home cage mice. However, we found no
effect of handling on either WT or R6/2 mice. Interestingly, when data from probe trials were pooled
throughout the experiment for analyses of main effects, we
found a significant interaction between sex and handling for
both platform proximity (F(1,141) = 21.540, p,0.001) and swim
speed (F(1,141) = 34.087, p,0.001). Handling had a beneficial
effect on the performance of female mice (p,0.001), but a
detrimental effect on the performance of males (p = 0.012;
Figure 6A), irrespective of genotype or playground exposure. Similarly,
handling
reduced
swim
speed
in
female
mice
(p = 0.002), but increased it in male mice (p,0.001; Figure 6B). Post
hoc
analyses
revealed
further
sex-specific
effects
of
enrichment. Morris Water Maze Traditional measures of performance in MWM probe trials
include percent times spent in the target quadrant and zone. PLoS ONE | www.plosone.org PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 3 Enrichment in R6/2 Mice Analysis of the percent time spent in the target quadrant revealed
a main effect of genotype (F(1,141) = 214.113, p,0.001). In all
probe trials, WT mice spent significantly more time in the target
quadrant than R6/2 mice (Figure 2A). There were no main effects
of sex, handling or playground exposure, either beneficial or
deleterious (not shown). This suggests that enrichment had no
influence on the performance of WT or R6/2 mice in the MWM
as measured by the time spent in the target quadrant. Thus, for
presentation, data from both sexes and all enrichment groups has
been combined for presentation (Figure 2). With the exception of
the probe trial after the first retention interval (session 2 probe 1),
WT mice displayed a significant preference for the target quadrant
in every probe trial (Figure 2B-F). This indicates learning of the
platform position as no preference for a particular quadrant was
observed in either WT or R6/2 mice when they were first exposed
to the MWM (Figure S1A). No preference for the target quadrant
was observed in R6/2 mice in any of the probe trials (Figure 2B-F). Interestingly, in the third MWM session, R6/2 mice showed a
significant preference for the quadrant right-adjacent to the target
quadrant (Figure 1E, F). Mice were placed into the pool from a
random starting position. However, the right-adjacent to the target
quadrant is the quadrant from which the experimenter ap-
proached to remove the mice from the pool after a trial, or to
guide them to the platform position during training. This suggests
some ability for spatial learning in the R6/2 mice, but they used a
strategy that the MWM is not designed to test. also measured proximity to the platform position in the probe
trials. An analysis of data from all probe trials revealed no main
effect of sex. Therefore, data from both sexes were combined for
the purpose of presentation (Figure 4). There was a main effect of
genotype. PLoS ONE | www.plosone.org Morris Water Maze Female R6/2 mice from all enrichment groups
showed better awareness of the platform position than the home
cage/no handling mice (home cage/handling: p,0.001, play-
ground/no handling: p = 0.03, playground/handling: p,0.001;
Figure 6E). This effect was not present in male R6/2 (Figure 6E)
or WT mice of either sex (Figure 6C). Thus, data from the
proximity analysis suggest that enrichment through access to
playgrounds or handling improves cognitive performance in
female mice only. A sex–specific effect of the enrichment
conditions was also found for swim speed in the MWM, where
both WT and R6/2 male mice increased their swim speed in
response to handling. This was found for the home cage (WT,
p = 0.001; R6/2, p = 0.041; Figure 6D, F) as well as for the
playground groups (WT, p = 0.004; R6/2, p = 0.044; Figure 6D,
F). Playground exposure without handling did not lead to
significant increases in swim speed of male mice compared to
home cage/no handling groups. Compared to male mice of
both genotypes, R6/2 females exhibited the opposite response
to enrichment conditions. They showed an increase in swim
speed in response to the playground condition but not in
response to handling. The positive effect of playground exposure Recent work has suggested that the proximity score (also known
as the Gallagher score) may be a more sensitive measure of
performance than these classical parameters [21]. Therefore, we PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 4 Enrichment in R6/2 Mice c
e t
6/
c Figure 2. Quadrant preference of mice during Morris water maze probe trials. Percent time spent in the target quadrant in all probe trials
shown for WT and R6/2 mice (A). Comparisons of percent time spent in all quadrants are shown for WT and R6/2 mice for all MWM probe trials (B-F
Note that data from both sexes and all experimental groups were pooled for each genotype. All data shown are means 6 s.e.m. Where error bars ar
not visible, they are obscured by symbols. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g002
PLoS ONE | www plosone org
5
February 2010 | Volume 5 | Issue 2 | e907 Figure 2. Quadrant preference of mice during Morris water maze probe trials. Morris Water Maze Percent time spent in the target quadrant in all probe trials is
shown for WT and R6/2 mice (A). Comparisons of percent time spent in all quadrants are shown for WT and R6/2 mice for all MWM probe trials (B-F). Note that data from both sexes and all experimental groups were pooled for each genotype. All data shown are means 6 s.e.m. Where error bars are
not visible, they are obscured by symbols. * p,0.05, ** p,0.01, *** p,0.001. Figure 2. Quadrant preference of mice during Morris water maze probe trials. Percent time spent in the target quadrant in all probe trials is
shown for WT and R6/2 mice (A). Comparisons of percent time spent in all quadrants are shown for WT and R6/2 mice for all MWM probe trials (B-F). Note that data from both sexes and all experimental groups were pooled for each genotype. All data shown are means 6 s.e.m. Where error bars are
not visible, they are obscured by symbols. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g002 February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 5 Enrichment in R6/2 Mice Enrichment in R6/2 Mice on swim speed of R6/2 females was found for both no handling
(p = 0.034) and handling groups (p = 0.018; Figure 6F). WT
female mice responded to handling with a decrease in swim
speed as compared to the home cage/no handling group, both
in
the
home
cage/handling
(p = 0.002)
and
playground/
handling groups (p = 0.010; Figure 6D). As the playground/no
handling WT females did not show a reduced swim speed
compared to the home cage/no handling group, this suggests
that the effect was specific for handling and does not apply to all
forms of enrichment (Figure 6D). Overall, the swim speed data
suggest sex and genotype–specific responses of mice to the
enrichment conditions. Handling We examined the amount of handling that was needed to keep
the mice awake during the enrichment period. As expected, home
cage
mice
needed
more
handling
than
playground
mice,
whether WT (F(1,72) = 27.618 p,0.001; Figure 8A) or R6/2
(F(1,72) = 172.766, p,0.001; Figure 8B). All mice needed more
handling at the end of the experiment than the beginning,
regardless of the playground enrichment condition (WT home
cage, p = 0.002; WT playground, p,0.001; R6/2 home cage,
p,0.001; R6/2 playground, p,0.001: Figure 8A, B). To compare
the increase in handling required over the course of the
experiment, data from the first (week 1) and the last (week 6)
week of the experiment were analysed (Figure 8C, D). R6/2 home
cage mice needed more handling than WT home cage mice both
in week 1 (p = 0.002) and week 6 (p,0.001; Figure 8C) of the
experiment. In the playground groups, this genotype difference
was not present in week 1 but was observed in week 6 (p = 0.021;
Figure 8C). Although all groups needed significantly more
handling in week 6 than in week 1 of the experiment, the need
for handling increased more strongly in R6/2 than in WT mice for
both home cage (p,0.001) and playground groups (p = 0.003;
Figure 8D). There was no difference in the increase in handling
needed between R6/2 groups, but WT playground mice showed a
greater increase in handling needed than the home cage group
(p = 0.003, Figure 8D). Body Weight y
g
Body weights of the mice were recorded until the last R6/2
mouse was killed due to ill health at 27 weeks of age (Figure 6). Data are presented up to 19 weeks for R6/2 mice, as beyond this
point the drop out of mice made the data difficult to analyse. Data
for WT mice are shown up to 27 weeks, when the experiment
finished. Analysis revealed the expected main effect of sex, with
males being heavier (F(1,134) = 159.148, p,0.001).There was also
an expected main effect of genotype as the R6/2 mice started to
lose weight from around 12 weeks of age. Group comparisons
revealed differences in the female R6/2 home cage (between 14.5
and 17 weeks of age) and playground groups (13.5 to 19 weeks of
age), where handling significantly reduced body weights compared
to the no handling group (home cage: p = 0.042, playground:
p = 0.06 Figure 9B, D). A similar negative effect of handling on
body weight for was found for male R6/2 mice in the home cage
groups between 9 and 16 weeks of age (p = 0.024; Figure 9A) but
not in the playground groups (Figure 9C). In the R6/2 male
handling groups, playground exposure led to an increase in body
weight between 14.5 and 19 weeks of age (Figure 9E). There were
no differences between any of the WT groups except in the male
handling groups, where the playground mice increased weight
significantly compared to the home cage mice, from 22 weeks
onwards (p = 0.027; Figure 9E). Although a similar tendency was
found for the male no handling groups, it did not reach statistical
significance (p = 0.096; Figure 9G). Daytime Activity y
y
The effect of enrichment on daytime activity measured during
the daily experimental period in the mice is shown in Figure 7. The behaviour of each mouse was scored as ‘‘active’’ or ‘‘inactive’’
in 15-minute intervals. Data were analysed on a day-by-day basis,
but for clarity only weekly averages are presented. In the no
handling groups, there were significantly higher levels of activity in
the playground groups compared to the home cage groups,
irrespective of genotype and sex (F(1,143) = 125.438, p,0.001;
Figure 7A). This suggests strongly that the playgrounds were
intrinsically stimulating, and remained so throughout the enrich-
ment period. Overall, activity of the mice was lowest in the home
cage/no handling groups, and highest in the playground/handling
group, regardless of genotype (Figure 7B). In the playground/
handling groups, R6/2 mice were more active than WT mice
(p,0.001; Figure 7B). Although the playgrounds stimulated
activity in the mice, the playground/no handling groups showed
the greatest decline in activity between weeks 1 and 6 of the
enrichment period. This was true for WT male (p,0.001;
Figure 7C), WT female (p = 0.008; Figure 7D), R6/2 male
(p,0.001; Figure 7E) and R6/2 female (p,0.001; Figure 7F)
mice. Female mice in the playground/handling groups also
showed a decline in activity during the enrichment period
(p,0.001; Figure 7D, F), an effect that was not seen in male
mice (Figure 7C, E). Survival We looked at the effect of EE on survival in R6/2 mice. Median
survival for all groups is presented in Table 1. In the home cage
groups, handling had no effect on the age at death in male mice,
but had a beneficial effect in female mice (p = 0.001; Table 2). In
the playground groups, handling had a detrimental effect on
survival in male mice (p,0.001; Table 3), but no effect in female
mice (Table 2). Both male and female playground/no handling
mice lived significantly longer than those in the home cage/no
handling groups (male: p = 0.002, female: p = 0.011; Table 2, 3). In the mice that were handled, there was no difference in survival
between home cage and playground groups of either sex
(Table 2, 3). February 2010 | Volume 5 | Issue 2 | e9077 Discussion Since EE was first shown to improve survival in the R6/2 mouse
model of HD [5], numerous studies have been conducted to
further investigate its effects in models of neurodegenerative
disease [7,8,13,14,22]. Most of these studies have used the R6/1
mouse, which has a repeat length of approximately 115 CAG
repeats, with a delayed onset of, and less severe, phenotype than
the R6/2 mouse. Studies using EE in the R6/2 mouse are far
fewer, because the early onset and severity of the phenotype makes
it much more challenging to show beneficial effects. However, as
we have already had success in improving the lifespan of these
mice through home cage enrichment [5], we wanted to see
whether we could also improve the cognitive dysfunction in R6/2
mice through access to additional enrichment. In this study, we examined the effect of two different types of EE
on the cognitive function, body weight and survival in R6/2 mice. We found a range of effects, both beneficial and detrimental, with
significant genotype and sex effects. First, we assessed cognitive
performance in probe trials in the WT task using the classical
methods of percent time spent in the target quadrant or zone. These showed the expected deficits in R6/2 as compared to WT
mice, and an absence of any beneficial (or detrimental) effects February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 6 Enrichment in R6/2 Mice Figure 3. Zone preference of mice during Morris water maze probe trials. Percent time spent in the target zone in all probe trials i
for WT and R6/2 mice (A). Data from both sexes and all experimental groups were pooled for each genotype. Comparisons of percent time spe
quadrants are shown for WT and R6/2 mice from home cage and playground groups for all MWM probe trials (B-F). Data from both sexes
as handling and no handling groups were pooled. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by s
* p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g003 Figure 3. Zone preference of mice during Morris water maze probe trials. Discussion Percent time spent in the target zone in all probe trials is shown
for WT and R6/2 mice (A) Data from both sexes and all experimental groups were pooled for each genotype Comparisons of percent time spent in a Figure 3. Zone preference of mice during Morris water maze probe trials. Percent time spent in the target zone in all probe trials is shown
for WT and R6/2 mice (A). Data from both sexes and all experimental groups were pooled for each genotype. Comparisons of percent time spent in all
quadrants are shown for WT and R6/2 mice from home cage and playground groups for all MWM probe trials (B-F). Data from both sexes as well
as handling and no handling groups were pooled. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by symbols. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g003 PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 7 Enrichment in R6/2 Mice Figure 4. Proximity of mice to platform position during Morris water maze probe trials. Proximity to platform position for hom
handling, home cage/handling, playground/no handling and playground/handling groups of WT (A) and R6/2 (B) mice in all MWM session
from both sexes pooled. Data from handling and no handling groups as well as from both sexes were pooled to compare home cage and
groups of WT (C) and R6/2 (D) mice. Data from both sexes and all experimental groups were pooled for WT and R6/2 mice (E). Grey shad
indicates period of enrichment. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by symbols. n.s. non
* p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g004
PLoS ONE | www.plosone.org
8
February 2010 | Volume 5 | Issue Figure 4. Proximity of mice to platform position during Morris water maze probe trials. Proximity to platform position for home cage/no
handling, home cage/handling, playground/no handling and playground/handling groups of WT (A) and R6/2 (B) mice in all MWM sessions, with data
from both sexes pooled. Data from handling and no handling groups as well as from both sexes were pooled to compare home cage and playground
groups of WT (C) and R6/2 (D) mice. PLoS ONE | www.plosone.org Discussion Data from both sexes and all experimental groups were pooled for WT and R6/2 mice (E). Grey shading in C, D
indicates period of enrichment. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by symbols. n.s. non-significant,
* p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g004 p
p
p
doi:10.1371/journal.pone.0009077.g004 PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 8 Enrichment in R6/2 Mice Figure 5. Swim speed of mice during Morris water maze probe trials. Swim speed for home cage/no handling, home
playground/no handling and playground/handling groups of WT (A) and R6/2 (B) mice in all MWM sessions, with data from both sexe
from handling and no handling groups as well as from both sexes were pooled to compare home cage and playground groups of WT (C
mice. Data from both sexes and all experimental groups were pooled for WT and R6/2 mice and are shown in (E). The percentage
floating in each MWM trial is shown for WT and R6/2 mice, with data from both sexes and all experimental groups pooled for each gen
shading in C, D indicates period of enrichment. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured b
non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g005 Figure 5. Swim speed of mice during Morris water maze probe trials. Swim speed for home cage/no handling, home cage/handling,
playground/no handling and playground/handling groups of WT (A) and R6/2 (B) mice in all MWM sessions, with data from both sexes pooled. Data
from handling and no handling groups as well as from both sexes were pooled to compare home cage and playground groups of WT (C) and R6/2 (D)
mice. Data from both sexes and all experimental groups were pooled for WT and R6/2 mice and are shown in (E). The percentage of time spent
floating in each MWM trial is shown for WT and R6/2 mice, with data from both sexes and all experimental groups pooled for each genotype (F). Grey
shading in C, D indicates period of enrichment. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g005 February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 9 Enrichment in R6/2 Mice Figure 6. Discussion Sex and handling interaction on Morris maze performance and swim speed. Platform proximity (A) and swim speed (B) d
were combined across all probe trials. Data are shown for male (blue bars) and female (red bars) groups for ‘handling’ and ‘no handling’ condit
with data from both genotypes as well as home cage and playgrounds groups pooled. Proximity to the platform position and swim speed combi
across probe trials are shown for all WT (C, D) and R6/2 (E, F) groups. Data for male and female mice are shown separately. All data shown are me
6 s.e.m. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g006 Figure 6. Sex and handling interaction on Morris maze performance and swim speed. Platform proximity (A) and swim speed (B) data
were combined across all probe trials. Data are shown for male (blue bars) and female (red bars) groups for ‘handling’ and ‘no handling’ conditions
with data from both genotypes as well as home cage and playgrounds groups pooled. Proximity to the platform position and swim speed combined
across probe trials are shown for all WT (C, D) and R6/2 (E, F) groups. Data for male and female mice are shown separately. All data shown are means
6 s.e.m. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g006 Figure 6. Sex and handling interaction on Morris maze performance and swim speed. Platform proximity (A) and swim speed (B) data
were combined across all probe trials. Data are shown for male (blue bars) and female (red bars) groups for ‘handling’ and ‘no handling’ conditions
with data from both genotypes as well as home cage and playgrounds groups pooled. Proximity to the platform position and swim speed combined
across probe trials are shown for all WT (C, D) and R6/2 (E, F) groups. Data for male and female mice are shown separately. All data shown are means
6 s.e.m. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g006 February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 10 Enrichment in R6/2 Mice arising from enrichment in the R6/2 mice. The Gallagher
proximity score has been demonstrated to be more sensitive for
detecting group differences than these traditional measures [21]. However, we again found no improvements over tim
groups. The most likely explanation for this findi
deficit in spatial learning apparent by 9 weeks of age
Figure 7. Discussion It is
possible that, in the male R6/2 mice, the lack of effect of
enrichment on proximity score, combined with increased swim
speed, is indicative of sex-specific increased stress caused by
handling. It would be interesting, in future experiments, to
measure levels of circulating corticosterone or testosterone to
explore this possibility further. Although EE did not improve the overall cognitive performance
of R6/2 mice in the MWM during the course of the study, it did
produce significant differences between groups. For example, all
mice in enriched female R6/2 groups performed significantly
better than those in the home cage/no handling group. This
suggests that female R6/2 mice may be more sensitive to the
beneficial effects of EE than the other groups. This suggestion is
reinforced by the finding that handling had beneficial effects on
the cognitive performance of female mice of both genotypes, while
having a negative effect in male mice. Interestingly, we found the
reverse effect with regard to swim speed, where handling produced
an increase in swim speed in male mice, but a decrease in females. While this increase in swim speed in males might at face value
suggest a beneficial effect, it may also be a response to increased
stress caused by handling, since it has been shown that stress in rats
and mice can cause an increase in swim speed in the MWM
[23,24]. It should be noted that stress in rats also caused a deficit in
MWM probe trial performance [25]. In addition, it has been
reported that a stress paradigm had opposite effects on MWM In order to keep the playgrounds as stimulating as possible, we
changed some of the toys every day to maintain an element of
novelty. This appeared to have the desired effect, as mice in the
playground/no handling groups were more active than mice in the
home cage/no handling groups throughout the experiment. We
found a graded effect of activity across the groups, with the least
active mice being the home cage/no handling groups, followed by
the home cage/handling, playground/no handling, and the most
active being the playground/handling groups. Notably, although
the playground groups were more active throughout the entire
experiment, the playground/no handling groups showed the
greatest decline in activity between weeks 1 and 6. Discussion Activity of mice during the enrichment period. Average daily activity scores are shown for weeks 1 to 6 of the exper
handling groups (A). Average activity scores throughout the whole experiment are presented for all groups of WT and R6/2 mice i
from male and female mice pooled. Average daily activity scores in weeks 1 to 6 of the experiment are shown for all WT male (C), WT f
male (E) and R6/2 female (F) groups. For key to symbols in (A), see (C), (D), (E) and (F). Symbols left of data series indicate signific
from week 1 to week 6. All data shown are mean 6 s.e.m. Where error bars are not visible, they are obscured by symbols. n.s. * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g007
Enrichme Figure 7. Activity of mice during the enrichment period. Average daily activity scores are shown for weeks 1 to 6 of the experiment for all no
handling groups (A). Average activity scores throughout the whole experiment are presented for all groups of WT and R6/2 mice in (B), with data
from male and female mice pooled. Average daily activity scores in weeks 1 to 6 of the experiment are shown for all WT male (C), WT female (D), R6/2
male (E) and R6/2 female (F) groups. For key to symbols in (A), see (C), (D), (E) and (F). Symbols left of data series indicate significance of decline
from week 1 to week 6. All data shown are mean 6 s.e.m. Where error bars are not visible, they are obscured by symbols. n.s. non-significant,
* p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g007 However, we again found no improvements over time in the R6/2
groups. The most likely explanation for this finding is that the
deficit in spatial learning apparent by 9 weeks of age, when MWM However, we again found no improvements over time in the R6/2
groups. The most likely explanation for this finding is that the
deficit in spatial learning apparent by 9 weeks of age, when MWM arising from enrichment in the R6/2 mice. The Gallagher
proximity score has been demonstrated to be more sensitive for
detecting group differences than these traditional measures [21]. PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 11 Enrichment in R6/2 Mice Figure 8. Discussion Amount of handling needed to keep the mice awake during the enrichment period. The average number of daily handling
events required by each mouse during weeks 1 to 6 of the experiment for home cage and playground groups in WT (A) and R6/2 (B) mice. Some of
these data are reproduced in (C) to allow comparisons between WT and R6/2 groups in home cage and playground conditions for the first and last
weeks of the experiment. (D) shows the increase in the number of daily handling events from week 1 to week 6 of the experiment that were required
by WT and R6/2 mice in home cage and playground groups. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by
symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g008 Figure 8. Amount of handling needed to keep the mice awake during the enrichment period. The average number of daily handling
events required by each mouse during weeks 1 to 6 of the experiment for home cage and playground groups in WT (A) and R6/2 (B) mice. Some of
these data are reproduced in (C) to allow comparisons between WT and R6/2 groups in home cage and playground conditions for the first and last
weeks of the experiment. (D) shows the increase in the number of daily handling events from week 1 to week 6 of the experiment that were required
by WT and R6/2 mice in home cage and playground groups. All data shown are means 6 s.e.m. Where error bars are not visible, they are obscured by
symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g008 training in the current study began, cannot be reversed by means
of EE. It might be worthwhile to begin both enrichment and
cognitive testing at an earlier age, to see if EE can prevent the
development of cognitive deficits, as has been shown in R6/1 mice
[14]. However, as R6/2 mice have been shown to have deficits in
the MWM from as early as 3.5 weeks of age [12], this may not be
possible. performance of male and female rats, with female rats deriving a
beneficial effect while males suffered detrimental effects [26]. PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 Discussion While this is in
part due to the fact that they were very active to start with and so
had further to decline, it does seem that the stimulatory effect of
the playgrounds was falling by the end of the study. In addition to
the decline in activity seen in all of the playground/no handling
groups, the female playground/handling groups also showed a
significant decline in activity between weeks 1 and 6, which February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 PLoS ONE | www.plosone.org 12 Enrichment in R6/2 Mice strongly suggests that even when the contents of the playgrounds
were regularly changed, female mice habituated to the play-
grounds faster than male mice. This adds more weight to the idea
that there are significant differen
that they respond to EE, and tha
be more beneficial to one sex tha
Figure 9. Body weights. Body weights were measured from 9.5 to 19 weeks for R6/2 and 9.5 to 27 weeks for W
and shown for home cage (A, B), playground (C, D), handling (E, F) and no handling (G, H) conditions. MWM1,
maze testing. Grey shaded areas represent the enrichment period. All data shown are mean 6 s.e.m. Where
obscured by symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g009 Figure 9. Body weights. Body weights were measured from 9.5 to 19 weeks for R6/2 and 9.5 to 27 weeks for WT groups. Data are separated by sex,
and shown for home cage (A, B), playground (C, D), handling (E, F) and no handling (G, H) conditions. MWM1, 2 and 3 are periods of Morris water
maze testing. Grey shaded areas represent the enrichment period. All data shown are mean 6 s.e.m. Where error bars are not visible, they are
obscured by symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g009 Figure 9. Body weights. Body weights were measured from 9.5 to 19 weeks for R6/2 and 9.5 to 27 weeks for WT groups. Data are separated by sex,
and shown for home cage (A, B), playground (C, D), handling (E, F) and no handling (G, H) conditions. MWM1, 2 and 3 are periods of Morris water
maze testing. Grey shaded areas represent the enrichment period. All data shown are mean 6 s.e.m. Discussion Median survival (days)
Sex
Group
No handling
Handling
Male
Home cage
141
156
Playground
163
152
Female
Home cage
156
177
Playground
176
158
doi:10.1371/journal.pone.0009077.t001 effects of EE on MWM learning and memory have previously
been reported for a mouse model of Down syndrome where EE
had beneficial effects on spatial learning in female mice but
deleterious effects in male mice [27]. The possibility that the stimulatory effect of the playgrounds was
declining over the course of the experiment is further supported by
the finding that, even though the playground mice were more
active and required less handling than home cage mice, by the end
of the study the amount of handling they needed had increased
significantly. The R6/2 mice needed a larger increase in handling
between weeks 1 and 6 than WT mice to keep them active during
the enrichment period, in both the home cages and playgrounds. This correlated with the onset of an overt phenotype in the R6/2
mice, although the mice were still capable of climbing and
running. It is possible that as part of their developing phenotype
with increasing age, R6/2 mice find their surroundings less
interesting than do their WT littermates and display a reduction in
voluntary activity. This could reflect an element of apathy. Apathy
has been shown to be a major component of the disease in patients
[28], and it becomes more severe with illness duration, and motor
and cognitive dysfunction [29,30,31]. There are currently no
reliable tests for apathy in rodents, but an apathy-like syndrome
has been identified and successfully treated in R6/2 mice [17]. It
would
be
interesting
to
apply
the
same
pharmacological
intervention to an EE study, to see whether improving the
circadian rhythm has added benefits in enriched mice. Sex-dependent differences in normal behaviour have been
reported in other rodent models of HD. In a rat model, male
animals display increased daytime activity at an earlier stage of
phenotype than female rats [36]. In the N171-82Q model, male
mice show poorer performance on the rotarod than female mice
[37]. In the YAC128 model, female mice live longer than male
mice [38]. A detailed examination of the 140 CAG knock-in
model of HD also revealed a number of sex differences, including
increased grooming and dark phase running in female mice, and
decreased climbing in male mice [39]. Discussion Where error bars are not visible, they are
obscured by symbols. n.s. non-significant, * p,0.05, ** p,0.01, *** p,0.001. doi:10.1371/journal.pone.0009077.g009 strongly suggests that even when the contents of the playgrounds
were regularly changed, female mice habituated to the play-
grounds faster than male mice. This adds more weight to the idea that there are significant differences between the sexes in the way
that they respond to EE, and that some forms of enrichment may
be more beneficial to one sex than the other. Similar sex – specific PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 13 Enrichment in R6/2 Mice although the circadian rhythms of R6/2 mice are disrupted, the
SCN itself appears to function normally in vitro [16]. This suggests
that the abnormal behavioural and molecular circadian rhythms
observed in R6/2 mice arise from dysfunction of brain circuitry
afferent to the SCN, rather than the pacemaker itself [16]. Although we could not measure circadian rhythms directly in this
experiment, we hypothesize that disruptions to the sleep-wake
patterns of these mice may have resulted from enrichment during
circadian day. These disruptions, together with developing deficits
in metabolism [32] and motor function [35], might have left the
R6/2 mice increasingly more tired and less rousable than their
WT littermates. It is possible, therefore, that the increasing
inactivity seen in the R6/2 mice was a result of decreased strength
and energy. This hypothesis is supported further by the body
weight data from this study, which showed weight loss in all R6/2
mice by the end of the enrichment period. It is interesting that
there was a tendency among R6/2 home cage mice of both sexes
for the no-handling groups to be heavier than handling groups
towards the end of the enrichment period. Interestingly, in the
male R6/2 handling groups, playground exposure led to increased
body weight at the end of the enrichment period, a tendency that
was not observed in female groups. These findings further support
a sex-specific mix of beneficial and detrimental effects of the two
types of enrichment. Table 1. Median survival times of R6/2 mice. References derived neurotrophic factor expression deficits in Huntington’s disease
transgenic mice. Neurosci 141: 569–584. derived neurotrophic factor expression deficits in Huntington’s disease
transgenic mice. Neurosci 141: 569–584. 1. The Huntington’s Disease Collaborative Research Group (1993) A novel gene
containing a trinucleotide repeat that is expanded and unstable on Huntington’s
disease chromosomes. Cell 72: 971–983. 1. The Huntington’s Disease Collaborative Research Group (1993) A novel gene
containing a trinucleotide repeat that is expanded and unstable on Huntington’s
disease chromosomes. Cell 72: 971–983. 14. Nithianantharajah J, Barkus C, Murphy M, Hannan AJ (2008) Gene-
environment interactions modulating cognitive function and molecular corre-
lates of synaptic plasticity in Huntington’s disease transgenic mice. Neurobiol
Dis 29: 490–504. 2. Fratiglioni L, Paillard-Borg S, Winblad B (2004) An active and socially
integrated lifestyle in late life might protect against dementia. Lancet Neurol 3:
343–353. 3. Sullivan FR, Bird ED, Alpay M, Cha JH (2001) Remotivation therapy and
Huntington’s disease. J Neurosci Nurs 33: 136–142. 15. Morton AJ, Wood NI, Hastings MH, Hurelbrink C, Barker RA, et al. (2005)
Disintegration of the sleep-wake cycle and circadian timing in Huntington’s
disease. J Neurosci 25: 157–163. 4. Zinzi P, Salmaso D, De Grandis R, Graziani G, Maceroni S, et al. (2007) Effects
of an intensive rehabilitation programme on patients with Huntington’s disease:
a pilot study. Clin Rehab 21: 603–613. 16. Pallier PN, Maywood ES, Zheng Z, Chesham JE, Inyushkin AN, et al. (2007)
Pharmacological imposition of sleep slows cognitive decline and reverses
dysregulation of circadian gene expression in a transgenic mouse model of
Huntington’s Disease. J Neurosci 27: 7869–7878. p
y
5. Carter RJ, Hunt MJ, Morton AJ (2000) Environmental stimulation increases
survival in mice transgenic for exon 1 of the Huntington’s disease gene. Mov Dis
15: 925–937. 17. Pallier PN, Morton AJ (2009) Management of sleep/wake cycles improves
cognitive function in a transgenic mouse model of Huntington’s disease. Brain
Res 1279: 90–98. 6. van Dellen A, Blakemore C, Deacon R, York D, Hannan AJ (2000) Delaying the
onset of Huntington’s in mice. Nature 404: 721–2. 7. Hockly E, Cordery PM, Woodman B, Mahal A, van Dellen A, et al. (2002)
Environmental enrichment slows disease progression in R6/2 Huntington’s
disease mice. Ann Neurol 51: 235–242. 18. Mangiarini L, Sathasivam K, Seller M, Cozens B, Harper A, et al. Discussion Survival comparisons using log-rank test between groups of male R6/2 mice. Home cage/no handling
Home cage/handling
Playground/no handling
Playground/handling
Home cage/no handling
x
n.s. **
x
Home cage/handling
n.s. x
x
n.s. Playground/no handling
**
x
x
***
Playground/handling
x
n.s. ***
x
*p,0.05, ** p,0.01, *** p,0.001, n.s. not significant, x comparison not valid. doi:10.1371/journal.pone.0009077.t003 *p,0.05, ** p,0.01, *** p,0.001, n.s. not significant, x comparison not valid. doi:10.1371/journal.pone.0009077.t003 survival in both male and female R6/2 mice, beneficial effects of
EE on cognition were seen in females only. Although the effects we
observed were not as marked as those reported in other EE studies,
there are two possible reasons for this. The first reason is that we
used R6/2 mice, which have an early, aggressive onset of
phenotype, and so have been rarely used in EE experiments. The second reason is that our mice are routinely kept in conditions
that would, in most labs, be considered ‘‘enriched’’ already. They
are group-housed, have plastic nest boxes, a range of bedding,
lowered water bottle spouts, and a mashed food supplement to
facilitate feeding and help maintain hydration. This home cage
enrichment raises the threshold for beneficial changes and makes
them less likely. Data from the current study, which show that
enhanced EE can produce a further improvement in cognitive
ability and survival, are very encouraging in the context of using
EE to improve the quality of life of HD patients. g
p
p
p
Found
at:
doi:10.1371/journal.pone.0009077.s001
(0.30
MB
TIF) Movie S1
Example of playground configuration. Found
at:
doi:10.1371/journal.pone.0009077.s002
(4.03
MB
MP4) Movie S2
Demonstration of the gentle handling used to keep
mice active. Found
at:
doi:10.1371/journal.pone.0009077.s003
(1.60
MB
MP4) Found
at:
doi:10.1371/journal.pone.0009077.s003
(1.60
MB
MP4) Author Contributions Conceived and designed the experiments: NIW EAS CJM JM. Performed
the experiments: NIW VC SM EAS CJM YLMA AD NW SMA JXF
TWL. Analyzed the data: NIW VC SM EAS CJM YLMA AD NW SMA
JXF TWL JM. Wrote the paper: NIW SM JM. Discussion These studies have shown
sex differences in phenotypically-altered behaviours, but ours is the
first to demonstrate that modulation of the environment also has
sex-dependent effects in R6/2 mice, with enrichment having
either positive or negative effects depending upon the sex of the
mouse. One unexpected finding is the trend towards changes in body
weights in WT mice, long after the end of the EE period. Male
WT mice that had been exposed to the playgrounds tended to
have higher weights than home cage mice (in both the handling
and no handling conditions) from around 21 weeks of age,
although this difference reached significance in the handling group
only. It is unclear what could have caused this, since by the time
the effect developed, the mice had been out of the playgrounds for
6 weeks. The need for an increased amount of handling to keep the R6/2
mice active as the experiment progressed may be due to a
developing dysfunction in their circadian rhythms. We have shown
that as R6/2 mice age, their circadian activity changes from a
pattern of discrete extended periods of activity and sleep, to a
constant level of very short periods of activity and inactivity [15]. It
is not clear what causes this change. The main mammalian
circadian oscillator is the suprachiasmatic nucleus (SCN) in the
hypothalamus. The hypothalamus controls a number of important
physiological functions, such as feeding and drinking, that are also
abnormal in R6/2 mice [32,33]. This may be caused by
hypothalamic neuronal degeneration/atrophy [34]. However, Results from this experiment have demonstrated clearly sex
differences in response to EE. While exposure to the playgrounds,
for 6 hours a day over a 6 week period, significantly improved Table 2. Survival comparisons using log-rank test between groups of female R6/2 mice. Home cage/no handling
Home cage/handling
Playground/no handling
Playground/handling
Home cage/no handling
x
**
*
x
Home cage/handling
**
x
x
n.s. Playground/no handling
*
x
x
n.s. Playground/handling
x
n.s. n.s. x
*p,0.05, ** p,0.01, *** p,0.001, n.s. not significant, x comparison not valid. doi:10.1371/journal.pone.0009077.t002
PLoS ONE | www.plosone.org
14
February 2010 | Volume 5 | Issue 2 | e9077 Table 2. Survival comparisons using log-rank test between groups of female R6/2 mice. Table 2. Survival comparisons using log-rank test between groups of female R6/2 mice. PLoS ONE | www.plosone.org 14 Enrichment in R6/2 Mice Table 3. Supporting Information Figure S1
Quadrant and zone preferences during first Morris
water maze (MWM) trial. Percentage times spent in each quadrant References van der Burg JMM, Bacos K, Wood NI, Lindqvist A, Wierup N, et al. (2008)
Increased metabolism in the R6/2 mouse model of Huntington’s disease. Neurobiol Dis 29: 41–51. g
y
25. Kim JJ, Lee HJ, Welday AC, Song EY, Cho J, et al. (2007) Stress-induced
alterations in hippocampal plasticity, place cells, and spatial memory. Proc Natl
Acad Sci USA 104: 18297–18302. 33. Wood NI, Goodman AO, van der Burg JM, Gazeau V, Brundin P, et al. (2008)
Increased thirst and drinking in Huntington’s disease and the R6/2 mouse. Brain Res Bull 76: 70–79. 26. Kitraki E, Kremmyda O, Youlatos D, Alexis M, Kittas C (2004) Spatial
performance and corticosteroid receptor status in the 21-day restraint stress
paradigm. Ann NY Acad Sci 1018: 323–327. 34. Peterse´n A, Gil J, Maat-Schieman ML, Bjo¨rkqvist M, Tanila H, et al. (2005)
Orexin loss in Huntington’s disease. Hum Mol Genet 14: 39–47. 35. Ribchester RR, Thomson D, Wood NI, Hinks K, Gillingwater TH, et al. (2004)
Progressive abnormalities in skeletal muscle and neuromuscular junctions of
transgenic mice expressing the Huntington’s disease mutation. Eur J Neurosci
20: 3092–3114. p
g
27. Martinez-Cue C, Baamonde C, Lumbreras M, Paz J, Davisson MT, et al. (2002)
Differential effects of environmental enrichment on behaviour and learning of
m-ale and female Ts65Dn mice, a model for Down syndrome. Behav Brain Res
134: 185–200. 36. Bode FJ, Stephan M, Wiehager S, Nguyen HP, Bjo¨rkqvist M, et al. (2009)
Increased numbers of motor activity peaks during light cycle are associated with
reductions in adrenergic a2-receptor levels in a transgenic Huntington’s disease
rat model. Behav Brain Res 205: 175–182. 28. Cummings JL (1995) Behavioral and psychiatric symptoms associated with
Huntington’s disease. In: Weiner WJ, Lang AE, eds. Behavioral neurology of
movement disorders. New York: Raven. pp 179–186. 29. Paulsen JS, Zhao H, Stout JC, Brinkmann RR, Guttman M, et al. (2001)
Clinical markers of early disease in persons near onset of Huntington’s disease. Neurol 57: 658–662. 37. Orr AL, Huang S, Roberts M, Reed JC, Li S, et al. (2008) Sex-dependent effect
of BAG1 in ameliorating motor deficits of Huntington disease transgenic mice. J Biol Chem 283: 16027–16036. 30. Thompson JC, Snowden JS, Craufurd D, Neary D (2002) Behavior in
Huntington’s disease: dissociating cognition-based and mood-based changes. J Neuropsych Clin Neurosci 14: 37–43. J
38. Van Raamsdonk JM, Pearson J, Rogers DA, Bissada N, Vogl AW, et al. References (1996)
Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a
progressive neurological phenotype in transgenic mice. Cell 87: 493–
506. 8. Spires TL, Grote HE, Varshney NK, Cordery PM, van Dellen A, et al. (2004)
Environmental enrichment rescues protein deficits in a mouse model of
Huntington’s disease, indiciating a possible disease mechanism. J Neurosci 24:
2270–2276. 19. Wood NI, Morton AJ (2003) Chronic lithium chloride treatment has variable
effects on motor behaviour and survival of mice transgenic for the Huntington’s
disease mutation. Brain Res Bull 61: 375–83. 9. Zuccato C, Ciammola A, Rigamonti D, Leavitt BR, Goffredo D, et al. (2001)
Loss of Huntingtin-mediated BDNF gene transcription in Huntington’s disease. Science 293: 493–498. 20. Wood NI, Pallier PN, Wanderer J, Morton AJ (2006) Systemic administration of
Congo red does not improve motor or cognitive function in R6/2 mice. Neurobiol Dis 25: 342–353. 10. Ferrer I, Goutan E, Marin C, Rey MJ, Ribalta T (2000) Brain-derived
neurotrophic factor in Huntington’s disease. Brain Res 866: 257–261. 21. Maei HR, Zaslavsky K, Teixeira CM, Frankland PW (2009) What is the most
sensitive measure of water maze probe test performance? Frontiers Integ
Neurosci 3: 1–9. 11. Brown J, Cooper-Kuhn CM, Kempermann G, Van Praag H, Winkler J, et al. (2003) Enriched environment and physical activity stimulate hippocampal but
not olfactory bulb neurogenesis. Eur J Neurosci 17: 2042–2046. 22. Schilling G, Savonenko AV, Coonfield ML, Morton JL, Vorovich E, et al. (2004)
Environmental, pharmacological, and genetic modulation of the HD phenotype
in transgenic mice. Exp Neurol 187(1): 137–49. 12. Lione LA, Carter RJ, Hunt MJ, Bates GP, Morton AJ, Dunnett SB (1999)
Selective discrimination learning impairments in mice expressing the human
Huntington’s disease mutation. J Neurosci 19: 10428–10437. 23. Avital A, Richter-Levin G (2005) Exposure to juvenile stress exacerbates the
behavioural consequences of exposure to stress in the adult rat. Int J Neuro-
psychopharm 8: 163–173. 13. Pang TYC, Stam NC, Nithianantharajah J, Howard ML, Hannan AJ (2006)
Differential effects of voluntary physical excercise on behavioral and brain- PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 February 2010 | Volume 5 | Issue 2 | e9077 15 Enrichment in R6/2 Mice 24. Buchanan JB, Sparkman NL, Chen J, Johnson RW (2008) Cognitive and
neuroinflammatory consequences of mild repeated stress are exacerbated in
aged mice. Psychoneuroendocrin 33: 755–765. 32. References (2005)
Loss of wild-type huntingtin influences motor dysfunction and survival in the
YAC128 mouse model of Huntington disease. Hum Mol Gen 14: 1379–1392. g
39. Dorner JL, Miller BR, Barton SJ, Brock TJ, Rebec GV (2007) Sex differences in
behavior and strial ascorbate release in the 140 CAG knock-in mouse model of
Huntington’s disease. Beh Brain Res 178: 90–97. p y
31. Baudic S, Maison P, Dolbeau G, Boisse MF, Bartolomeo P, et al. (2006)
Cognitive impairment related to apathy in early Huntington’s disease. Dement
Geriatr Cogn Disord 14: 316–321. PLoS ONE | www.plosone.org February 2010 | Volume 5 | Issue 2 | e9077 16
|
https://openalex.org/W3159138597
|
https://publications.aston.ac.uk/id/eprint/42634/2/Changes_in_theta_and_alpha_oscillatory_signatures_of_attentional_control.pdf
|
English
| null |
Changes in theta and alpha oscillatory signatures of attentional control in older and middle age
|
European journal of neuroscience/EJN. European journal of neuroscience
| 2,021
|
cc-by
| 18,841
|
R E S E A R C H R E P O R T R E S E A R C H R E P O R T K E Y W O R D S ageing, attentional control, brain oscillations, magnetoencephalography, middle age Received: 20 December 2020 |
Revised: 9 April 2021 |
Accepted: 23 April 2021 Received: 20 December 2020 |
Revised: 9 April 2021 |
Accepted: 23 April 2021 Received: 20 December 2020 |
Revised: 9 April 2021 |
Accepted: 23 April 2021
DOI: 10.1111/ejn.15259 | 1
wileyonlinelibrary.com/journal/ejn Changes in theta and alpha oscillatory signatures of attentional
control in older and middle age | Carol Holland1,4 | Klaus Kessler1,2 | Hongfang Wang2 | Carol Holland1,4 | Klaus Kessler1,2 Eleanor Huizeling1,2,3 1Aston Research Centre for Healthy Ageing,
Aston University, Birmingham, UK
2Institute of Health and Neurodevelopment,
Aston University, Birmingham, UK
3Max Planck Institute for Psycholinguistics,
Nijmegen, Netherlands
4Centre for Ageing Research, Division of
Health Research, Lancaster University,
Lancaster, UK 1Aston Research Centre for Healthy Ageing,
Aston University, Birmingham, UK
2Institute of Health and Neurodevelopment,
Aston University, Birmingham, UK
3Max Planck Institute for Psycholinguistics,
Nijmegen, Netherlands
4Centre for Ageing Research, Division of
Health Research, Lancaster University,
Lancaster, UK Abstract
Background: Recent behavioural research has reported age-related changes in the
costs of refocusing attention from a temporal (rapid serial visual presentation) to a
spatial (visual search) task. Using magnetoencephalography, we have now compared
the neural signatures of attention refocusing between three age groups (19–30, 40–
49 and 60+ years) and found differences in task-related modulation and cortical lo-
calisation of alpha and theta oscillations. Efficient, faster refocusing in the youngest
group compared to both middle age and older groups was reflected in parietal theta
effects that were significantly reduced in the older groups. Residual parietal theta
activity in older individuals was beneficial to attentional refocusing and could reflect
preserved attention mechanisms. Slowed refocusing of attention, especially when a
target required consolidation, in the older and middle-aged adults was accompanied
by a posterior theta deficit and increased recruitment of frontal (middle-aged and
older groups) and temporal (older group only) areas, demonstrating a posterior to
anterior processing shift. Theta but not alpha modulation correlated with task per-
formance, suggesting that older adults' stronger and more widely distributed alpha
power modulation could reflect decreased neural precision or dedifferentiation but
requires further investigation. Our results demonstrate that older adults present with
different alpha and theta oscillatory signatures during attentional control, reflecting
cognitive decline and, potentially, also different cognitive strategies in an attempt to
compensate for decline. Correspondence
Klaus Kessler, Life and Health Sciences,
Psychology, Aston University, Birmingham,
UK, B4 7ET. Email: k.kessler@aston.ac.uk
Eleanor Huizeling, Max Planck Institute for
Psycholinguistics, Wundtlaan 1, Nijmegen,
The Netherlands, 6525 XD. Email: eleanor.huizeling@mpi.nl
Funding information
The Rees Jeffreys Road Fund; School of
Life and Health Sciences, Aston University;
The Wellcome Trust; Dr Hadwen Trust for
Humane Research
Edited by: John Foxe 2 | HUIZELING et al. attention in both time and space (Coull & Nobre, 1998;
Fu et al., 2005; Gross et al., 2004; Madden et al., 2007;
Nagamatsu et al., 2013). In addition to finding overlapping
activation for temporal and spatial attention, Coull and Nobre
(1998) found distinct subpatterns of activation for the two
types of attention. The latter suggests that the human brain
might have to be “retuned” when switching from a tempo-
ral to a spatial focus of attention (and vice versa), a dynamic
process that could be particularly affected by age-related de-
cline. For our current study, we therefore expected fronto-
parietal networks in conjunction with occipital areas to reveal
age-related changes (see Table 1 H1a–c, H4a–c, H5a–c). To
complicate matters, findings are inconsistent as to whether
ageing results in reduced activity in these cortical attention
networks (Cabeza, 2002; Madden & Gottlob, 1997; Madden
et al., 2002; Ross et al., 1997) or more widely distributed
activity across the cortex (Adamo, Westerfield, Haist, &
Townsend, 2003; Lague-Beauvais et al., 2013; Madden
et al., 2007; Nagamatsu et al., 2013). has emerged that aims to find out which areas of cogni-
tion remain high-functioning for longer and can be utilised
to support lesser preserved processes. Here we aimed to
understand how flexible refocusing of attention in time
and space might be affected in middle and older age and
whether certain processing elements, such as bottom-up
stimulus-driven processing, are affected more strongly than
others, such as top-down attentional control (or vice versa). A further aim was to investigate whether preserved func-
tioning could be recruited to support more affected pro-
cessing elements. Age-related deterioration of performance has been re-
ported separately for temporal as well as spatial selective at-
tention (Bennett et al., 2012; Foster et al., 1995; Humphrey
& Kramer, 1997; Lahar et al., 2001; Lee & Hsieh, 2009;
Maciokas & Crognale, 2003; Nagamatsu et al., 2013; Plude
& Doussard-Roosevelt, 1989). More specifically, beyond a
general slowing with increased age, an age-related decline in
spatial attention has been found when a serial visual search
is required but not when the target is salient and “pops-out”
of the visual display (Bennett et al., 2012; Foster et al., 1995;
Humphrey & Kramer, 1997; Nagamatsu et al., 2013; Plude
& Doussard-Roosevelt, 1989). 2 | Furthermore, older adults are
slower at processing visual stimuli (Ball et al., 2006; Rubin
et al., 2007) and display an increased magnitude of the “at-
tentional blink” effect. The attentional blink effect is when,
for up to 500 ms after detecting a (first) target in a rapid se-
rial visual presentation (RSVP) stream, there is a reduced
ability to detect a second target (Raymond et al., 1992). This effect is stronger and lasts for longer with increased
age (Lahar et al., 2001; Lee & Hsieh, 2009; van Leeuwen
et al., 2009; Maciokas & Crognale, 2003; Shih, 2009), which,
again, cannot be explained by general slowing alone (Lee &
Hsieh, 2009; Maciokas & Crognale, 2003). ,
;
g
,
)
One view is that ageing leads to increased activity across
the cortex due to dedifferentiation of cognitive mechanisms
(Cabeza, 2002). Such a view is compatible with theories of
impaired neural inhibition with increased age (Shih, 2009),
which could result in difficulties in reaching raised acti-
vation thresholds (Adamo et al., 2003; Aydin et al., 2013). Inhibition has been strongly linked to alpha oscillations
(8–12 Hz), including task-related modulations in amplitude
and phase (Capotosto et al., 2009; van Diepen et al., 2015,
2019; Foxe et al., 1998; Hanslmayr et al., 2005, 2007; Jensen
& Mazaheri, 2010; Klimesch et al., 2007; Rohenkohl &
Nobre, 2011; Sauseng et al., 2005; Thut et al., 2006; Worden
et al., 2000; Yamagishi et al., 2003). It has been reported that
older adults do not modulate alpha oscillations to the same ex-
tent as younger adults (Deiber et al., 2013; Hong et al., 2015;
Pagano et al., 2015; Vaden et al., 2012). This seems to be
particularly the case in anticipation of a visual target (Deiber
et al., 2013; Zanto et al., 2011). However, reduced modula-
tion of alpha oscillations does not seem to consistently result
in impaired performance. Older individuals have been found
to successfully inhibit visual information despite a lack of
alpha modulation (Vaden et al., 2012), possibly indicating
the implementation of alternative neural mechanisms, whilst
alpha might become a mere indicator for progressing dedif-
ferentiation. However, the aforementioned research that pres-
ents alpha oscillations as a primary candidate for attentional
gating has predominantly been conducted with young adults,
mostly under the age of 30 years. It is therefore unclear to
what extent attentional processes in young adults generalise
to attention mechanisms in older participants. 1 |
INTRODUCTION However, recent findings suggest that older adults are able
to efficiently recruit alternative cognitive mechanisms
when performing cognitive tasks (Cabeza et al., 2018;
Daselaar et al., 2015; Park & Reuter-Lorenz, 2009; Reuter-
Lorenz & Park, 2014). An important trajectory of research However, recent findings suggest that older adults are able
to efficiently recruit alternative cognitive mechanisms
when performing cognitive tasks (Cabeza et al., 2018;
Daselaar et al., 2015; Park & Reuter-Lorenz, 2009; Reuter-
Lorenz & Park, 2014). An important trajectory of research Eur J Neurosci. 2021;00:1–24.
| 1
wileyonlinelibrary.com/journal/ejn
1 |
INTRODUCTION
Over past decades, the predominant view of age-related
changes in cognitive function was that of a continuous
decline (Dempster, 1992; Salthouse, 1996; West, 1996). However, recent findings suggest that older adults are able
to efficiently recruit alternative cognitive mechanisms
when performing cognitive tasks (Cabeza et al., 2018;
Daselaar et al., 2015; Park & Reuter-Lorenz, 2009; Reuter-
Lorenz & Park, 2014). An important trajectory of research
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd
Eleanor Huizeling, Carol Holland was contributed equally to this work. Over past decades, the predominant view of age-related
changes in cognitive function was that of a continuous
decline (Dempster, 1992; Salthouse, 1996; West, 1996). Eur J Neurosci. 2021;00:1–24.
| 1
wileyonlinelibrary.com/journal/ejn
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited and is not used for commercial purposes. © 2021 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd
Eleanor Huizeling, Carol Holland was contributed equally to this work. Eur J Neurosci. 2021;00:1–24. 2 | Occipital and
parietal
Kolev et al., (2002)
5c
Desync: Y < M
b. Frontal
Kolev et al., (2002)
Abbreviations: ACC, anterior cingulate cortex; b, bilateral; DV, dependent variable; FEF, frontal eye fields; H, hypothesis; M, middle-age; MFG, Middle frontal gyrus;
N/A, not applicable; O, old; rDLPFC, right dorsolateral prefrontal cortex; RSVP, rapid serial visual presentation; RT, response time; VS, visual search; Y, young. Abbreviations: ACC, anterior cingulate cortex; b, bilateral; DV, dependent variable; FEF, frontal eye fields; H, hypothesis; M, middle-age; MFG, Middle frontal gyrus;
N/A, not applicable; O, old; rDLPFC, right dorsolateral prefrontal cortex; RSVP, rapid serial visual presentation; RT, response time; VS, visual search; Y, young. However, inconsistent with a simple formulation of the
PASA hypothesis of ageing (Davis et al., 2008), theta modu-
lations (3–7 Hz) along the frontal midline have been reported
to diminish with increasing age—in both resting state and
task-related conditions (Cummins & Finnigan, 2007; Reichert
et al., 2016; van de Vijver et al., 2014). Theta oscillations
are associated with a broad array of tasks measuring exec-
utive function and cognitive control (Cavanagh et al., 2009;
Cavanagh & Frank, 2014; Demiralp & Başar, 1992; Green &
McDonald, 2008; Min & Park, 2010; Sauseng et al., 2010). Although one recent magnetoencephalography (MEG) study
found decreased theta with increased age—in left frontal eye
fields (FEF), right dorsolateral prefrontal cortex (DLPFC) and
right postcentral gyrus—in a visual processing task (Wiesman
& Wilson, 2019), age-related reductions in frontal midline
theta have most commonly been observed in memory recall
tasks and during resting state and were mostly recorded with
electroencephalography (EEG; Cummins & Finnigan, 2007;
Reichert et al., 2016; van de Vijver et al., 2014). Alternative
evidence suggests that theta power decreases from childhood
throughout adulthood, which could reflect increased experi-
ence and reduced cognitive effort, yet increases again later in
life (Gómez et al., 2013). Consistent with these findings and
a PASA hypothesis of ageing (Davis et al., 2008), Gazzaley
et al., (2008) found an increase in frontal midline theta power In support of alternative processing strategies as a
reason for more widely distributed brain activity in older
age, there is evidence to suggest that older adults are
able to compensate for attentional deficits with top-down
control of attention, such as utilising cues more effec-
tively than younger people in selective attention tasks
(McLaughlin & Murtha, 2010; Neider & Kramer, 2011;
Watson & Maylor, 2002). 2 | The present
study sets out to shed further light on whether changes in
alpha oscillations could be indicative of dedifferentiation or
reduced inhibition in older age groups and explain deficits in
attentional focusing. Abilities in switching between temporal and spatial
attention have remained underinvestigated (Callaghan
et al., 2017), despite dynamic refocusing of attention poten-
tially being crucial for everyday activities such as driving
(Callaghan et al., 2017; Huizeling et al., 2020; Torrens-
Burton et al., 2020). Our recent findings show that older age
groups are less efficient at switching from a temporal to a
spatial focus of attention (Callaghan et al., 2017). The aim of
the current study was to investigate the neural patterns that
reflect age-related changes in the ability to refocus or real-
locate attention from time to space. Using a paradigm devel-
oped in our recent behavioural work (Callaghan et al., 2017),
we compared three age groups on their ability to switch from
a standard temporal attention task, which required the iden-
tification of a single target in a stream of distractors (RSVP),
to allocating attention spatially and to identify a target in a
visual search (VS) task. Overlapping brain networks across occipital, frontal, pa-
rietal and motor regions have been implicated in directing 3 HUIZELING et al. |
TABLE 1
Hypotheses
Time
DV
H
no. H
Location
Citation
RSVP
Alpha
1a
Desync: Y > O
b. Occipital and
parietal
Deiber et al., (2013) and Zanto et al., (2011)
1b
Desync: Y > M
b. Occipital and
parietal
Kolev et al., (2002)
1c
Sync: Y > O
b. Occipital and
parietal
Vaden et al., (2012)
VS
RT
2a
No-Switch <Switch
N/A
Callaghan et al., (2017)
2b
Y = M
N/A
Callaghan et al., (2017)
2c
Y < O
N/A
Callaghan et al., (2017)
Switch-Costs
3a
Switch Target Y < M
N/A
Callaghan et al., (2017)
3b
Switch Target Y < O
N/A
Callaghan et al., (2017)
Theta
4a
Y > O
Frontal midline, MFG,
FEF, rDLPFC
Cummins and Finnigan (2007); van de Vijver
et al., (2014) and Wiesman and Wilson (2019)
4b
Y < O
b. Frontal and ACC
Madden et al., (2007)
4c
Y? M
b. Frontal and ACC? N/A
Alpha
5a
Desync: Y > O
b. Occipital and
parietal
Deiber et al., (2013) and Zanto et al., (2011)
5b
Desync: Y > M
b. 4 | The aim of the current study was to investigate the oscilla-
tory patterns that reflect age-related changes in the ability to
switch from allocating attention in time, to allocating atten-
tion spatially. Specifically, we compared age groups on their
ability to switch from identifying a single target in an RSVP
stream (spatially focal but temporally changing), to identify-
ing a target in a VS display (spatially distributed but tempo-
rally unchanging). The cost of switching from the temporal
attention task to the spatial attention task was manipulated
by altering the position of the target in the RSVP stream. As
in Callaghan et al., (2017), the RSVP target was (a) the first
item in the stream, which behaved as a No-Switch condition,
because the participant was no longer required to attend to
the RSVP stream once they had identified the target; (b) to-
wards the end of the RSVP stream (Switch Target condition)
or (c) absent from the RSVP stream (Switch No-Target con-
dition), which each (both b and c) behaved as Switch condi-
tions, because the participant was required to attend to the
RSVP stream until (near to) the end of the stream. Based on
Callaghan et al., (2017), we expected faster RTs in the No-
Switch compared to the two Switch conditions and greater
costs of switching in the older and middle-aged groups in
comparison to the younger group, (especially in the Switch
Target condition; Table 1 H3a–b). We hypothesised that, in response to the VS display, there
could be an increase in frontal theta activity with increased
age (Table 1, H4b), either reflecting beneficial, additional
top-down processing (Davis et al., 2008; Fabiani et al., 2006;
Gazzaley et al., 2008; Madden, 2007) or merely reflecting
dedifferentiation (Cabeza, 2002). The former would be re-
flected in an improvement in performance with increased
theta modulation. Conversely, in the latter, no such improve-
ment in performance would be observed with increased cor-
tical recruitment. Alternatively, reduced theta power could
be observed over the midline, as demonstrated in previous
EEG studies (Cummins & Finnigan, 2007; van de Vijver
et al., 2014), or in bilateral middle frontal gyrus (MFG),
FEF and right DLPFC, as demonstrated through MEG and
functional magnetic resonance imaging (fMRI; Madden
et al., 2007; Wiesman & Wilson, 2019). 4 | HUIZELING et al. 10–15 Hz) alpha oscillations during visual processing com-
pared to younger adults, although only in increased phase-
locking and ongoing power and not in prestimulus alpha
power, or alpha power compared to baseline, in which there
were no age group differences (Kolev et al., 2002). More
recently, Reuter et al., (2019) found reduced event-related
potential latencies and amplitudes for middle-compared to
both older- and younger-age groups during visual processing
and attentional control. In the current work, it was hypothe-
sised that the middle-age group could either begin to reflect
a similar pattern to the older group, for example, a posterior-
anterior shift in processing resources (Davis et al., 2008;
Kolev et al., 2002) or show efficient processing signatures
(Reuter et al., 2019), more similar to the younger group (see
Table 1 H1b, H4c, H5b–c). in older adults when implementing a visual attention task,
which could reflect an increase in the implementation of
top-down attentional guidance. However, it remains unclear
whether such increased activity is beneficial for performance
or rather a further indication of dedifferentiation and lack of
neural precision. In light of the aforementioned inconsistencies and com-
peting theoretical accounts, we set out to clarify whether
impaired attentional control (refocusing from a temporal to
a spatial task) in older adults (Callaghan et al., 2017) is char-
acterised by an increased spread of activation or a reduced
activation across cortical networks. Based on the reviewed
findings, our primary focus of investigation was centred on
modulations of alpha and theta frequency bands. We used
MEG to increase spatial resolution over previous EEG stud-
ies, whilst achieving the necessary temporal resolution for
frequency-specific analysis, thus, allowing for oscillatory
analysis in source space. Based on the reviewed literature, it was expected that
older adults would display age-related differences in alpha
modulation during RSVP and VS processing, either through
a weaker alpha power decrease (Table 1 H1a, H5a; Deiber
et al., 2013; Zanto et al., 2011) that could be indicative of
reduced target processing, or through a weaker alpha power
increase (Table 1 H1c; Vaden et al., 2012) that could be indic-
ative of reduced distractor suppression. We expected to ob-
serve age differences in alpha power modulation in bilateral
parietal and occipital visual attention regions (see Table 1
H1a–c, H5a–c). 2 | As proposed by the “Scaffolding
Theory of Ageing and Cognition” (STAC; Park & Reuter-
Lorenz, 2009; Reuter-Lorenz & Park, 2014), successful
compensatory cognitive strategies are likely to recruit ad-
ditional neural resources, which could be reflected by a
wider distribution of brain activity—prominently involving
brain areas related to top-down control. Accordingly, the
“posterior to anterior shift in ageing hypothesis” (PASA;
Davis et al., 2008) proposes that there is a compensa-
tory shift in activity towards frontal regions in conjunc-
tion with declines in occipital sensory processing, which
has accrued supporting evidence (Buckner et al., 2000;
Cabeza et al., 2004; Davis et al., 2008; Grady, 2000;
Huettel et al., 2001; Madden, 2007; Madden et al., 2002;
Ross et al., 1997). Crucially, increased frontal activity has
been shown to correlate with decreased occipital activity
(Cabeza et al., 2004; Davis et al., 2008) and improved task
performance (Davis et al., 2008; Madden, 2007). 2.2 The attention switching paradigm from (Callaghan
et al., 2017) was adapted for use with MEG (see Figure 1). The major change to the MEG paradigm was to reduce the
number of conditions whilst increasing the number of trials in
each condition (for the required signal-to-noise ratio for MEG
analysis), by focusing only on pop-out VS, since Callaghan
et al., (2017) had reported ceiling effects for Switch-Costs in
serial VS. On each experimental trial, participants attended
to an RSVP stream first before switching to a pop-out VS
display. Each trial consisted of a fixation cross, presented for
2,000 ms, followed by the RSVP stream, which was imme-
diately followed by the VS display. E-Prime 2.0 Professional
(Psychology Software Tool. Inc.) was used on a windows PC
to present stimuli, record responses and send triggers to the
MEG through a parallel port (at the onsets of RSVP, target [if
applicable] and VS display, as well as upon response to VS). Stimuli were back-projected onto a screen inside a magneti-
cally shielded room (MSR) approximately 86 cm in front of
the participant at a resolution of 1,400 × 1,050. All stimuli
were presented in black (RGB 0-0-0) on a grey background
(RGB 192-192-192). Sixty-three participants in three age groups (19–30, 40–49
and 60+ years; see Table 2 for details) were included in the
final analysis. An age range of 40–49 years was selected for
the middle-age group, so as to be an equal number of years
apart from the youngest and oldest groups. Whilst an age
range of 40–49 years might not cover the full range of mid-
dle age, it is representative of middle age and avoids debates
about the exact start and end age of middle age. Participants
with visual impairments, photosensitive epilepsy and a his-
tory of brain injury or stroke were excluded from participa-
tion. 2.1 | Participants Participants were recruited from Aston University staff and
students and the community. Participants aged over 60 years
were also recruited from the Aston Research Centre for
Healthy Ageing (ARCHA) participation panel. Participants
provided written informed consent before participating
and were screened for contraindications to having an MRI
or MEG scan and received standard payment according to
local rules. The research was approved by Aston University
Research Ethics Committee (#776) and complied with the
Declaration of Helsinki. 4 | Whilst temporal attention manifests as a sharp focus of
attention to a single location, and a strong inhibition of the
surrounding locations, an efficient switch to spatial atten-
tion is expected to require a rapid release of these inhibition
processes, combined with a sharpening of attention to the
surrounding locations that were previously inhibited. We ex-
pected higher level attentional control regions, such as the
frontal cortex and anterior cingulate cortex (ACC; Table 1
H4a–c), as well as parietal cortex (Table 1 H1a–c, H5a–c),
to be involved in coordinating such attentional control, as
well as alpha synchronisation (for inhibition; Table 1 H1c), In addition to comparing younger and older aged adults,
we compared performance in a middle-aged group. Visual
attention processing is understudied in middle age. However,
there is some evidence to suggest that attentional con-
trol is already less efficient in middle age compared to in
young adults (Callaghan et al., 2017; Georgiou-Karistianis
et al., 2006; Huizeling et al., 2020; Zhou, Fan, Lee, Wang,
& Wang, 2011). The current study provides valuable novel
insights into neural oscillatory signatures in middle age
during attention switching. Middle-aged adults have been
shown to display a posterior-anterior shift in ongoing (upper; 5 HUIZELING et al. scored over the 87 cut-off for possible cognitive impairment
on the Addenbrookes Cognitive Examination 3 (ACE-3;
Noone, 2015). The ACE-3 consists of a series of short tasks
that provide measures of language, memory, attention, flu-
ency and visuospatial abilities. In total, 73 participants were
tested, but six participants were excluded from analysis due
to low performance accuracy and/or too noisy MEG data re-
sulting in fewer than 30 out of 80 trials remaining for one
or more conditions after data preprocessing. These six par-
ticipants included one individual aged 40–49 years and five
participants aged 60+ years. Two participants withdrew from
the study, and in two data sets, there was a recording error. Demographics for the remaining 63 participants are pre-
sented in Table 2. desynchronisation (for enhanced attention; Table 1 H1a–b,
H5a–c) and theta increases (for increased processing; Table 1
H4a–c). 2.2 ISI Inter
stimulus interval similarity to certain numbers, letters I, O and S were excluded
from the stream. Letters K and Z were targets defined for
the VS task and were therefore also not employed as distrac-
tors in the RSVP. It should be noted that the current RSVP
task differs from a standard attentional blink paradigm as the
RSVP stream only contained a maximum of a single target. the first item in the stream (No-Switch condition) or the
target was either the seventh or ninth item in the stream
(Switch Target condition) or absent from the stream (Switch
No-Target condition). Illustrations of the RSVP stream and
of the VS display are presented in Figure 1. There were 80
trials of each of the three conditions (No-Switch/Switch
Target/Switch No-Target), with a total of 240 trials. To pro-
vide the opportunity for breaks, trials were divided into 10
blocks. Trials were randomised within blocks. Participants
completed 24 practise trials before starting the experimental
trials. The VS display consisted of eight letters presented in a
circle around a fixation cross in the centre of the screen, in-
cluding seven distractors and one target. Participants were
instructed to keep their eyes fixed on the cross at the centre
of the screen, whilst they completed the VS and to respond
as quickly as possible. The target letter was always either a
“K” or a “Z” and distractors were always a “P.” rendering a
“pop-out” VS, conforming to effects observed by Callaghan
et al., (2017; see Section 1 for details). Stimuli were pre-
sented in font size 20 pt (0.50 × 0.50 cm, 0.52°), and the cen-
tre of each stimulus was 2.3 cm (2.40°) from the centre of the
fixation cross. Participants pressed a button with their right
index finger once they had identified the VS target. Note that
conforming to Callaghan et al., (2017), this button press did
not discriminate between K or Z but merely indicated that the
participant had identified the target on that trial. Participants'
RTs to press this button were recorded and allowed for a more
accurate and less variable search time estimate than a dis-
criminative response (for detailed discussion, see Callaghan
et al., 2017). For MEG it had the added benefit that this re-
sponse did not trigger different neural motor patterns (e.g.,
for different finger taps). 2.2 All participants in the 60+ years group (60–82 years) TABLE 2
Participant demographics
Age group (years)
19–30 (n = 20)
40–49 (n = 20)
60+
(n = 23)
Age (years)
Mean
24.6
44.95
68.61
SD
2.96
3.28
5.43
Sex
Male
08
07
10
Female
12
13
13
Handedness
Right
16
19
22
Left
04
01
01
ACE-3
Mean
n/a
n/a
95.5
SD
n/a
n/a
2.69
Note: This table presents the demographics for each age group, including
participants' mean age, the number of participants who are male and female, the
number of participants who are left and right handed, in addition to the mean
ACE-3 scores for the 60+ years group. TABLE 2
Participant demographics The RSVP stream consisted of a rapidly changing stream
of letters in the centre of the display. There were 10 items
in each RSVP stream, each presented for 100 ms with no
interstimulus interval. Stimuli were presented in font size
30pt (0.75 × 0.75 cm, 0.78°). On two thirds of the trials,
one of the items in the stream was a target, namely, a digit
(1/2/3/4/6/7/8/9), which participants were expected to detect
and memorise for report at the end of the trial (after the VS). The target could be either the first stimulus of the stream (re-
moving the need to attend to the stream) or the seventh or
ninth item in the stream of 10 stimuli. In the remaining one
third of the trials, the RSVP contained only letters and no
target digit. Due to its visual similarity to the letter S, “5”
was excluded from the pool of targets. Based on their visual 6 |
HUIZELING et al. FIGURE 1
Illustration of trial
structure and stimulus examples. The rapid
serial visual presentation (RSVP) stream
illustration (left) displays a Switch Target
RSVP stream (a target digit at position 7 in
the RSVP). Each trial consisted of a fixation
cross (2,000 ms) followed by an RSVP
stream immediately followed by a pop-out
visual search (VS) display (right). ISI Inter
stimulus interval 6 | HUIZELING et al. FIGURE 1
Illustration of trial
structure and stimulus examples. The rapid
serial visual presentation (RSVP) stream
illustration (left) displays a Switch Target
RSVP stream (a target digit at position 7 in
the RSVP). Each trial consisted of a fixation
cross (2,000 ms) followed by an RSVP
stream immediately followed by a pop-out
visual search (VS) display (right). 2.2 Subsequently, participants pressed
a button to indicate whether it was a “K” (right index finger
response) or a “Z” (left index finger response) in the display. Participants were then prompted to indicate whether they had
seen a target digit in the RSVP stream (yes: right index finger
response; no: left index finger response). If a digit was cor-
rectly detected in the RSVP stream, participants then pressed
the button that corresponded with the number that they saw. Participants wore earphones through which a “ding” sound
was played after a correct response and a chord sound was
played after an incorrect response. MEG data were recorded with a 306-channel Elekta
Neuromag system (Vectorview; Elekta) in a MSR at a sam-
pling rate of 1,000 Hz. The 306 sensors were made up of 102
triplets incorporating one magnetometer and two orthogonal
planar gradiometers. Data were recorded in two halves within
the same session. Head position was recorded continuously throughout data
acquisition via the location of five HPI coils. Three HPI coils
were positioned across the participant's forehead and one
on each mastoid. The position of each HPI coil, three fidu-
cial points and 300–500 points evenly distributed across the
head surface were recorded prior to the MEG recording with
Polhemus Fastrak head digitisation. A T1 structural MRI
was obtained for each participant and acquired using a 3T
Siemens MAGNETOM Trio MRI scanner with a 32-channel
head coil. 2.3.1 | Response times To investigate the direction of task-related change
oscillatory power in each age group, thereby improving
terpretability of subsequent source level effects, we c
pared “active” task periods (3–5 Hz: 550–1,550 ms rela
to RSVP onset; 10–14 Hz: 450–950 ms and 1,000–1,500
relative to RSVP onset; see Source level analysis section
details) to a baseline period (3–5 Hz: −1,500 to −500
10–14 Hz: −1,000 to −500 ms). Conditions were collap
to obtain the average across all conditions. Two-tailed
pendent t tests were carried out to compare the active
periods with a baseline period separately for each age gro
Multiple comparisons were corrected for using nonparam
ric cluster permutations (Maris & Oostenveld, 2007), w
2,000 permutations (cluster alpha = 0.05). HUIZELING et al. No-Target) was a within subjects factor and age group (19–
30, 40–49 and 60+ years) was a between subjects factor. Multiple comparisons were corrected for with Bonferroni
correction. For data cleaning prior to sensor level analysis, noisy sensors
were interpolated with the average of neighbouring sensors. Independent components analyses (ICA) were implemented
for each participant, across all conditions, and components
with eye blink or heartbeat signatures were removed from
the data. The data were expected to violate assumptions of equality
of variance due to increases in interindividual variability with
age (Hale et al., 1988; Morse, 1993), yet there is evidence to
support that ANOVA is robust to violations of homogene-
ity of variance (Budescu, 1982). Where Mauchly's Test of
Sphericity was significant, indicating that the assumption of
sphericity had been violated, Greenhouse–Geisser corrected
statistics were reported. Time-frequency analysis was carried out on signals from
the planar gradient representation of 102 gradiometer pairs
using a Hanning taper from 2–30 Hz (for every 1 Hz), with
four cycles per time window in stages of 50 ms. For each
participant, trials were averaged within each condition (No-
Switch/Switch Target/Switch No-Target). To interpret the age group × RSVP condition interactions,
“Switch-Costs” were calculated as the percentage differ-
ence in RTs between Switch Target and No-Switch condi-
tions (Target Switch-Costs) and between Switch No-Target
and No-Switch conditions (No-Target Switch-Costs) for each
individual. As interaction effects were already shown to be
statistically significant in the ANOVA, Restricted Fisher's
Least Significant Difference test was applied and corrections
for multiple comparisons were not conducted (Snedecor &
Cochran, 1967). 2.3.1 | Response times Where Levene's test for equality in variance
was significant (p < .05) when computing t tests, “Equality of
variance not assumed” statistics were reported. To investigate the direction of task-related changes in
oscillatory power in each age group, thereby improving in-
terpretability of subsequent source level effects, we com-
pared “active” task periods (3–5 Hz: 550–1,550 ms relative
to RSVP onset; 10–14 Hz: 450–950 ms and 1,000–1,500 ms
relative to RSVP onset; see Source level analysis section for
details) to a baseline period (3–5 Hz: −1,500 to −500 ms;
10–14 Hz: −1,000 to −500 ms). Conditions were collapsed
to obtain the average across all conditions. Two-tailed de-
pendent t tests were carried out to compare the active task
periods with a baseline period separately for each age group. Multiple comparisons were corrected for using nonparamet-
ric cluster permutations (Maris & Oostenveld, 2007), with
2,000 permutations (cluster alpha = 0.05). 2.3.2 | MEG MEG data were preprocessed in Elekta software using
MaxFilter (temporal signal space separation, tSSS, 0.98 cor-
relation; Taulu & Hari, 2009) to remove noise from sources
inside and outside the sensor array. Seventeen participants
displayed magnetic interference from dental work and so a
tSSS correlation of 0.90 was applied instead. This included
five participants from the 19–30 years group, six from the 40–
49 years group and six from the 60+ years group. Movement
correction was applied to one participant in the 40–49 years
group due to head movement (>7 mm). 2.3.1 | Response times Participants' median VS RTs (ms) on trials where both VS and
RSVP responses were correct were extracted. Participants'
proportions of correct VS target identifications and RSVP
target identifications were also extracted. Differences in median VS RTs between age groups and
RSVP conditions were analysed in a 3 × 3 mixed ANOVA,
where RSVP condition (No-Switch/Switch Target/Switch To manipulate the cost of switching, the position of the
target in the RSVP stream that preceded the VS was either HUIZELING et al. No-Target) was a within subjects factor and age group (19–
30, 40–49 and 60+ years) was a between subjects factor. Multiple comparisons were corrected for with Bonferroni
correction. The data were expected to violate assumptions of equality
of variance due to increases in interindividual variability with
age (Hale et al., 1988; Morse, 1993), yet there is evidence to
support that ANOVA is robust to violations of homogene-
ity of variance (Budescu, 1982). Where Mauchly's Test of
Sphericity was significant, indicating that the assumption of
sphericity had been violated, Greenhouse–Geisser corrected
statistics were reported. To interpret the age group × RSVP condition interactions,
“Switch-Costs” were calculated as the percentage differ-
ence in RTs between Switch Target and No-Switch condi-
tions (Target Switch-Costs) and between Switch No-Target
and No-Switch conditions (No-Target Switch-Costs) for each
individual. As interaction effects were already shown to be
statistically significant in the ANOVA, Restricted Fisher's
Least Significant Difference test was applied and corrections
for multiple comparisons were not conducted (Snedecor &
Cochran, 1967). Where Levene's test for equality in variance
was significant (p < .05) when computing t tests, “Equality of
variance not assumed” statistics were reported. 2.3.2 | MEG
MEG data were preprocessed in Elekta software using
2.3.3 | Sensor level analysis
For data cleaning prior to sensor level analysis, noisy sen
were interpolated with the average of neighbouring sens
Independent components analyses (ICA) were implemen
for each participant, across all conditions, and compon
with eye blink or heartbeat signatures were removed f
the data. Time-frequency analysis was carried out on signals f
the planar gradient representation of 102 gradiometer p
using a Hanning taper from 2–30 Hz (for every 1 Hz), w
four cycles per time window in stages of 50 ms. For e
participant, trials were averaged within each condition (
Switch/Switch Target/Switch No-Target). 2.3.4 | Source level analysis For source localisation using spatial filters (beamformers),
noisy sensors were excluded rather than interpolated and ICA
was not implemented to remove eye blinks and cardio arte-
facts. Due to size restrictions of the MEG data file, each data
set was recorded in two halves within the same session and
were therefore MaxFiltered separately prior to concatenating
the data, which could lead to different components being re-
moved in each half of data (see MaxFilter details in above
MEG section). To reduce potential artefacts due to applying
MaxFiltering to the two halves of data separately, a principle
components analysis was implemented to reduce data dimen-
sionality to components that accounted for 99% of the vari-
ance. The participant remained in the scanner, and the door
to the MSR remained shut between recording the two halves
of data. Data were read into the Matlab® toolbox Fieldtrip version
20151004 (Oostenveld et al., 2011), with Matlab® 2015a,
band-pass filtered between 0.5 and 85 Hz and epoched from
3.5 s preceding VS onset (i.e., 2.5s preceding RSVP stream
onset) to 2.0 s after the onset of the VS display. Trials were
visually inspected for artefacts, and any noisy trials were
removed. Fieldtrip version 20161031 was used for further
analysis. Trials with incorrect responses were excluded. After ex-
cluding inaccurate and noisy trials, the mean number of trials
that remained for each condition was 68.93 (SD = 6.58) for
the 19–30 years group, 67.97 (SD = 7.51) for the 40–49 years
group and 60.36 (SD = 9.85) for the 60+ years group. Participants with fewer than 30 trials were excluded from the
analysis (see Methods: Participants section). Using an in-house Matlab script and Elekta software MRI
Lab, individual MRIs were aligned with the sensor array, by
aligning the individual's MRI with the fiducial positions and
head shape that were recorded with Polhemus Fastrak head
digitisation. Individual single-shell head-models (5-mm vox-
els) were created from the coregistered MRIs. Head-models HUIZELING et al. 8 plots in Figures 5, 6 and 8–11 were plotted in BrainNet
Viewer 1.63 (Xia et al., 2013). were normalised to MNI space (Montreal Neurological
Institute template). were normalised to MNI space (Montreal Neurological
Institute template). 2.3.4 | Source level analysis p
)
To identify the cortical generators of sensor level fre-
quency modulations, we extracted time-frequency tiles from
the time frequency representations (TFRs) in Figure 3, se-
lecting 3–5 Hz with a time window of 550–1,550 ms (relative
to RSVP onset) and 10–14 Hz with a time window of 450–
950 and 1,000–1,500 ms (relative to RSVP onset) for theta
and alpha frequencies, respectively. Note that this does not
inflate type-1 error rates, as the selection was not made by
contrasting conditions or age groups but rather on the over-
all pattern across all conditions and groups. A 3–5 Hz theta
range (selected from inspection of Figure 3) is lower than a
typical theta band of 4–7 Hz and overlaps with typical delta
frequency (0–4 Hz), however, is in line with early accounts
of a theta response (3–6 Hz) to visual stimuli (Demiralp &
Başar, 1992). Similarly, 10–14 Hz is higher than a typical
alpha range of 8–12 Hz but is consistent with a range in which
effects have previously been found in visual processing stud-
ies (Vaden et al., 2012). Moreover, the overlap between alpha
and theta frequency ranges was minimised in order to capture
distinct processing. To estimate theta frequency (centred at
4 Hz), we required a time window of 1,000 ms. Given that
the average VS RT of the younger participants was 550 ms
in the No-Switch condition, we avoided selecting a time win-
dow that went beyond 550 ms after VS onset (1,550 ms after
RSVP onset), which would be contaminated with processing
of, and response to, the follow-up question, “Was the letter a
K or a Z?”. To estimate alpha frequency (centred at 12 Hz),
we required a time window of 500 ms. For alpha, it was there-
fore possible to select two separate time windows (theta did
not allow for such temporal resolution) to capture the two
distinct phases of each trial (temporal and spatial tasks). The
first alpha time window (450–950 ms) was selected to cap-
ture processing of the end of the RSVP stream and the prepa-
ration to switch whilst avoiding any spectral leakage from
the onset of the VS display. This window included the RSVP
target in the Switch Target condition, which was presented at
either 700 or 900 ms. 2.3.4 | Source level analysis The second alpha time window (1,000–
1,500 ms) was time-locked to the onset of the VS display to
capture VS processing immediately after the switch. Two-tailed dependent t tests were carried out to compare
each of the Switch conditions (Switch Target/Switch No-
Target) with the No-Switch condition separately for each age
group. Multiple comparisons were corrected for with non-
parametric cluster permutations (Maris & Oostenveld, 2007). Second level analysis was carried out by comparing
Switch-Costs at the group level (Bögels et al., 2014; Wang
et al., 2016). For each participant, the No-Switch condition
was subtracted from each of the Switch conditions separately. These differences were entered into two two-tailed indepen-
dent cluster permutation t tests (2,000 permutations) to com-
pare age groups (19–30 years vs. 40–49 years/19–30 years vs. 60+ years). To explore the relationship between behavioural perfor-
mance and power changes in theta and alpha frequencies
in the two older groups (to better understand cognitive de-
cline), differences in power (at peaks of the main significant
clusters of the source analysis) between Switch Target and
No-Switch conditions, in theta and alpha power, were en-
tered into one-sided Spearman's correlation analyses with
behavioural RT Target Switch-Costs. Power change was ex-
tracted from single voxels in which the strongest effect of
switching was observed (as per the t-statistics presented in
Figures 5, 8 and 10a). Coordinates of peaks were visually
inspected to ensure they were indeed in the centre of the most
prominent clusters. As no significant age group differences
were found in No-Target Switch-Costs, we focused only on
correlations between power change in the Switch Target con-
dition (compared to No-Switch) and Target Switch-Costs in
RT. Similarly, as the youngest group showed no significant
difference in RTs between Switch and No-Switch conditions,
and because we were specifically interested in understanding
impaired switching performance in the two older groups, we
focused on correlations in only the middle- and older-aged
groups. To investigate possible correlations between be-
haviour and residual activity in regions shown to be involved
in younger but not older groups, power change at the younger
group's cluster peaks were additionally entered into the older
groups' correlation analyses. Bonferroni correction was used
to adjust the level of alpha to control for the number of cor-
relations (the number of correlations is described below). | |
9 Target conditions is presented in Figure S1. Group means of
participants' median VS RTs are presented in Figure 2. For the correlation of Target Switch-Costs and alpha
power change during the RSVP window, one (parietal) ROI
was chosen from the source level analysis of the 19–30 years
group, and one ROI was chosen for each of the older and
middle-aged groups (from the source level analysis pre-
sented in Figure 8). This resulted in four correlations, with
two for each (older) age group (i.e., 60+ years: parietal and
superior temporal gyrus; 40–49 years: parietal and posterior
parietal). Target conditions is presented in Figure S1. Group means of
participants' median VS RTs are presented in Figure 2. The 3 × 3 (RSVP condition × age group) mixed ANOVA
on participants' median VS RTs revealed a significant main
effect of age (F(2, 60) = 11.36, p < .001, η²p = .28), a sig-
nificant main effect of RSVP condition (F(2, 120) = 35.21,
p < .001, η²p = .37) and a significant interaction between age
and RSVP condition (F(4, 120) = 7.05, p < .001, η²p = .19). Post hoc comparisons revealed that the main effect of age
resulted from significantly slower RTs in the 60+ years group
in comparison to both the 19–30 (p < .001) and 40–49 years
(p = .029) groups. There was no significant difference be-
tween the 19–30 and 40–49 years groups (p > .10). For the correlation of Target Switch-Costs with alpha
power change during the VS window, one (inferior frontal
gyrus; IFG) ROI was chosen from the source level analysis
from the 19–30 years group, and one ROI was chosen for
each of the older and middle-aged groups (from the source
level analysis presented in Figure 10). This resulted in four
correlations, with two for each (older) age group (i.e., 60+
years: IFG and cerebellum; 40–49 years: IFG and cerebel-
lum). Coordinates for the selected peaks can be found in
Table S1–S3 in the supporting information. The main effect of RSVP condition resulted from signifi-
cantly slower RTs in both the Switch Target (p < .001) and
Switch No-Target (p < .001) conditions in comparison to the
No-Switch condition. There was no significant difference in
RTs between the Switch Target and Switch No-Target condi-
tions (p > .10). | To investigate the hypothesis that there would be signifi-
cantly greater Switch-Costs in both the 40–49 and 60+ years
groups in comparison to the 19–30 years group and to in-
terpret the interaction between age and RSVP condition, in-
dependent t tests were carried out comparing Switch-Costs
across age groups. Please refer to Methods for a description
of how Switch-Costs were calculated for each participant. Means and SDs of participants' Switch-Costs are presented
in Table 3. 2.3.4 | Source level analysis Frequency band specific Dynamic Imaging of Coherent
Sources (DICS; Gross et al., 2001) beamformers (spatial
filters; 2% lambda regularisation) were calculated based
on cross-spectral densities obtained from the fast Fourier
transform (FFT) of signals from 204 gradiometers using a
Hanning taper, spectral smoothing of ±2 Hz and 2.0 s of data
padding. No baseline correction was applied, and conditions
were directly compared instead. Note that although group
differences were also present in the beta frequency band (15–
25 Hz), our hypotheses focused on alpha and theta bands
based on the previous literature (see Introduction). Surface For the correlation of theta power change with Target
Switch-Costs, one (parietal) region of interest (ROI) was
chosen from the source level analysis (presented in Figure 5)
from the 19–30 years group (reflecting residual activity in
the older groups), in addition to two ROIs from each of the
older and middle-aged groups. This resulted in six correla-
tions for theta power in total, with three for each (older) age
group (i.e., 60+ years: parietal, MFG and temporal lobe; 40–
49 years: parietal, ACC and occipital lobe). HUIZELING et al. 3.2 | MEG results Frequencies from 2–30 Hz were explored, and TFRs are
shown in Figure 3, averaged over a group of posterior sen-
sors for visualisation purposes only. Although a similar pat-
tern was seen across frontal sensors (see Figure S3), here we
present posterior sensors due to the improved signal-to-noise
ratio compared to frontal sensors. Note that although group
differences may also be present in the beta frequency band
(15–25 hz), our hypotheses focused on alpha and theta bands
based on previous literature (see Section 1) and we therefore
omitted beta in our analysis. TABLE 3
Means and SDs of Switch-Costs for each age group
Age group (years)
19–30 (n = 20)
40–49 (n = 20)
60+ (n = 23)
Target Switch-Costs
Mean
4.02
19.67
26.65
SD
12.72
15.78
15.67
No-target Switch-Costs
Mean
12.59
17.29
17.98
SD
15.24
15.66
18.43 3
Means and SDs of Switch-Costs for each age group 3.1 | Response times: switch-costs All groups correctly identified over 96% of VS targets in all
three conditions. Thus, no further analysis was carried out
on VS accuracy. All groups correctly identified over 73% of
RSVP targets in both RSVP conditions. RSVP accuracy was
unrelated to the aims and hypotheses of the current study, and
no further analysis was carried out on RSVP accuracy. The
proportion of correct RSVP target identifications in the two Switch-Costs, when the target was presented towards the
end of the RSVP (Switch Target), were significantly greater
in both the 40–49 (df = 38, t = −3.45, p < .001) and 60+
(df = 41, t = −5.15, p < .001) years groups in comparison to FIGURE 2
Group means of
participants' median visual search
(VS) response times (RTs). Vertical
bars represent the SE. The figure was
created in R (R Core Team, 2018) using
the RainCloudPlots package (Allen
et al., 2021) 10 10 HUIZELING et al. the 19–30 years group. There were no significant age group
differences in Switch-Costs (p > .10), when no target was
presented in the RSVP (Switch No-Target). the 19–30 years group. There were no significant age group
differences in Switch-Costs (p > .10), when no target was
presented in the RSVP (Switch No-Target). used to switch when target consolidation was required. To im-
prove our understanding of the neurocognitive processing used
to switch between modalities of attention across the three age
groups, in the following sections, we will investigate group dif-
ferences in task-related oscillatory signatures. The RT results replicated findings from Callaghan
et al., (2017) by demonstrating deficits in switching in both the
40–49 years and 60+ years groups in comparison to the 19–
30 years group. Consistent with Callaghan et al., (2017), greater
Switch-Costs in the older age groups were only significant
when participants were required to process a target digit before
switching. When there was no target in the RSVP stream, older
participants seemed better able to switch from the temporal to
the spatial attention task, suggesting either an increased de-
mand for more processing resources or differences in strategies | (p = .020) and the 60+ years group (p = .075), although the
latter did not reach significance. These positive clusters ad-
ditionally extended to occipital cortex, resulting from lower
theta in the Switch Target condition in comparison to the
No-Switch condition in the older groups but not the younger
group. In the 60+ years group, greater theta power increases
in the parietal region were associated with decreased Target
RT-Switch-Costs (r = −0.53, p = .005). Importantly, due
to reduced parietal theta in the 60+ years group overall
(Figure 5b), the coordinates for the parietal correlation effect
were adopted from the 19–30 years group, in order to specif-
ically investigate whether residual theta power in the oldest
participants would be beneficial for attention switching. No
such correlations were observed for the middle-aged group
(p > .10 uncorrected). VS display, relative to baseline, in a time window of 550–
1,550 ms, in all age groups, which was significantly greater
than baseline in the 19–30 and 40–49 years groups. Statistical
results comparing theta power in Switch Target and No-
Switch conditions and exploring the interaction between
RSVP condition and age group are presented in Figure 5 (for
details, see Methods, Section 2). Figure 5 illustrates that all age groups displayed a signifi-
cantly higher theta increase in the Switch Target condition
in comparison to the No-Switch condition, which localised
to superior and inferior parietal gyri, occipital gyri and the
MFG in the 19–30 years group, bilateral frontal cortex and
the ACC in the 40–49 years group and the superior frontal
gyrus, temporal gyri and the cerebellum in the 60+ years
group (Figure 5a). In summary, the 19–30 years group dis-
played higher theta in parietal regions, and the two older
groups demonstrated extensive frontal recruitment. The
correlation between increased left MFG theta power and
decreased Switch-Costs in the 60+ years group (r = −0.40,
p = .029) did not reach significance using a more stringent
alpha level of p < .008 after Bonferroni correction to control
for the number of tests performed (n = 6). The 60+ years
group additionally displayed higher temporal lobe theta. The
40–49 years group additionally presented with a posterior
(occipital/cerebellar) negative cluster, which reflects lower
theta in the Switch Target condition in comparison to the No-
Switch condition, although this did not reach significance in
a two-sided test (p = .033). 3.2.2 Age-group comparisons of differences between Switch
Target and No-Switch conditions, which are presented in
Figure 5b, confirmed that the higher theta increase in the
Switch Target condition was greater in the 19–30 years group
in parietal regions in comparison to the 40–49 years group There was a nonsignificant increase in alpha power in rela-
tion to baseline in the 450–950 ms time window (relative
to RSVP onset) in the 19–30 years group (see Figure 7;
also Figure 3). In contrast, the 60+ years group showed a | Note that the spread of source
power to the centre of the brain in medial slices in Figures 5,
8 and 9 is likely to be a result of spatial leakage, a known
challenge in the spatial resolution of MEG source analysis,
particularly towards the centre of the brain, where the signal-
to-noise ratio is lower and source estimation is less precise
(Hillebrand & Barnes, 2002). Figure 6a reveals that there was no significant differ-
ence between Switch No-Target and No-Switch conditions
in theta frequency in the 19–30 years group, suggesting that
the differences observed in theta between Switch Target and
No-Switch conditions in this age group (see Figure 5) were
a result of processing the RSVP target in the Switch Target
condition. In contrast, both the 40–49 and 60+ years groups again
display negative clusters that localise to the occipital lobes,
indicating deficient theta increases in the Switch No-Target
condition, a finding that cannot be due to RSVP target pro-
cessing. The 60+ years group again showed higher theta in
the Switch No-Target condition in comparison to the No-
Switch condition that localised to frontal regions and the left
temporal lobe. However, group differences did not reach sig-
nificance for a two-sided test (Figure 6b). 3.2.1 | Theta power The TFRs in Figure 3 and sensor level analysis in Figure 4
illustrate that there was a theta increase in response to the FIGURE 3
Time frequency
representations (TFRs) present power
in relation to a baseline period of −0.6
to −0.01 s in a group of four posterior
gradiometer pairs (gradiometer pair
positions are illustrated as black dots
on an empty topographical plot of the
magnetoencephalography (MEG) helmet,
top-right corner of the figure). The onset of
the rapid serial visual presentation (RSVP)
stream occurred at 0.0 s. Black lines placed
over TFRs indicate the onset of the visual
search (VS) display, and RSVP target onset
occurred at either 0.7 or 0.9 s FIGURE 3
Time frequency
representations (TFRs) present power
in relation to a baseline period of −0.6
to −0.01 s in a group of four posterior
gradiometer pairs (gradiometer pair
positions are illustrated as black dots
on an empty topographical plot of the
magnetoencephalography (MEG) helmet,
top-right corner of the figure). The onset of
the rapid serial visual presentation (RSVP)
stream occurred at 0.0 s. Black lines placed
over TFRs indicate the onset of the visual
search (VS) display, and RSVP target onset
occurred at either 0.7 or 0.9 s FIGURE 3
Time frequency
representations (TFRs) present power
in relation to a baseline period of −0.6
to −0.01 s in a group of four posterior
gradiometer pairs (gradiometer pair
positions are illustrated as black dots
on an empty topographical plot of the
magnetoencephalography (MEG) helmet,
top-right corner of the figure). The onset of
the rapid serial visual presentation (RSVP)
stream occurred at 0.0 s. Black lines placed
over TFRs indicate the onset of the visual
search (VS) display, and RSVP target onset
occurred at either 0.7 or 0.9 s FIGURE 3
Time frequency
representations (TFRs) present power
in relation to a baseline period of −0.6
to −0.01 s in a group of four posterior
gradiometer pairs (gradiometer pair
positions are illustrated as black dots
on an empty topographical plot of the
magnetoencephalography (MEG) helmet,
top-right corner of the figure). The onset of
the rapid serial visual presentation (RSVP)
stream occurred at 0.0 s. Black lines placed
over TFRs indicate the onset of the visual
search (VS) display, and RSVP target onset
occurred at either 0.7 or 0.9 s 11 HUIZELING et al. 11 FIGURE 4
Effects in theta (3–
5 Hz) when contrasting Switch period
(550–1,550 ms; collapsed across all three
rapid serial visual presentation [RSVP]
conditions) to the baseline period (−1,500
to −500 ms), for each age group. Sensor
topographies present t-statistics of
significant clusters (*p < .025, ***p < .001;
positive clusters denoted in red) care. The spatial distribution of alpha oscillatory effects that
are visible in Figure 8a, along with the lack of significant
group differences in direct group comparisons (Figure 8b),
suggest that this group's pattern of alpha oscillations, was
closer to the younger group. significant decrease in alpha power in relation to baseline in
the same time window, and the 40–49 years group showed no
significant difference (see Figure 7; also Figure 3). There was
a significant decrease in alpha power compared to baseline
in the VS time window (1,000–1,500 ms relative to RSVP
onset) in all age groups (see Figure 7; also Figure 3). Figure 8
presents the statistical results that compare alpha power in
Switch Target and No-Switch conditions (panel a), as well
as the interaction between RSVP condition and age group
(panel b). significant decrease in alpha power in relation to baseline in
the same time window, and the 40–49 years group showed no
significant difference (see Figure 7; also Figure 3). There was
a significant decrease in alpha power compared to baseline
in the VS time window (1,000–1,500 ms relative to RSVP
onset) in all age groups (see Figure 7; also Figure 3). Figure 8
presents the statistical results that compare alpha power in
Switch Target and No-Switch conditions (panel a), as well
as the interaction between RSVP condition and age group
(panel b). Group comparisons of differences highlighted that the
higher alpha in the Switch Target condition in comparison
to the No-Switch condition was significantly greater in the
60+ years groups in comparison to the 19–30 years group,
as is reflected by the widely distributed negative cluster in
Figure 8b, spanning frontal, parietal and temporal areas. However, the different origins of this group effect should be
kept in mind, when interpreting the result, since younger par-
ticipants revealed an alpha increase during the RSVP, whilst
older participants presented with an alpha decrease (see
Figures 3 and 7). There was no significant difference between
the 19–30 and 40–49 years groups (p > .10). There were no
significant correlations between the change in alpha power at
cluster peaks (during the RSVP window) and Target Switch-
Costs (all p > .10 uncorrected). As stated above, the spread
of source power to the centre of the brain in medial slices in
Figures 5, 8 and 9 is a result of spatial leakage. HUIZELING et al. 12 All age groups show significantly higher alpha power
in the Switch Target condition in comparison to the No-
Switch condition during the RSVP stream, which localised
primarily to parietal regions in the young and middle-aged
groups and was widely distributed across the cortex in the
60+ years group (Figure 8a). The 60+ years groups displayed
higher frontal lobe alpha, and both the 40–49 and 60+ years
groups displayed higher temporal lobe alpha in the Switch
Target condition in comparison to the No-Switch condition
(Figure 8a). Figures 3 and 7 suggest that, in the 19–30 years group, this
difference in alpha resulted from an alpha increase through-
out the RSVP stream that was higher in the Switch Target
condition than the No-Switch condition. In contrast, in the
60+ years group, higher alpha in the Switch Target condi-
tion resulted from a greater alpha decrease in the No-Switch
condition than the Switch Target condition throughout RSVP
presentation (Figure 3). Given that no significant change in
alpha power in relation to baseline was detectable in the 40–
49 years group (see Figure 7) at sensor level, the source level
alpha effects in this age group should be interpreted with Similar to the Switch Target versus No-Switch contrast, all
age groups show significantly higher alpha in the Switch No-
Target condition in comparison to the No-Switch condition in
the RSVP time window, which localised to parietal regions
in all age groups and was widely distributed across the cortex
in the 60+ years group. In the 40–49 years group, the distri-
bution extended primarily into the ventral processing stream
in occipito-temporal cortex. In the 60+ years group the wider
distribution also comprised frontal and prefrontal areas. FIGURE 5
Effects in theta (3–5 Hz)
when contrasting Switch Target and No-
Switch conditions in each age group (a),
and when exploring the Switch Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01,
***p < .001; positive clusters denoted in
red; negative clusters denoted in blue). For
unthresholded effects see Figure S2 FIGURE 5
Effects in theta (3–5 Hz)
when contrasting Switch Target and No-
Switch conditions in each age group (a),
and when exploring the Switch Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01,
***p < .001; positive clusters denoted in
red; negative clusters denoted in blue). For
unthresholded effects see Figure S2 FIGURE 5
Effects in theta (3–5 Hz)
when contrasting Switch Target and No-
Switch conditions in each age group (a),
and when exploring the Switch Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01,
***p < .001; positive clusters denoted in
red; negative clusters denoted in blue). For
unthresholded effects see Figure S2 13 HUIZELING et al. 13 Similar to the pattern seen when comparing Switch Target
and No-Switch conditions in Figure 8, lower alpha in the No-
Switch condition in comparison to the Switch No-Target con-
dition appears to have resulted from a greater alpha increase
in the Switch No-Target condition in the 19–30 years group
and a greater alpha decrease in the No-Switch condition in
the 60+ years group (see Figures 3 and 7), which is import-
ant to consider when interpreting intragroup and intergroup
effects. Switch No-Target condition (Figure 11a). The 40–49 years
group showed a greater alpha decrease in occipital cortex in
the Switch No-Target condition compared to the No-Switch
condition. The cluster in the 40–49 years group also extends
to the cerebellum; however, the estimation of sources close
to the edge of the sensor array can be poor and should be
interpreted with caution, as this could be a result of spatial
leakage. Group comparisons revealed that the higher alpha in the
Switch No-Target condition in comparison to the No-Switch
condition was significantly higher in the 60+ years group in
comparison to the 19–30 years group, as is reflected by the
negative clusters in Figure 9b. Group differences between
the 19–30 and 40–49 years groups did not reach significance
(p = .056). Whilst alpha effects were contained to parietal
regions in the 19–30 years group, in the 60+ years group the
higher alpha effects were both stronger and more widely dis-
tributed across the cortex. 4 |
DISCUSSION 4 In our previous work, we demonstrated that older adults find
refocusing attention from time to space more difficult than
younger adults (Callaghan et al., 2017). In the current study,
we replicated these results and found that the older (60+) as
well as the middle-aged (40–49) group had increased Switch-
Costs compared to the younger (19–30) group, as reflected by
disproportionately increased RTs when required to refocus at-
tention from a temporal RSVP task (when it included a target)
to a spatial VS task. Age group differences cannot be attributed
to spatial attention deficits, as age group differences are typi-
cally absent for pop-out VS (beyond general slowing; Bennett
et al., 2012; Foster et al., 1995; Humphrey & Kramer, 1997;
Nagamatsu et al., 2013; Plude & Doussard-Roosevelt, 1989),
which was used here. The primary aim of the current study was
to investigate the age-related changes in neural mechanisms In response to VS onset, the 19–30 years group displayed
a greater alpha decrease in the No-Switch compared to the
Switch Target condition in frontal cortex (Figure 10a). In con-
trast, the 40–49 years and 60+ years groups show a greater
alpha decrease in the Switch Target condition compared to
the No-Switch condition in parietal cortex and cerebellum. Group comparisons demonstrated that such group differences
were significant (Figure 10b). There were no significant cor-
relations between the change in alpha power at cluster peaks
(during the VS window) and Target Switch-Costs (p > .10
uncorrected). In response to VS onset, both the 19–30 years and
60+ years groups displayed a greater alpha decrease in left
frontal and parietal cortex in the No-Switch compared to the FIGURE 6
Effects in theta (3–5 Hz)
when contrasting Switch No-Target and
No-Switch conditions in each age group (a),
and when exploring the Switch No-Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (**p < .01, **p < .001;
positive clusters denoted in red; negative
clusters denoted in blue) was a theta increase after VS onset in all age groups, which
is typically seen during the processing of a visual stimulus
(e.g., Demiralp & Başar, 1992; Wiesman & Wilson, 2019). By comparing theta across Switch and No-Switch condi-
tions, we can investigate the effect switching has on process-
ing the VS display, as illustrated in Figure 12. We observed
increased theta in the Switch conditions relative to the No-
Switch condition in all age groups. In the youngest group,
the dominant pattern of theta oscillations was an increase in
parietal theta. In the two older groups, the dominant pattern
was a theta increase in frontal (middle-aged and older group)
and temporal (older group) regions, accompanied by weaker
occipital theta in the Switch compared to No-Switch condi-
tions (a schematic of these results is presented in Figure 12). However, only differences in parietal and occipital theta were
significantly different from younger adults in the middle-aged
group in the direct group comparisons (Switch Target condi-
tion), effects that were observed as nonsignificant trends in
the oldest group. Increased temporal lobe theta in the oldest
group compared to the youngest group was again observed
as a nonsignificant trend in the group comparisons (Switch
No-Target). that may underlie this difficulty in refocusing attention from
events changing in time to stimuli distributed spatially. We
aimed to determine whether changes in attention refocusing
are characterised by a reduced activation across cortical net-
works or an increased spread of activation, which could reflect
either increased compensation or dedifferentiation. Also consistent with Callaghan et al., (2017), RTs of the
60+ years group were slower overall in comparison to the
19–30 years group. On the other hand, RTs of the 40–49 and
19–30 years groups did not significantly differ, implying that
the 40–49 years group found the baseline No-Switch condition
no more demanding than younger adults. However, the 40–
49 years group again presented significantly higher Switch-
Costs than the 19–30 years group, suggesting that they found
the Switch Target condition disproportionality more demanding
than the No-Switch condition, contrasting with the 19–30 years
group. The 40–49 years group indeed seems to represent an
intermediate stage of ageing, where some aspects of attentional
control function at a similar level to younger adults, whereas
other aspects coincide more with patterns observed in older
adults, as observed in both RTs and neural oscillations. Conforming to our hypotheses based on previous reports
(Cummins & Finnigan, 2007; Deiber et al., 2013; Gazzaley
et al., 2008; Vaden et al., 2012; van de Vijver et al., 2014),
we indeed observed modulations of theta and alpha oscilla-
tory power (Figures 3–11). The enhanced spatial resolution
of MEG compared to EEG allowed us to go beyond the previ-
ous literature to investigate group differences in source space. Our findings do not support previous findings of a reduc-
tion in frontal midline theta power, as indicated by several
previous EEG reports (Cummins & Finnigan, 2007; Reichert
et al., 2016; van de Vijver et al., 2014). An increase in frontal
midline theta with increased age has, instead, previously been
observed by Gazzaley et al., (2008). Although the observed
correlation between reduced Switch-Costs and higher MFG
theta power did not reach significance with a conservative
Bonferroni correction, due to insufficient power, and thus
cannot be regarded as reliable at the current stage, the strength
of the correlation (with a medium effect size, of r = −0.40;
Cohen, 1992) indicates that it is worthy to guide further 4.1 | Theta The hypothesis that there would be reduced theta power with
increased age (Table 1 H4a) was partially supported. There 14 HUIZELING et al. 14 FIGURE 7
Effects in alpha (10–14 Hz) when contrasting an rapid serial visual presentation (RSVP) window (450–950 ms) and the visual
search (VS) onset window (1,000–1,500 ms) to the baseline period (−1,000 to −500 ms), for each age group, collapsed across conditions. Sensor
topographies present t-statistics of significant clusters (**p < .01, ***p < .001; positive clusters denoted in red; negative clusters denoted in blue) Both the 40–49 and 60+ years groups showed signifi-
cantly lower occipital and cerebellar theta in Switch condi-
tions (compared to the No-Switch condition), a difference
that was not present in the 19–30 years group (see schematic
of the three age groups in Figure 12). Although no significant
posterior negative cluster was seen in the 60+ years group in
the Switch Target comparison, this could be due to the lim-
ited sensitivity of cluster permutation analyses when localis-
ing both positive and negative clusters. Indeed, when plotting
t-statistics across the entire cortex, a similar (nonsignificant)
negative cluster is also visible in the Switch Target condi-
tion compared to the No-Switch condition (see Figure S2). Significant group differences were observed in a small part
of the occipital cortex between the young and middle-aged
group, an effect that was observed as a nonsignificant trend
in older adults. Theta activity (difference between Switch
Target and No-Switch) in this region revealed a negative cor-
relation with RT-Switch-Costs in the Switch Target condition
(r = −0.44, p = .018) in the 60+ years group. Weaker poste-
rior theta in the two Switch conditions may be linked to age-
related increases in VS RTs in these conditions. Interestingly,
group differences in occipital theta were observed in only
the Switch Target condition (and not the Switch No-Target The 19–30 years group showed higher Switch Target re-
lated theta in parietal regions in comparison to the two older
age groups (although the latter was only a trend). Posterior
parietal activity is usually observed during enhanced atten-
tion in young adults (Coull & Nobre, 1998; Li et al., 2013;
Madden et al., 2007). Increased parietal theta in the current
task seems to be affected by RSVP target processing and/
or storage, as no significant difference in theta was seen be-
tween Switch No-Target and No-Switch conditions in the 19–
30 years group (Figure 6a). From the TFRs in Figure 3, the
onset of the (posterior) theta response seems time-locked to
the onset of the VS display, rather than the RSVP target, sug-
gesting that RSVP target processing influenced subsequent
VS processing. Moreover, residual parietal theta in the old-
est group was related to lower Switch-Costs (Switch Target),
further suggesting that RSVP target processing may influ-
ence subsequent switching. Importantly, the parietal source
coordinates were adopted from a theta effect in the young-
est group. FIGURE 7
Effects in alpha (10–14 Hz) when contrasting an rapid serial visual presentation (RSVP) window (450–950 ms) and the visual
search (VS) onset window (1,000–1,500 ms) to the baseline period (−1,000 to −500 ms), for each age group, collapsed across conditions. Sensor
topographies present t-statistics of significant clusters (**p < .01, ***p < .001; positive clusters denoted in red; negative clusters denoted in blue) FIGURE 7
Effects in alpha (10–14 Hz) when contrasting an rapid serial visual presentation (RSVP) window (450–950 ms) and the visual
search (VS) onset window (1,000–1,500 ms) to the baseline period (−1,000 to −500 ms), for each age group, collapsed across conditions. Sensor
topographies present t-statistics of significant clusters (**p < .01, ***p < .001; positive clusters denoted in red; negative clusters denoted in blue) 15 HUIZELING et al. 15 investigation and future replications. We cannot rule out that
increased frontal theta power reflects alternative, beneficial
processing, instead of mere dedifferentiation (Cabeza, 2002)
or lack of neural precision (Shih, 2009; Welford, 1981). Overall, theta power findings partially support that older
adults might predominantly recruit executive, top-down re-
sources during attentional control (Davis et al., 2008; Fabiani
et al., 2006; Madden, 2007), as well as compensatory mod-
els of ageing such as STAC (Park & Reuter-Lorenz, 2009;
Reuter-Lorenz & Park, 2014) and PASA (Davis et al., 2008),
which proposes a posterior to anterior shift with increasing
age. theta activity in older individuals, which resembles parietal
theta activity in the young group, is beneficial to attentional
switching in these older individuals, and reflects the mainte-
nance of attention mechanisms (Cabeza et al., 2018; Nyberg
et al., 2012; Park & Reuter-Lorenz, 2009; Reuter-Lorenz &
Park, 2014). This then could be complemented by compen-
satory MFG recruitment in theta but requires confirmation in
future work. theta activity in older individuals, which resembles parietal
theta activity in the young group, is beneficial to attentional
switching in these older individuals, and reflects the mainte-
nance of attention mechanisms (Cabeza et al., 2018; Nyberg
et al., 2012; Park & Reuter-Lorenz, 2009; Reuter-Lorenz &
Park, 2014). This then could be complemented by compen-
satory MFG recruitment in theta but requires confirmation in
future work. Thus, it appears that stronger residual parietal FIGURE 8
Effects in alpha (10–14 Hz)
during rapid serial visual presentation
(RSVP) when contrasting Switch Target
and No-Switch conditions in each age group
(a), and when exploring the Switch Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (***p < .001; positive
clusters denoted in red; negative clusters
denoted in blue) HUIZELING et al. window appeared to be very similar when the target was both
present (Switch Target) and absent (Switch No-Target) from
the RSVP stream, across all groups. In contrast, age group
differences in Switch-Costs were only observed in the Switch
Target condition. It therefore seems unlikely that a deficit in
temporal attention alone can explain difficulties in switching. Furthermore, there is some evidence to suggest that increased
Switch-Costs are also observed when switching from spatial
to temporal attention (Jefferies et al., 2015), but future work
should aim to provide further confirmatory evidence. maintenance of task goals or storing targets in working mem-
ory (e.g., silent vocalisations could facilitate either target re-
call or the maintenance of task goals). Anecdotally, whilst
collecting behavioural data outside of the scanner (Callaghan
et al., 2017), the first author noticed that older participants
have a tendency to speak quietly to themselves during the
task (e.g., whispering the target). It could be that a similar
strategy was applied silently in the MEG scanner (in which
they were instructed not to speak and keep their face still and
relaxed). However, such an explanation is speculative and re-
quires further investigation. The middle-aged group presented an intermediate pattern
of alpha power at sensor level (Figure 3), where no signifi-
cant difference in alpha power from baseline was detectable
(collapsed across all conditions; see Figure 7), which differed
from a significant alpha increase in the younger adults and
a significant alpha decrease in the older adults. In source
space (RSVP time window), the pattern of switch-costs in
the middle-aged group was again somewhere between the
younger and older groups, where alpha modulation was addi-
tionally observed in the temporal lobe, but this wider distri-
bution did not reach significance in direct group comparisons
(Figures 8 and 9). FIGURE 11
Effects in alpha (10–
14 Hz) during visual search (VS) when
contrasting Switch No-Target and No-
Switch conditions in each age group (a),
and when exploring the Switch No-Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01;
positive clusters denoted in red; negative
clusters denoted in blue) FIGURE 11
Effects in alpha (10–
14 Hz) during visual search (VS) when
contrasting Switch No-Target and No-
Switch conditions in each age group (a),
and when exploring the Switch No-Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01;
positive clusters denoted in red; negative
clusters denoted in blue) FIGURE 9
Effects in alpha
(10–14 Hz) during rapid serial visual
presentation (RSVP) when contrasting
Switch No-Target and No-Switch conditions
in each age group (a), and when exploring
the Switch No-Target condition × age
interaction (b). The colour bar displayed in
panel a applies to both (a) and (b). Source
plots present t-statistics of significant
clusters (***p < .001; positive clusters
denoted in red; negative clusters denoted
in blue) FIGURE 10
Effects in alpha (10–
14 Hz) during visual search (VS) when
contrasting Switch Target and No-Switch
conditions in each age group (a), and when
exploring the Switch Target condition × age
interaction (b). The colour bar displayed in
panel a applies to both (a) and (b). Source
plots present t-statistics of significant
clusters (*p < .025, **p < .01, ***p < .001;
positive clusters denoted in red; negative
clusters denoted in blue) condition) in the group comparison statistics, consistent with
the pattern of RT-Switch-Cost results. These findings further
suggest that switching is particularly impaired after RSVP
target processing. Reduced activity in the occipital lobe is
consistent with previous findings of age-related reductions
in visual cortex activity during visual processing and, more
generally, with the PASA hypothesis (Davis et al., 2008;
Huettel et al., 2001; Madden et al., 2002; Ross et al., 1997;
Ross et al., 1997). The temporal lobe activity in the 60+ years group could
indicate strategies to complete the task, such as episodic
memory encoding (Schacter & Wagner, 1999) and/or silent
vocalisation (Graves et al., 2007; Hickok & Poeppel, 2007;
Hocking & Price, 2009; Smith et al., 1998). A lack of correla-
tion with reduced RT-Switch-Costs means that these theta
sources could also reflect dedifferentiation. However, an al-
ternative possibility is that they reflect strategies to support
task processes unrelated to the speed of switching, such as the HUIZELING et al. 17 FIGURE 11
Effects in alpha (10–
14 Hz) during visual search (VS) when
contrasting Switch No-Target and No-
Switch conditions in each age group (a),
and when exploring the Switch No-Target
condition × age interaction (b). The colour
bar displayed in panel a applies to both (a)
and (b). Source plots present t-statistics of
significant clusters (*p < .025, **p < .01;
positive clusters denoted in red; negative
clusters denoted in blue) 4.2 | Alpha Nonsignificant: ns 18 |
HUIZELING et al. HUIZELING et al. 18 FIGURE 12
Schematic of the timeline of expected cognitive processes during the attention switching task. Switching (presented in yellow)
may have delayed attention towards the visual search (VS) (presented in purple) after attending to the rapid serial visual presentation (RSVP)
stream (presented in blue). RSVP target consolidation (presented in red) may have further delayed switching when overlapping with switching
(Switch Target) but not when the target was presented first (No-Switch). Topographical plots at the bottom represent approximate illustrations of
the pattern of observed results (based on Figures 5, 6 and 8–11a). In addition, we have included schematic alpha power topographical plots for
the RSVP period, to emphasise our observation that the young group showed an alpha increase during the RSVP (presented in red), whilst the
oldest group presented with an alpha decrease (presented in blue; based on Figures 3 and 7). Nonsignificant: ns FIGURE 12
Schematic of the timeline of expected cognitive processes during the attention switching task. Switching (presented in yellow)
may have delayed attention towards the visual search (VS) (presented in purple) after attending to the rapid serial visual presentation (RSVP)
stream (presented in blue). RSVP target consolidation (presented in red) may have further delayed switching when overlapping with switching
(Switch Target) but not when the target was presented first (No-Switch). Topographical plots at the bottom represent approximate illustrations of
the pattern of observed results (based on Figures 5, 6 and 8–11a). In addition, we have included schematic alpha power topographical plots for
the RSVP period, to emphasise our observation that the young group showed an alpha increase during the RSVP (presented in red), whilst the
oldest group presented with an alpha decrease (presented in blue; based on Figures 3 and 7). Nonsignificant: ns (Switch Target) and parietal (Switch No-Target) cortex could
also indicate differences in relation to presence (Switch
Target) versus absence (Switch No-Target), respectively, of a
target in the preceding RSVP. have fewer processing resources available to enhance atten-
tion to the VS display. This is further supported by the alpha
power decrease in the 60+ group during the RSVP (collapsed
across all conditions; see Figure 7), relative to baseline. An il-
lustration of this timeline is presented in Figure 12. However,
such an explanation is merely speculative, and future research
should aim to thoroughly investigate this hypothesis. 4.2 | Alpha As anticipated, there were age-related changes in task related
alpha modulation during both the RSVP and VS time win-
dows (Table 1 H1a–c, H5a–c). During the RSVP window, in-
stead of showing an alpha increase to inhibit irrelevant visual
information (Vaden et al., 2012), the 60+ years age group
showed an alpha decrease. This stronger and widely dis-
tributed alpha desynchronization (Figures 3 and 7–9) could
reflect an enhanced attention strategy rather than an inhibi-
tion strategy. A lack of alpha synchronisation is in line with
the hypothesis of reduced inhibition in older adults (Adamo
et al., 2003). Such group differences in temporal attention
strategies may have impaired the older groups' ability to effi-
ciently switch to the spatial attention task, as fewer attentional
resources were available to refocus attention (illustrated in
Figure 12). In other words, it is possible that slower switch-
ing resulted from a deficit in temporal attention. However,
the pattern of alpha power modulation during the RSVP time During the VS window, all groups displayed an alpha de-
synchronization in relation to baseline. In the younger group,
the alpha desynchronization was greater in the No-Switch
compared to both Switch conditions but predominantly local-
ised to frontal cortex in the Switch Target condition and left
parietal cortex in the Switch No-Target contrast. It could be
that this greater alpha desynchronization in frontal and pari-
etal regions reflects increased resources available to attend to
the VS in the No-Switch condition compared to the Switch
conditions. In addition, the distinct localisations to frontal 18 |
HUIZELING et al. FIGURE 12
Schematic of the timeline of expected cognitive processes during the attention switching task. Switching (presented in yellow)
may have delayed attention towards the visual search (VS) (presented in purple) after attending to the rapid serial visual presentation (RSVP)
stream (presented in blue). RSVP target consolidation (presented in red) may have further delayed switching when overlapping with switching
(Switch Target) but not when the target was presented first (No-Switch). Topographical plots at the bottom represent approximate illustrations of
the pattern of observed results (based on Figures 5, 6 and 8–11a). In addition, we have included schematic alpha power topographical plots for
the RSVP period, to emphasise our observation that the young group showed an alpha increase during the RSVP (presented in red), whilst the
oldest group presented with an alpha decrease (presented in blue; based on Figures 3 and 7). 4.2 | Alpha In response to VS presentation, there was a greater alpha
decrease in the Switch Target condition compared to the No-
Switch condition in the two older groups, whereas the oppo-
site was seen in the younger group (illustrated as a schematic
in Figure 12). In the RSVP time window that preceded the
VS, only the youngest group showed a trend for an increase
in alpha power relative to baseline. It could be that successful
inhibition of the RSVP stream in the No-Switch condition is
only effectively implemented in the younger group, making
more processing resources available to attend to the VS dis-
play in the No-Switch condition. In contrast, the middle-aged
and older groups may fail to inhibit the irrelevant distractors
in the RSVP stream after processing the RSVP Target (which
was the first item in the RSVP in the No-Switch condition) and Importantly, in the group comparison statistics, group
differences in alpha power during VS processing only
reached significance in the Switch Target contrast and not
the Switch No-Target contrast. This is similar to both the
RT-Switch-Cost results, where significant group differences
in Switch-Costs were observed only in the Switch Target
condition, and the theta results, where between-group sta-
tistics revealed group differences only in the Switch Target
condition—albeit only as a trend in the oldest group. These
findings all align to suggest that switching from the temporal
to the spatial attention task was more difficult with increased 19 HUIZELING et al. 19 | age only when there was a target embedded within the pre-
ceding RSVP stream. et al., (2017) found no correlation between measures of exec-
utive control and Switch-Costs. These findings suggest that
age-related changes in Switch-Costs in the current paradigm
reflect attention-specific effects, rather than executive con-
trol more generally. Both theta and alpha signatures revealed widely distrib-
uted processing networks in older participants compared to
the youngest group and predominantly displayed a strong
propensity towards frontal involvement (see Figure 12). However, alpha modulations did not reveal significant cor-
relations with behavioural Switch-Costs, possibly support-
ing an interpretation in terms of increased neural noise
(Shih, 2009; Welford, 1981). However, it should be noted
that decreased alpha amplitudes with age might hamper
correlational analysis due to a reduction in signal strength. 4.2 | Alpha Previous literature has shown that prestimulus alpha desyn-
chronization no longer predicts successful stimulus process-
ing in older age (Deiber et al., 2013) as it does in younger
adults (Sauseng et al., 2005). It could be that, in older age,
alpha oscillations no longer reliably gate sensory processing
(Jensen & Mazaheri, 2010) or enhance attention to visual
stimuli (Capotosto et al., 2009; Foxe et al., 1998; Hanslmayr
et al., 2005, 2007; Klimesch et al., 2007; Rohenkohl &
Nobre, 2011; Sauseng et al., 2005; Thut et al., 2006; Worden
et al., 2000; Yamagishi et al., 2003), possibly placing more
demand on top-down attentional control regions. The major-
ity of literature that supports alpha as a sensory gating mecha-
nism has been conducted in younger adults. It is possible that
such findings do not generalise to older age groups if pro-
cessing mechanisms become increasingly altered with age. The current findings call into question whether prestimulus
alpha desynchronisation predicts successful target stimulus
processing in middle age. There is some evidence to suggest
that prestimulus alpha power is unchanged in middle-aged
compared to younger adults (Kolev et al., 2002); however,
further research is needed to make firm conclusions. trol more generally. General attention switching effects also cannot account
for the observed Switch-Costs in the current task. It was only
when the temporal attention task became more demanding,
and the participant was required to process a target, that age
group differences in Switch-Costs emerged. This tentatively
supports that age group differences in Switch-Costs cannot
be explained by attention switching alone, as one may then
expect to see age group differences in attention switching
in both Switch conditions. Instead, it seems that processing
relevant information (i.e., the target) is required to delay at-
tention refocusing in older age. Arguably, the Switch Target
condition is more ecologically valid, since, in the real world,
attention tends to switch between relevant stimuli, rather than
requiring a “pure” switch from temporal to spatial attention. A limitation of the analysis was that, by investigating
oscillatory effects in source space, our temporal resolution
was constrained by our frequency bands (e.g., to 1,000 ms
for theta frequency). This analysis pipeline was chosen based
on our hypotheses drawn from the previous literature (see
Table 1). 4.2 | Alpha Although the statistical analyses were constrained
to certain length time windows, a general overview of the
temporal dynamics of the effects is displayed in the TFRs in
Figure 3. 5 |
CONCLUSIONS 5 We have replicated the behavioural findings of Callaghan
et al., (2017), observing age-related declines in the ability
to switch from temporal to spatial attention. Difficulties in
refocusing attention between time and space seem to be ac-
companied by a deficit in theta power modulation in pari-
etal, occipital and cerebellar regions. Older and middle-aged
adults' brains seem to partially attempt to compensate for this
posterior theta deficit by recruiting a more extensive frontal
network in Switch compared to No-Switch conditions, pos-
sibly reflecting increased top-down attentional control. The
60+ years group showed recruitment of the temporal lobes,
possibly reflecting task strategies such as episodic memory
encoding or silent vocalisation. Efficient (low) Switch-Costs
in the youngest group were reflected by parietal theta effects
that were absent in both older groups. However, residual pa-
rietal theta in the oldest group was related to reduced Switch-
Costs. Thus, resemblance with efficient processing in the
young brain appears to be beneficial for older brains. During
the RSVP time window, older adults showed an alpha de-
synchronization instead of synchronisation, possibly reflect-
ing an enhanced attention strategy rather than an inhibition REFERENCES Adamo, M., Westerfield, M., Haist, F., & Townsend, J. (2003). Cross-
modal shifting attention in healthy aging: An fMRI study. Society
for Neuroscience Abstract Viewer and Itinerary Planner, Abstract
No. 933.938. Allen, M., Poggiali, D., Whitaker, K., Marshall, T., van Langen, J., &
Kievit, R. (2021). Raincloud plots: a multi-platform tool for robust
data visualization. Wellcome Open Research, 4, 63. https://doi. org/10.12688/wellcomeopenres.15191.2 Aydin, S., Strang, N. C., & Manahilov, V. (2013). Age-related deficits in
attentional control of perceptual rivalry. Vision Research, 77, 32–40. https://doi.org/10.1016/j.visres.2012.11.010 ORCID Eleanor Huizeling
https://orcid. org/0000-0001-7338-5966 Eleanor Huizeling
https://orcid. org/0000-0001-7338-5966 and K.K.; Supervision and Funding Acquisition: C.H. and K.K. and K.K.; Supervision and Funding Acquisition: C.H. and K.K. strategy, as well as a stronger and more extensive task-related
alpha power modulation across the cortex. In contrast to theta
oscillations, alpha power modulations were not correlated
with Switch-Costs, indicating that increases in the extent of
power modulation could merely reflect dedifferentiation and/
or reduced neural precision in older participants. strategy, as well as a stronger and more extensive task-related
alpha power modulation across the cortex. In contrast to theta
oscillations, alpha power modulations were not correlated
with Switch-Costs, indicating that increases in the extent of
power modulation could merely reflect dedifferentiation and/
or reduced neural precision in older participants. ACKNOWLEDGEMENTS Bögels, S., Barr, D. J., Garrod, S., & Kessler, K. (2014). Conversational
interaction in the scanner: Mentalizing during language processing
as revealed by MEG. Cerebral Cortex, 25(9), 3219–3234. https://
doi.org/10.1093/cercor/bhu116 This research was supported by funding from The Rees
Jeffreys Road Fund and by the School of Life and Health
Sciences at Aston University. Scanning costs were supported
by The Wellcome Trust Lab for MEG Studies and the Dr
Hadwen Trust for Humane Research. In addition, we would
like to thank colleagues at the Aston Brain Centre for assist-
ing with MRI data acquisition. Buckner, R. L., Snyder, A. Z., Sanders, A. L., Raichle, M. E., & Morris,
J. C. (2000). Functional brain imaging of young, nondemented, and
demented older adults. Journal of Cognitive Neuroscience, 12, 24–
34. https://doi.org/10.1162/089892900564046 Budescu, D. V. (1982). The power of the F test in normal populations
with heterogeneous variances. Educational and Psychological
Measurement, 42(2), 409–416. https://doi.org/10.1177/0013164482
04200202 DATA AVAILABILITY STATEMENT The datasets collected and analysed during the current study
are available from the corresponding author on reasonable
request. ral attention task. Overall, our results demonstrate that older adults may
partially compensate for declines in attentional flexibility
with the recruitment of theta-related neural mechanisms. These findings have important implications for future
work, as they raise the question as to whether this recruit-
ment can be enhanced with cognitive training programmes
to improve compensation for functional decline. Improving
older adults' attentional flexibility could improve their
performance in everyday functions such as driving, where
one is required to quickly switch between fast changing
events in multiple surrounding locations (e.g., Huizeling
et al., 2020). ETHICS APPROVAL AND CONSENT TO
PARTICIPATE https://doi.org/10.1016/j.visres.2012.11.010 Ball, K. K., Roenker, D. L., Wadley, V. G., Edwards, J. D., Roth, D. L.,
McGwin, G., … Dube, T. (2006). Can high-risk older drivers be
identified through performance-based measures in a department of
motor vehicles setting? Journal of the American Geriatrics Society,
54(1), 77–84. https://doi.org/10.1111/j.1532-5415.2005.00568.x Participants provided written informed consent before par-
ticipating and were screened for contraindications to having
an MRI or MEG scan and received standard payment ac-
cording to local rules. The research was approved by Aston
University Research Ethics Committee (#776) and complied
with the Declaration of Helsinki. Bennett, I. J., Motes, M. A., Rao, N. K., & Rypma, B. (2012). White
matter tract integrity predicts visual search performance in young
and older adults. Neurobiology of Aging, 33(2), 433.e21–433.e31. https://doi.org/10.1016/j.neurobiolaging.2011.02.001 CONFLICT OF INTEREST The authors declare no conflict of interest. Cabeza, R. (2002). Hemispheric asymmetry reduction in older adults:
The HAROLD model. Psychology and Aging, 17(1), 85–100. https://doi.org/10.1037//0882-7974.17.1.85 PEER REVIEW The peer review history for this article is available at https://
publons.com/publon/10.1111/ejn.15259. The pattern of group differences in RT-Switch-Costs was
further reflected in alpha and theta results, where group dif-
ferences were predominantly seen when the temporal atten-
tion task involved target processing shortly before the switch,
rather than when there was no target in the preceding tempo-
ral attention task. 4.3 | Limitations One limitation of the current task design is that it does not
clearly distinguish between switching from temporal to spa-
tial attention and attention switching in general. It is unlikely
that age-related increases in Switch-Costs in the current task
reflect declines in more general executive control processes. Older adults have been shown to have preserved “local”
task switching performance compared to younger adults
(Wasylyshyn et al., 2011). Local task switching is typically
defined as the cost in RT of Switch trials compared to No-
Switch trials (similar to Switch-Costs in the current task)
and is thought to reflect the executive control in switching
from one task set to another. It therefore seems unlikely that
increased Switch-Costs in the current task could be attrib-
uted to a decline in executive function. Consistent with pre-
served executive control of switching in older adults, using
the same switching task as the current paradigm, Callaghan 20 20 HUIZELING et al. AUTHOR CONTRIBUTIONS Journal of Neural Engineering, 10(6), 066006. https://doi. org/10.1088/1741-2560/10/6/066006 Cohen, J. (1992). A power primer. Psychological Bulletin, 112(1), 155. https://doi.org/10.1037/0033-2909.112.1.155 Grady, C. L. (2000). Functional brain imaging and age-related changes
in cognition. Biological Psychology, 54(1–3), 259–281. https://doi. org/10.1016/s0301-0511(00)00059-4 Coull, J. T., & Nobre, A. C. (1998). Where and when to pay attention:
The neural systems for directing attention to spatial locations and
to time intervals as revealed by both PET and fMRI. Journal of
Neuroscience, 18(18), 7426–7435. https://doi.org/10.1523/JNEUR
OSCI.18-18-07426.1998 Graves, W. W., Grabowski, T. J., Mehta, S., & Gordon, J. K. (2007). A
neural signature of phonological access: Distinguishing the effects
of word frequency from familiarity and length in overt picture nam-
ing. Journal of Cognitive Neuroscience, 19(4), 617–631. https://doi. org/10.1162/jocn.2007.19.4.617 Cummins, T. D. R., & Finnigan, S. (2007). Theta power is reduced in
healthy cognitive aging. International Journal of Psychophysiology,
66(1), 10–17. https://doi.org/10.1016/j.ijpsycho.2007.05.008 Green, J. J., & McDonald, J. J. (2008). Electrical neuroimaging reveals
timing of attentional control activity in human brain. PLoS Biology,
6(4), 730–738. https://doi.org/10.1371/journal.pbio.0060081 Daselaar, S. M., Iyengar, V., Davis, S. W., Eklund, K., Hayes, S. M.,
& Cabeza, R. E. (2015). Less wiring, more firing: Low-performing
older adults compensate for impaired white matter with greater
neural activity. Cerebral Cortex, 25(4), 983–990. https://doi. org/10.1093/cercor/bht289 6(4), 730–738. https://doi.org/10.1371/journal.pbio.00600 Gross, J., Kujala, J., Hämäläinen, M., Timmermann, L., Schnitzler,
A., & Salmelin, R. (2001). Dynamic imaging of coherent sources:
Studying neural interactions in the human brain. Proceedings of
the National Academy of Sciences, 98(2), 694–699. https://doi. org/10.1073/pnas.98.2.694 Davis, S. W., Dennis, N. A., Daselaar, S. M., Fleck, M. S., & Cabeza, R. (2008). Qué PASA? The posterior-anterior shift in aging. Cerebral
Cortex, 18(5), 1201–1209. https://doi.org/10.1093/cercor/bhm155 Deiber, M.-P., Ibanez, V., Missonnier, P., Rodriguez, C., &
Giannakopoulos, P. (2013). Age-associated modulations of cerebral
oscillatory patterns related to attention control. NeuroImage, 82,
531–546. https://doi.org/10.1016/j.neuroimage.2013.06.037 Gross, J., Schmitz, F., Schnitzler, I., Kessler, K., Shapiro, K., Hommel,
B., & Schnitzler, A. (2004). Modulation of long-range neural syn-
chrony reflects temporal limitations of visual attention in humans. Proceedings of the National Academy of Sciences of the United
States of America, 101(35), 13050–13055. https://doi.org/10.1073/
pnas.0404944101 Demiralp, T., & Başar, E. (1992). Theta rhythmicities follow-
ing
expected
visual
and
auditory
targets. International
Journal
of
Psychophysiology, 13(2),
147–160. https://doi. org/10.1016/0167-8760(92)90054-F Hale, S., Myerson, J., Smith, G. A., & Poon, L. W. (1988). Age, variabil-
ity, and speed: Between-subjects diversity. Psychology and Aging,
3(4), 407. https://doi.org/10.1037/0882-7974.3.4.407 Dempster, F. N. (1992). AUTHOR CONTRIBUTIONS Writing-Review & Editing: E.H., H.W., C.H. and K.K.;
Conceptualization and Methodology: E.H., C.H. and
K.K.; Formal Analysis, Visualization, Data Collection
and Curation and Project Administration: E.H.; Software:
E.H. and H.W.; Writing-Original Draft Preparation: E.H. Cabeza, R., Albert, M., Belleville, S., Craik, F. I. M., Duarte, A., Grady, C. L.,
… Rajah, M. N. (2018). Maintenance, reserve and compensation: The
cognitive neuroscience of healthy ageing. Nature Reviews Neuroscience,
19(11), 701–710. https://doi.org/10.1038/s41583-018-0068-2 21 HUIZELING et al. 21 Cabeza, R., Daselaar, S. M., Dolcos, F., Prince, S. E., Budde, M., &
Nyberg, L. (2004). Task-independent and task-specific age effects
on brain activity during working memory, visual attention and
episodic retrieval. Cerebral Cortex, 14(4), 364–375. https://doi. org/10.1093/cercor/bhg133 Aging and Cognition, 2(4), 279–299. https://doi.org/10.1080/13825
589508256604 Aging and Cognition, 2(4), 279–299. https://doi.org/10.1080/13825
589508256604 Foxe, J. J., Simpson, G. V., & Ahlfors, S. P. (1998). Parieto-occipital ∼1
0 Hz activity reflects anticipatory state of visual attention mecha-
nisms. NeuroReport, 9(17), 3929–3933. Fu, S. M., Greenwood, P. M., & Parasuraman, R. (2005). Brain mech-
anisms of involuntary visuospatial attention: An event-related po-
tential study. Human Brain Mapping, 25(4), 378–390. https://doi. org/10.1002/hbm.20108 Callaghan, E., Holland, C., & Kessler, K. (2017). Age-related changes
in the ability to switch between temporal and spatial attention. Frontiers in Aging Neuroscience, 9, 28. https://doi.org/10.3389/
fnagi.2017.00028 Gazzaley, A., Clapp, W., Kelley, J., McEvoy, K., Knight, R. T., &
D'Esposito, M. (2008). Age-related top-down suppression deficit in
the early stages of cortical visual memory processing. Proceedings
of the National Academy of Sciences, 105(35), 13122–13126. https://doi.org/10.1073/pnas.0806074105 Capotosto, P., Babiloni, C., Romani, G. L., & Corbetta, M. (2009). Frontoparietal cortex controls spatial attention through modulation
of anticipatory alpha rhythms. The Journal of Neuroscience, 29(18),
5863–5872. https://doi.org/10.1523/JNEUROSCI.0539-09.2009 Cavanagh, J. F., Cohen, M. X., & Allen, J. J. B. (2009). Prelude to
and resolution of an error: EEG phase synchrony reveals cogni-
tive control dynamica during action monitoring. The Journal of
Neuroscience: The Official Journal of the Society for Neuroscience,
29(1), 98–105. https://doi.org/10.1523/JNEUROSCI.4137-08.2009 Georgiou-Karistianis, N., Tang, J., Mehmedbegovic, F., Farrow, M.,
Bradshaw, J., & Sheppard, D. (2006). Age-related differences
in cognitive function using a global local hierarchical paradigm. Brain Research, 1124, 86–95. https://doi.org/10.1016/j.brain
res.2006.09.070 Cavanagh, J. F., & Frank, M. J. (2014). Frontal theta as a mechanism
for cognitive control. Trends in Cognitive Sciences, 18(8), 414–421. https://doi.org/10.1016/j.tics.2014.04.012 Gómez, C., Pérez-Macías, J. M., Poza, J., Fernández, A., & Hornero, R. (2013). Spectral changes in spontaneous MEG activity across the
lifespan. 22 | 22 22 HUIZELING et al. Madden, D. J., & Gottlob, L. R. (1997). Adult age differences in
strategic and dynamic components of focusing visual attention. Aging Neuropsychology and Cognition, 4(3), 185–210. https://doi. org/10.1080/13825589708256647 Hickok, G., & Poeppel, D. (2007). The cortical organization of speech
processing. Nature Reviews Neuroscience, 8, 393. https://doi. org/10.1038/nrn2113 Hillebrand, A., & Barnes, G. R. (2002). A quantitative assessment of the
sensitivity of whole-head MEG to activity in the adult human cor-
tex. Neuroimage, 16(3, Part A), 638–650. https://doi.org/10.1006/
nimg.2002.1102 Madden, D. J., Spaniol, J., Whiting, W. L., Bucur, B., Provenzale, J. M., Cabeza, R., … Huettel, S. A. (2007). Adult age differences in
the functional neuroanatomy of visual attention: A combined fMRI
and DTI study. Neurobiology of Aging, 28(3), 459–476. https://doi. org/10.1016/j.neurobiolaging.2006.01.005 Hocking, J., & Price, C. J. (2009). Dissociating verbal and nonverbal
audiovisual object processing. Brain and Language, 108(2), 89–96. https://doi.org/10.1016/j.bandl.2008.10.005 Madden, D. J., Turkington, T. G., Provenzale, J. M., Denny, L. L.,
Langley, L. K., Hawk, T. C., & Coleman, R. E. (2002). Aging and
attentional guidance during visual search: Functional neuroanatomy
by positron emission tomography. Psychology and Aging, 17(1), 24–
43. https://doi.org/10.1037//0882-7974.17.1.24 Hong, X. F., Sun, J. F., Bengson, J. J., Mangun, G. R., & Tong, S. B. (2015). Normal aging selectively diminishes alpha lateralization
in visual spatial attention. NeuroImage, 106, 353–363. https://doi. org/10.1016/j.neuroimage.2014.11.019 Maris, E., & Oostenveld, R. (2007). Nonparametric statistical testing
of EEG-and MEG-data. Journal of Neuroscience Methods, 164(1),
177–190. https://doi.org/10.1016/j.jneumeth.2007.03.024 Huettel, S. A., Singerman, J. D., & McCarthy, G. (2001). The effects
of aging upon the hemodynamic response measured by func-
tional MRI. NeuroImage, 13(1), 161–175. https://doi.org/10.1006/
nimg.2000.0675 McLaughlin, P. M., & Murtha, S. J. E. (2010). The effects of age and
exogenous support on visual search performance. Experimental
Aging Research, 36(3), 325–345. https://doi.org/10.1080/03610
73x.2010.484752 Huizeling, E., Wang, H., Holland, C., & Kessler, K. (2020). Age-related
changes in attentional refocusing during simulated driving. Brain
Sciences, 10(8), 530. https://doi.org/10.3390/brainsci10080530 Min, B.-K., & Park, H.-J. (2010). Task-related modulation of anterior
theta and posterior alpha EEG reflects top-down preparation. BMC
Neuroscience, 11(1), 1–8. https://doi.org/10.1186/1471-2202-11-79 Humphrey, D. G., & Kramer, A. F. (1997). Age differences in visual
search for feature, conjunction, and triple-conjunction targets. Psychology and Aging, 12(4), 704–717. https://doi.org/10.1037//0
882-7974.12.4.704 Morse, C. K. (1993). Does variability increase with age? An archival
study of cognitive measures. Psychology and Aging, 8(2), 156. 22 | https://doi.org/10.1037/0882-7974.8.2.156 Jefferies, L., Roggeveen, A., Enns, J., Bennett, P., Sekuler, A., & Di
Lollo, V. (2015). On the time course of attentional focusing in older
adults. Psychological Research Psychologische Forschung, 79(1),
28–41. https://doi.org/10.1007/s00426-013-0528-2 Nagamatsu, L. S., Munkacsy, M., Liu-Ambrose, T., & Handy, T. C. (2013). Altered visual-spatial attention to task-irrelevant information
is associated with falls risk in older adults. Neuropsychologia, 51(14),
3025–3032. https://doi.org/10.1016/j.neuropsychologia.2013.10.002 Jensen, O., & Mazaheri, A. (2010). Shaping functional architecture by
oscillatory alpha activity: Gating by inhibition. Frontiers in Human
Neuroscience, 4, 186. https://doi.org/10.3389/fnhum.2010.00186 Neider, M. B., & Kramer, A. F. (2011). Older adults capitalize on con-
textual information to guide search. Experimental Aging Research,
37(5), 539–571. https://doi.org/10.1080/0361073x.2011.619864 Klimesch, W., Sauseng, P., & Hanslmayr, S. (2007). EEG alpha oscil-
lations: The inhibition–timing hypothesis. Brain Research Reviews,
53(1), 63–88. https://doi.org/10.1016/j.brainresrev.2006.06.003 Noone,
P. (2015). Addenbrooke’s
cognitive
examination-III. Occupational Medicine, 65(5), 418–420. https://doi.org/10.1093/
occmed/kqv041 Kolev, V., Yordanova, J., Basar-Eroglu, C., & Basar, E. (2002). Age
effects on visual EEG responses reveal distinct frontal alpha net-
works. Clinical Neurophysiology, 113(6), 901–910. https://doi. org/10.1016/S1388-2457(02)00106-2 Nyberg, L., Lovden, M., Riklund, K., Lindenberger, U., & Backman, L. (2012). Memory aging and brain maintenance. Trends in Cognitive
Sciences, 16(5), 292–305. https://doi.org/10.1016/j.tics.2012.04.005 Lague-Beauvais, M., Brunet, J., Gagnon, L., Lesage, F., & Bherer, L. (2013). A fNIRS investigation of switching and inhibition during the
modified Stroop task in younger and older adults. NeuroImage, 64,
485–495. https://doi.org/10.1016/j.neuroimage.2012.09.042 Oostenveld, R., Fries, P., Maris, E., & Schoffelen, J.-M. (2011). FieldTrip:
Open source software for advanced analysis of MEG, EEG, and in-
vasive electrophysiological data. Computational Intelligence and
Neuroscience, 2011, 1–9. https://doi.org/10.1155/2011/156869 Lahar, C. J., Isaak, M. I., & McArthur, A. D. (2001). Age differences in
the magnitude of the attentional blink. Aging, Neuropsychology, and
Cognition, 8(2), 149–159. https://doi.org/10.1076/anec.8.2.149.842 Pagano, S., Fait, E., Monti, A., Brignani, D., & Mazza, V. (2015). Electrophysiological correlates of subitizing in healthy aging. PLoS
One,
10(6),
e0131063. https://doi.org/10.1371/journ
al.pone.0131063 Lee, T.-Y., & Hsieh, S. (2009). The limits of attention for visual per-
ception and action in aging. Aging Neuropsychology and Cognition,
16(3), 311–329. https://doi.org/10.1080/13825580902741351 Park, D. C., & Reuter-Lorenz, P. (2009). The adaptive brain: Aging and
neurocognitive scaffolding. Annual Review of Psychology, 60(1),
173–196. https://doi.org/10.1146/annurev.psych.59.103006.093656 Li, L., Gratton, C., Fabiani, M., & Knighta, R. T. (2013). Age-related
frontoparietal changes during the control of bottom-up and top-down Li, L., Gratton, C., Fabiani, M., & Knighta, R. T. (2013). AUTHOR CONTRIBUTIONS The rise and fall of the inhibitory mech-
anism: Toward a unified theory of cognitive development
and aging. Developmental Review, 12(1), 45–75. https://doi. org/10.1016/0273-2297(92)90003-K Hanslmayr, S., Aslan, A., Staudigl, T., Klimesch, W., Herrmann,
C. S., & Bäuml, K.-H. (2007). Prestimulus oscillations predict
visual perception performance between and within subjects. NeuroImage, 37(4), 1465–1473. https://doi.org/10.1016/j.neuro
image.2007.07.011 Fabiani, M., Low, K. A., Wee, E., Sable, J. J., & Gratton, G. (2006). Reduced suppression or labile memory? Mechanisms of ineffi-
cient filtering of irrelevant information in older adults. Journal of
Cognitive Neuroscience, 18(4), 637–650. https://doi.org/10.1162/
jocn.2006.18.4.637 Hanslmayr, S., Klimesch, W., Sauseng, P., Gruber, W., Doppelmayr, M.,
Freunberger, R., & Pecherstorfer, T. (2005). Visual discrimination
performance is related to decreased alpha amplitude but increased
phase locking. Neuroscience Letters, 375(1), 64–68. https://doi. org/10.1016/j.neulet.2004.10.092 Foster, J. K., Behrmann, M., & Stuss, D. T. (1995). Aging and visual
search: Generalized cognitive slowing or selective deficit in attention? and
Performance,
18(3),
849. https://doi.org/10.1037/009
6-1523.18.3.849 Thut, G., Nietzel, A., Brandt, S. A., & Pascual-Leone, A. (2006). alpha-Band electroencephalographic activity over occipital cortex
indexes visuospatial attention bias and predicts visual target de-
tection. Journal of Neuroscience, 26(37), 9494–9502. https://doi. org/10.1523/jneurosci.0875-06.2006 Reichert, J. L., Kober, S. E., Witte, M., Neuper, C., & Wood, G. (2016). Age-related effects on verbal and visuospatial memory
are mediated by theta and alpha II rhythms. International Journal
of Psychophysiology, 99, 67–78. https://doi.org/10.1016/j.ijpsy
cho.2015.11.004 Torrens-Burton, A., Hanley, C. J., Wood, R., Basoudan, N., Norris, J. E., Richards, E., & Tales, A. (2020). Lacking pace but not preci-
sion: Age-related information processing changes in response to a
dynamic attentional control task. Brain Sciences, 10(6), 390. https://
doi.org/10.3390/brainsci10060390 Reuter, E.-M., Vieluf, S., Koutsandreou, F., Hübner, L., Budde, H.,
Godde, B., & Voelcker-Rehage, C. (2019). A non-linear relationship
between selective attention and associated ERP markers across the
lifespan. Frontiers in Psychology, 10, 30. https://doi.org/10.3389/
fpsyg.2019.00030 doi.org/10.3390/brainsci10060390 Vaden, R. J., Hutcheson, N. L., McCollum, L. A., Kentros, J., &
Visscher, K. M. (2012). Older adults, unlike younger adults, do
not modulate alpha power to suppress irrelevant information. NeuroImage, 63(3), 1127–1133. https://doi.org/10.1016/j.neuro
image.2012.07.050 Reuter-Lorenz, P. A., & Park, D. C. (2014). How does it STAC
up? Revisiting the scaffolding theory of aging and cognition. Neuropsychology Review, 24(3), 355–370. https://doi.org/10.1007/
s11065-014-9270-9 van de Vijver, I., Cohen, M. X., & Ridderinkhof, K. R. (2014). Aging
affects medial but not anterior frontal learning-related theta os-
cillations. Neurobiology of Aging, 35(3), 692–704. https://doi. org/10.1016/j.neurobiolaging.2013.09.006 Rohenkohl, G., & Nobre, A. C. (2011). Alpha oscillations related to
anticipatory attention follow temporal expectations. Journal of
Neuroscience, 31(40), 14076–14084. https://doi.org/10.1523/jneur
osci.3387-11.2011 van Diepen, R. M., Cohen, M. X., Denys, D., & Mazaheri, A. (2015). Attention and temporal expectations modulate power, not phase,
of ongoing alpha oscillations. Journal of Cognitive Neuroscience,
27(8), 1573–1586. https://doi.org/10.1162/jocn_a_00803 Ross, M. H., Yurgelun-Todd, D. A., Renshaw, P. F., Maas, L. C.,
Mendelson, J. H., Mello, N. K., … Levin, J. M. (1997). Age-
related reduction in functional MRI response to photic stim-
ulation. Neurology, 48(1), 173–176. https://doi.org/10.1212/
WNL.48.1.173 Van Diepen, R. M., Foxe, J. J., & Mazaheri, A. (2019). The functional
role of alpha-band activity in attentional processing: The current
zeitgeist and future outlook. Current Opinion in Psychology, 29,
229–238. https://doi.org/10.1016/j.copsyc.2019.03.015 Rubin, G. S., Ng, E. S., Bandeen-Roche, K., Keyl, P. M., Freeman, E. E., & West, S. K. (2007). A prospective, population-based study of
the role of visual impairment in motor vehicle crashes among older
drivers: The SEE study. Investigative Ophthalmology and Visual
Science, 48(4), 1483. https://doi.org/10.1167/iovs.06-0474 van Leeuwen, S., Müller, N. G., & Melloni, L. (2009). Age effects
on attentional blink performance in meditation. Consciousness
and
Cognition,
18(3),
593–599. https://doi.org/10.1016/j. concog.2009.05.001 Salthouse, T. A. (1996). The processing-speed theory of adult age dif-
ferences in cognition. Psychological Review, 103(3), 403. https://
doi.org/10.1037/0033-295X.103.3.403 Wang, H., Callaghan, E., Gooding-Williams, G., McAllister, C., &
Kessler, K. (2016). Rhythm makes the world go round: An MEG-
TMS study on the role of right TPJ theta oscillations in embodied
perspective taking. Cortex, 75, 68–81. https://doi.org/10.1016/j. cortex.2015.11.011 Sauseng, P., Griesmayr, B., Freunberger, R., & Klimesch, W. (2010). Control mechanisms in working memory: A possible func-
tion of EEG theta oscillations. Neuroscience & Biobehavioral
Reviews,
34(7),
1015–1022. https://doi.org/10.1016/j.neubi
orev.2009.12.006 Wasylyshyn, C., Verhaeghen, P., & Sliwinski, M. J. (2011). Aging and
task switching: A meta-analysis. Psychology and Aging, 26(1), 15–
20. https://doi.org/10.1037/a0020912 Sauseng, P., Klimesch, W., Stadler, W., Schabus, M., Doppelmayr, M.,
Hanslmayr, S., … Birbaumer, N. (2005). A shift of visual spatial
attention is selectively associated with human EEG alpha activity. European Journal of Neuroscience, 22(11), 2917–2926. https://doi. org/10.1111/j.1460-9568.2005.04482.x Watson, D. G., & Maylor, E. A. (2002). Aging and visual marking:
Selective deficits for moving stimuli. Psychology and Aging, 17(2),
321–339. https://doi.org/10.1037/0882-7974.17.2.321 Welford, A. T. (1981). Signal, noise, performance, and age. Human
Factors,
23(1),
97–109. https://doi.org/10.1177/0018720881
02300109 Schacter, D. L., & Wagner, A. D. (1999). Medial temporal lobe activa-
tions in fMRI and PET studies of episodic encoding and retrieval. Hippocampus, 9(1), 7–24. https://doi.org/10.1002/(sici)1098-1063 West, R. L. (1996). An application of prefrontal cortex function theory
to cognitive aging. Psychological Bulletin, 120(2), 272–292. https://
doi.org/10.1037/0033-2909.120.2.272 Shih, S.-I. (2009). Using the attention cascade model to probe cog-
nitive aging. Psychology and Aging, 24(3), 550–562. https://doi. org/10.1037/a0016724 doi.org/10.1037/0033-2909.120.2.272 Wiesman, A. I., & Wilson, T. W. (2019). The impact of age and
sex on the oscillatory dynamics of visuospatial processing. NeuroImage, 185, 513–520. https://doi.org/10.1016/j.neuro
image.2018.10.036 Smith, E. E., Jonides, J., Marshuetz, C., & Koeppe, R. A. (1998). Components of verbal working memory: Evidence from neuroim-
aging. Proceedings of the National Academy of Sciences, 95(3),
876–882. Worden, M. S., Foxe, J. J., Wang, N., & Simpson, G. V. (2000). Anticipatory biasing of visuospatial attention indexed by retinoto-
pically specific alpha-band electroencephalography increases over
occipital cortex. Journal of Neuroscience, 20(6), art. no.-RC63. 22 | Age-related
frontoparietal changes during the control of bottom-up and top-down
attention: An ERP study. Neurobiology of Aging, 34(2), 477–488. attention: An ERP study. Neurobiology of Aging, 34(2), 477–488. Plude, D. J., & Doussard-Roosevelt, J. A. (1989). Aging, selective atten-
tion and feature integration. Psychology and Aging, 4(1), 98–105. https://doi.org/10.1037//0882-7974.4.1.98 Maciokas, J. B., & Crognale, M. A. (2003). Cognitive and atten-
tional changes with age: Evidence from attentional blink defi-
cits. Experimental Aging Research, 29(2), 137–153. https://doi. org/10.1080/03610730390176095 R Core Team. (2018). R: A language and environment for statistical
computing. R Foundation for Statistical Computing. Madden, D. J. (2007). Aging and visual attention. Current
Directions in Psychological Science, 16(2), 70–74. https://doi. org/10.1111/j.1467-8721.2007.00478.x Raymond, J. E., Shapiro, K. L., & Arnell, K. M. (1992). Temporary
suppression of visual processing in an RSVP task: An attentional
blink? Journal of Experimental Psychology: Human Perception 23 HUIZELING et al. 23 Snedecor, G. W., & Cochran, W. (1967). Statistical methods (pp. 593). Iowa State Univ. Taulu, S., & Hari, R. (2009). Removal of magnetoencephalographic ar-
tifacts with temporal signal-space separation: Demonstration with
single-trial auditory-evoked responses. Human Brain Mapping,
30(5), 1524–1534. https://doi.org/10.1002/hbm.20627 Xia, M., Wang, J., & He, Y. (2013). BrainNet Viewer: A network visual-
ization tool for human brain connectomics. PLoS One, 8(7), e68910. https://doi.org/10.1371/journal.pone.0068910 24 | 24 24 HUIZELING et al. Yamagishi, N., Callan, D. E., Goda, N., Anderson, S. J., Yoshida, Y.,
& Kawato, M. (2003). Attentional modulation of oscillatory activ-
ity in human visual cortex. NeuroImage, 20(1), 98–113. https://doi. org/10.1016/S1053-8119(03)00341-0 SUPPORTING INFORMATION Additional supporting information may be found online in
the Supporting Information section. Zanto, T. P., Pan, P., Liu, H., Bollinger, J., Nobre, A. C., & Gazzaley, A. (2011). Age-related changes in orienting attention in time. Journal
of Neuroscience, 31(35), 12461–12470. https://doi.org/10.1523/
jneurosci.1149-11.2011 How to cite this article: Huizeling E, Wang H,
Holland C, Kessler K. Changes in theta and alpha
oscillatory signatures of attentional control in older
and middle age. Eur J Neurosci. 2021;00:1–24. https://doi.org/10.1111/ejn.15259 Zhou, S.-S., Fan, J., Lee, T. M. C., Wang, C.-Q., & Wang, K. (2011). Age-related differences in attentional networks of alerting and ex-
ecutive control in young, middle-aged, and older Chinese adults. Brain and Cognition, 75(2), 205–210. https://doi.org/10.1016/j. bandc.2010.12.003
|
https://openalex.org/W4205822792
|
https://www.researchsquare.com/article/rs-41871/v2.pdf?c=1631869562000
|
English
| null |
Calcium-sensitive receptor expression differs between primary and secondary hyperparathyroidism
|
Research Square (Research Square)
| 2,020
|
cc-by
| 4,417
|
Calcium-sensitive receptor expression differs
between primary and secondary
hyperparathyroidism Aiping Song
China-Japan Friendship Hospital Honglei Zhang
China-Japan Friendship Hospital Bo Pang
Centers for Disease Control and Prevention 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. Research article Keywords: Calcium-sensitive receptor, secondary parathyroidism, primary hyperparathyroidism
Posted Date: September 14th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-41871/v2 Page 1/14 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 2/14 Abstract Background: Calcium-sensitive receptor (CASR) plays an important role in the pathogenesis and
progression of secondary hyperparathyroidism (SHPT). The purpose of this study is to examine the
protein and gene expression characteristics of CASR in SHPT. Methods: Immunohistochemistry and real-time PCR were used to detect and compare the expression of
CASR protein and genes in SHPT and primary hyperparathyroidism (PHPT) tissues. Results: CASR protein was down-regulated in SHPT and PHPT compared with normal parathyroid tissues
(2.42±0.5 vs. 3.2±0.62, P<0.05; 1.8±0.83 vs. 3.2±0.62, P<0.05). Further, SHPT tissue showed higher
expression of both CASR protein (2.42±0.5 vs. 1.8±0.83, P<0.05) and CASR gene (0.29±0.23 vs. 0.01±0.12,
P<0.05) than PHPT tissue, respectively. Conclusion: The expression of CASR protein and gene in SHPT is higher than that in PHPT. This feature
provides a theoretical basis and further ideas for studying the mechanism of CASR down-regulation. Conclusion: The expression of CASR protein and gene in SHPT is higher than that in PHPT. This feature
provides a theoretical basis and further ideas for studying the mechanism of CASR down-regulation. RNA isolation and real-time reverse transcription quantitative PCR In all, 31 SHPT and 16 PHPT tissue specimens were stored at 80°C until RNA isolation. Total RNA was
isolated using the TaKaRa MiniBEST Universal RNA Extraction Kit (TaKaRa Biochemicals, Osaka, Japan). The first-strand cDNA extraction was synthesized using the PirmeScript ™ 1st Strand cDNA Synthesis Kit
(TaKaRa Biochemicals). ABI 7500 FAST real-time PCR detection system (ABI, CASRisbad, CA, USA) and
SYBR Premix Ex Taq ™ (TaKaRa Biochemicals) were used for real-time quantitative PCR analysis. The
CASR primer sequences and internal reference gene sequences used for real-time reverse transcription
quantitative PCR are shown in Table 1. The specificity of the PCR product was verified by melting curve
analysis. The mRNA expression of the target gene CASR was measured with 2-△ct: △ct = CASR ct value -
β-actin ct value. Immunohistochemistry Immunohistochemical staining of CASR was performed in 31, 20, and 20 tissue specimens of SHPT,
PHPT, and normal parathyroid tissue, respectively. Staining assessed the parathyroid adenomas, nodular
hyperplasia glands, and normal glands. The parathyroid tissues of 71 patients with formalin-fixed paraffin
were evaluated. Mouse CASR monoclonal antibody (Thermo Fisher, MA1-934) was diluted 1:1500. Formalin-embedded tissue showing the best histological features was selected and sliced into 4-µm-thick
slices with a slicer, and the sections were stained with the EnVision/HRP method using an automatic
immunostainer. Microscopic evaluations were performed under 20× and 40× microscopes. A total of 500
cells were counted (five different regions, 100 cells each) to evaluate the percentage of CASR expression. CASR expression was quantified as follows: cell membrane and cytoplasm staining, CASR staining was
weak at +, moderate at ++, strong at +++, and very strong at ++++. Observers were not involved while
immunohistochemistry was in progress. expression of proteins and genes in secondary hyperthyroidism, compared with primary
hyperparathyroidism, provides a theoretical basis and ideas for studying the mechanism of CASR down-
regulation in SHPT. expression of proteins and genes in secondary hyperthyroidism, compared with primary
hyperparathyroidism, provides a theoretical basis and ideas for studying the mechanism of CASR down-
regulation in SHPT. Patients The specimens of this study were collected from patients who underwent surgery at the China-Japan
Friendship Hospital from 2013 to 2016. Both SHPT and PHPT were confirmed by pathological diagnosis. Normal parathyroid tissue was obtained from parathyroid tissue that was accidentally removed during
thyroidectomy. Background Secondary hyperparathyroidism (SHPT) is a common comorbidity of chronic kidney disease (CKD). It
occurs early in the progression of renal insufficiency and is an adaptation mechanism of the body to help
maintain the mineral balance. SHPT is characterized by phosphorus retention, hyperphosphatemia,
elevated parathyroid hormone (PTH) levels, increased fibroblast growth factor 23 (FGF23), 1,25-
dihydroxyvitamin D deficiency, hypocalcemia, decreased intestinal calcium absorption, and poor
expression of calcium-sensitive receptor (CASR) and vitamin D receptor (VDR)[1]. SHPT is the leading
cause of death and cardiovascular events in patients with CKD. The main determinants of parathyroid
dysfunction in CKD are CASR and VDR. CASR directly regulates the secretion of PTH, and CASR and VDR
signaling pathways affect the transcription of PTH genes, expression of PTH mRNA, and proliferation of
parathyroid glands. After a long disease course and end-stage renal disease (ESRD), the parathyroid
glands show changes from diffuse hyperplasia to nodular hyperplasia, along with a reduction in CASR
and VDR; however, these mechanisms remain poorly understood. Primary hyperparathyroidism (PHPT) is a disease caused by increased secretion of PTH due to tumor-like
hyperplasia of one or more parathyroid glands. Parathyroid adenoma is the most common cause. Sporadic adenoma accounts for 85% of PHPT, and hereditary adenoma accounts for 10–20%. Genetically
related genes include the MEN1 and RET genes. The molecular mechanism of sporadic parathyroid
tumors is not clear. Some clinical studies have revealed that germ cell and somatic gene mutations are
likely related, including CASR[2]. Koh et al.[3] found that CASR is one of the genes that play an important
role in parathyroid adenoma. Published literature has shown that both SHPT and PHPT are associated with deceased CASR gene and
protein expression, but there are fewer reports on the differences between the two. The different Page 3/14 Page 3/14 expression of proteins and genes in secondary hyperthyroidism, compared with primary
hyperparathyroidism, provides a theoretical basis and ideas for studying the mechanism of CASR down-
regulation in SHPT. Result We analyzed CASR protein expression in 71 tissue specimens (31 SHPT, 20 PHPT, and 20 normal
parathyroid tissue). The CASR expression in SHPT was lower than that in normal parathyroid tissue
(2.42±0.5 vs. 3.2±0.62, P<0.05) and higher than that in PHPT (2.42±0.5 vs. 1.8±0.83, P<0.05). The CASR
expression of normal parathyroid tissue was higher than both SHPT and PHPT tissues (P<0.05) (Table 2,
Figures 1–4). The expression in thyroid tissue was negative (Figure 5). We analyzed CASR mRNA
expression in 47 cases (31 SHPT and 16 PHPT,). The expression of CASR mRNA in SHPT was higher than
that in PHPT (0.29±0.23 vs. 0.01±0.12, P<0.05) (Table 3 and Figure 6). Table 1. Primers used for realtimePCR Table 1. Primers used for realtimePCR
基因
正向引物
反向引物
CASR[4]
CGGGGTACCTTAAGCACCTACGGCATCTAA
GCTCTAGAGTTAACGCGATCCCAAAGGGCTC
β-
actin[3]
ACTCTTCCAGCCTTCCTTCC
CAGGAGGAGCAATGATCTTG Table 2. CASR protein expression in different parathyroid tissues
Quantity
Expression value mean ± SD
P value
SHPT
31
2.42±0.5
＜0.05
PHPT
20
1.8±0.83
＜0.05
Normal parathyroid
20
3.2±0.62
＜0.05 Table 2. CASR protein expression in different parathyroid tissues Page 5/14
Table 3. T test for SHPT and PHPT CASR mRNA expression（realtimePCR 2-△ct）
数量
CASR/β-actin
2-△ct (χ̅ ±sd)
P值
SHPT
31
0.29±0.23
＜0.05
PHPT
16
0.01±0.12
＜0.05
Discussion Table 3. T test for SHPT and PHPT CASR mRNA expression（realtimePCR 2-△ct）
数量
CASR/β-actin
2-△ct (χ̅ ±sd)
P值
SHPT
31
0.29±0.23
＜0.05
PHPT
16
0.01±0.12
＜0.05 3. T test for SHPT and PHPT CASR mRNA expression（realtimePCR 2-△ct） sample t-test. Values were expressed in the form of mean±standard deviation. P≤0.05 was considered to
indicate statistical significance sample t-test. Values were expressed in the form of mean±standard deviation. P≤0.05 was considered to
indicate statistical significance Statistical methods The results were analyzed statistically using the SPSS software. Quantitative data between multiple
groups were analyzed by ANOVA. Quantitative data between two groups were analyzed by independent Page 4/14 Page 4/14 Discussion The human CASR gene is located on chromosome 3q13.3-21 and is abundantly expressed in the
parathyroid gland, kidney, and C cells near the thyroid follicles. CASR is a G protein-coupled receptor that is
sensitive to extracellular calcium and plays a key role in maintaining calcium balance. Under normal
circumstances, calcium-activated CASR triggers the mitogen-activated protein kinase C (MAPK) cascade,
promotes the synthesis of phospholipase A2, and production of arachidonic acid, which ultimately
reduces the synthesis and secretion of PTH[5]. CASR activation can also inhibit parathyroid cell
proliferation, 1,25-dihydroxyvitamin D3 synthesis, and renal calcium reabsorption. SHPT is a chronic progressive disease that is common in patients with CKD and has a poor prognosis,
especially for those undergoing hemodialysis. In the United States, the prevalence of CKD patients with
SHPT ranges from 2 to 5 million, with 30–50% patients with ESRD having SHPT[6]. According to the
results of Dialysis Outcomes and Practice Patterns Study (DOPPS), 27% patients with ESRD have higher
parathyroid hormone levels than recommended by the Kidney Disease Outcome Quality Initiative (KDOQI)
[7]. The consequence of SHPT is a disorder of bone metabolism including high-transport bone disease, which
can reduce bone mass and is often accompanied by bone pain and fractures; an estimated 40–87% of
dialysis patients are affected by this[8]. Extra-skeletal manifestations include calcification of soft tissues
and blood vessels and increased risk of cardiovascular disease, and may lead to very high cardiovascular
mortality in dialysis patients. In fact, >50% patients with CKD die of cardiovascular disease, and patients
with CKD receiving dialysis are 10 times more likely to develop cardiovascular disease and die than the
general population[9]. According to current treatments, a significant proportion of patients have insufficient control of PTH,
phosphorus, and/or calcium levels, and their range often exceeds the recommended values[10]. Data from
DOPPS indicate that in patients receiving hemodialysis over 180 days, the risk of cardiovascular and all-
cause death is greater when the levels of calcium, phosphorus, and PTH exceed 10 mg/dL, 7 mg/dL, and
600 pg/mL, respectively. The risks are likewise increased in patients with combinations of these high-risk
categories.[11, 12] At present, the treatment of SHPT should follow three steps: reducing the absorption of phosphorus
through dietary restriction or using phosphate binders; controlling vitamin D metabolites on PTH, and the
use of calcimimetics[13]. Discussion Ritter's research on uremic rat
models found that the decrease in CASR in uremic rats was mainly detected in the hyperparathyroidism
area[20]. Yano et al.[21] also confirmed this observation in proliferative human parathyroid tissue. In the
present study, we found that in the same tissue with SHPT, the expression of CASR in areas with
significant nodular hyperplasia was relatively weak at the protein level. Ritter et al.[22] pointed out that the
decrease in CASR content occurred after hyperparathyroidism, so it is not the initial event of SHPT
development, rather may be the result of proliferation. They found that after parathyroid hyperplasia in
uremia rats, limiting phosphate can reverse the down regulation of parathyroid CASR [20], and FGF23 will
directly affect parathyroid proliferation and/or differential regulation of CASR and VDR expression, which
may be the direction of future research[23]. CASR protein was down-regulated in both SHPT and PHPT compared with normal parathyroid tissues
(2.42±0.5 vs. 3.2±0.62, P<0.05; 1.8±0.83 vs. 3.2±0.62, P<0.05). Further, SHPT tissue showed higher
expression of both CASR protein (2.42±0.5 vs. 1.8±0.83, P<0.05) and CASR mRNA (0.29±0.23 vs. Similarly, a decrease in CASR expression also occurred in PHPT, when parathyroid gland proliferation was
not triggered by external factors rather by gene-related abnormal stimulation[24]. Signaling pathways from
CASR or controlling CASR activity may be altered in proliferating parathyroid cells. Cell proliferation may
trigger a series of events that directly or indirectly lead to the down-regulation of CASR in surrounding
cells, and transforming growth factor-alpha (TGF-α)[25], acidic fibroblast growth factor (acidic-FGF)[26],
endothelin-1 (ET-1)[27], cyclin D1[28], parathyroid hormone-related peptide (PTHrP)[29], and c-myc[30] may
play a role in this process. At present, the mechanism of down-regulation of CASR expression in SHPT is not very clear. One reason is
the lack of an effective control group. It is difficult to obtain parathyroid tissue of the normal population as
a negative control, and the parathyroid tissue from animal models cannot fully reflect the true condition of
the human body. Primary parathyroid tissue provides a good positive control. The study of the differences
between SHPT and PHPT provides further effective research ideas. Under the influence of multiple factors,
SHPT development undergoes a gradual process, from diffuse to nodular to tertiary hyperplasia. CASR
downregulation occurs at this stage. The mechanism of CASR downregulation is the key to exploring the
pathogenesis and progression of SHPT. Discussion Researchers have generally recognized the basic role of CASR in regulating PTH
secretion and emphasized the potential therapeutic value of drugs that regulate CASR activity in
parathyroid tissue[14, 15]. The emergence of new types of calcimimetics such as cinacalcet and
etelcalcetide brings new hope for treatment of SHPT. The target of these drugs is to increase CASR, but
there are also bottlenecks such as drug side effects, high cost, resistance, and poor response. At present, the treatment of SHPT should follow three steps: reducing the absorption of phosphorus
through dietary restriction or using phosphate binders; controlling vitamin D metabolites on PTH, and the
use of calcimimetics[13]. Researchers have generally recognized the basic role of CASR in regulating PTH
secretion and emphasized the potential therapeutic value of drugs that regulate CASR activity in
parathyroid tissue[14, 15]. The emergence of new types of calcimimetics such as cinacalcet and
etelcalcetide brings new hope for treatment of SHPT. The target of these drugs is to increase CASR, but
there are also bottlenecks such as drug side effects, high cost, resistance, and poor response. Parathyroidectomy is usually the last treatment strategy after failed drug treatment, but there are problems
such as surgical complications, recurrence of hyperparathyroidism, severe hypocalcemia, and hypokinetic
bone disease. The goal of treatment is to maintain serum calcium, serum phosphorus, and PTH within
acceptable target ranges[16]. Given the limitations of SHPT treatment standards, the PTH index of many
patients does not reach the ideal range [17]. Parathyroidectomy is usually the last treatment strategy after failed drug treatment, but there are problems
such as surgical complications, recurrence of hyperparathyroidism, severe hypocalcemia, and hypokinetic
bone disease. The goal of treatment is to maintain serum calcium, serum phosphorus, and PTH within
acceptable target ranges[16]. Given the limitations of SHPT treatment standards, the PTH index of many
patients does not reach the ideal range [17]. Page 6/14 Page 6/14 CASR protein was down-regulated in both SHPT and PHPT compared with normal parathyroid tissues
(2.42±0.5 vs. 3.2±0.62, P<0.05; 1.8±0.83 vs. 3.2±0.62, P<0.05). Further, SHPT tissue showed higher
expression of both CASR protein (2.42±0.5 vs. 1.8±0.83, P<0.05) and CASR mRNA (0.29±0.23 vs. 0.01±0.12, P<0.05) than PHPT tissue, respectively. Many scholars have reported that the down-regulation
of CASR expression in secondary parathyroidism is an important factor in the pathogenesis of SHPT, but
the cause and mechanism of CASR down-regulation are unclear. Discussion It is unknown whether it due to CASR
itself or external factors. Koh et al. observed that in primary parathyroidism, abnormal elevation of RGS5
has the effect of down-regulating CASR[3]. Mizobuchi et al.[18] found that GCM2 can regulate the
expression level of CASR in in vitro experiments. Brown et al.[19] found that the expression of CASR mRNA
and protein is often more severely suppressed in the nodular region of rats with SHPT; however, it was
unclear whether this difference was due to the higher cell proliferation rate. Ritter's research on uremic rat
models found that the decrease in CASR in uremic rats was mainly detected in the hyperparathyroidism
area[20]. Yano et al.[21] also confirmed this observation in proliferative human parathyroid tissue. In the
present study, we found that in the same tissue with SHPT, the expression of CASR in areas with
significant nodular hyperplasia was relatively weak at the protein level. Ritter et al.[22] pointed out that the
decrease in CASR content occurred after hyperparathyroidism, so it is not the initial event of SHPT
development, rather may be the result of proliferation. They found that after parathyroid hyperplasia in
uremia rats, limiting phosphate can reverse the down regulation of parathyroid CASR [20], and FGF23 will
directly affect parathyroid proliferation and/or differential regulation of CASR and VDR expression, which
may be the direction of future research[23]. CASR protein was down-regulated in both SHPT and PHPT compared with normal parathyroid tissues
(2.42±0.5 vs. 3.2±0.62, P<0.05; 1.8±0.83 vs. 3.2±0.62, P<0.05). Further, SHPT tissue showed higher
expression of both CASR protein (2.42±0.5 vs. 1.8±0.83, P<0.05) and CASR mRNA (0.29±0.23 vs. 0.01±0.12, P<0.05) than PHPT tissue, respectively. Many scholars have reported that the down-regulation
of CASR expression in secondary parathyroidism is an important factor in the pathogenesis of SHPT, but
the cause and mechanism of CASR down-regulation are unclear. It is unknown whether it due to CASR
itself or external factors. Koh et al. observed that in primary parathyroidism, abnormal elevation of RGS5
has the effect of down-regulating CASR[3]. Mizobuchi et al.[18] found that GCM2 can regulate the
expression level of CASR in in vitro experiments. Brown et al.[19] found that the expression of CASR mRNA
and protein is often more severely suppressed in the nodular region of rats with SHPT; however, it was
unclear whether this difference was due to the higher cell proliferation rate. Discussion Despite the differences in pathogenesis of SHPT and PHPT, there
are some similarities in clinical manifestations. Both conditions are associated with decreased CASR
expression. The present understanding is that decrease in CASR in PHPT is more related to its genetic
factors. The decrease of CASR in SHPT is related to the proliferation of parathyroid glands, but the Page 7/14 Page 7/14 mechanism of how CASR decreases during the proliferation process is unknown, and there may be
changes in gene levels similar to that found in PHPT. mechanism of how CASR decreases during the proliferation process is unknown, and there may be
changes in gene levels similar to that found in PHPT. Our study compared the expression of CASR in SHPT and PHPT and found that although CASR was
down-regulated in both conditions, the degree of down-regulation in CASR expression in SHPT was not as
obvious as in primary parathyroidism. This indicates that the down regulation of CASR in primary
parathyroidism may occur early and severely, and changes in gene levels or signaling pathways may play
a role in the difference between the two. Research around this difference will provide further ideas for
identifying new targets to increase CASR expression. Conclusion SHPT is a common complication in patients undergoing dialysis for CKD, which can lead to decreased
quality of life, increased cardiovascular events, and increased mortality. There is currently no particularly
effective treatment for SHPT. CASR expression is down-regulated at the onset of SHPT and plays an
important role in its progression. Our research found that although CASR was down-regulated in both
SHPT and PHPT, the degree of CASR reduction was different at both the protein and gene levels. By further
analyzing this difference, we can find the cause and mechanism of CASR down-regulation, which presents
new perspectives for the treatment of SHPT. Consent for publication Not applicable. Ethics approval and consent to participate This study has been approved by China Japan Friendship Hospital ethics committee. Availability of data and materials The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Competing interests The authors declare that they have no conflict of interest. Acknowledgements Not applicable. Authors' contributions YM collected the clinical information and drafted the manuscript. LJ, SXLand JHY supported the data
collection, interpretation of the data, and writing of the manuscript. SAP and ZHL carried out
Immunohistochemical studies and evaluated the results. PB and lx carried out PCR study and evaluated
the results.LY, ZL and HLP reviewed the draft and made critical modifications. Funding This study is funded by Beijing Municipal Science & Technology Commission (grant No. Z191100006619014). Page 8/14 References [1]. Uchiyama, T., et al., Hypermethylation of the CaSR and VDR genes in the parathyroid glands in chronic
kidney disease rats with high-phosphate diet. Hum Cell, 2016. 29(4): p. 155-61. [1]. Uchiyama, T., et al., Hypermethylation of the CaSR and VDR genes in the parathyroid glands in chronic
kidney disease rats with high-phosphate diet. Hum Cell, 2016. 29(4): p. 155-61. [2]. Sengul, A.G., et al., Clinical Impact of p27(Kip1) and CaSR Expression on Primary Hyperparathyroidism. Endocr Pathol, 2018. 29(3): p. 250-258. [3]. Koh, J., et al., Regulator of G protein signaling 5 is highly expressed in parathyroid tumors and inhibits
signaling by the calcium-sensing receptor. Mol Endocrinol, 2011. 25(5): p. 867-76. [4]. Sanders, J.L., et al., Extracellular calcium-sensing receptor expression and its potential role in
regulating parathyroid hormone-related peptide secretion in human breast cancer cell lines. Endocrinology,
2000. 141(12): p. 4357-64. [5]. Brennan, S.C. and A.D. Conigrave, Regulation of cellular signal transduction pathways by the
extracellular calcium-sensing receptor. Curr Pharm Biotechnol, 2009. 10(3): p. 270-81. [6]. Joy, M.S., P.C. Karagiannis and F.W. Peyerl, Outcomes of secondary hyperparathyroidism in chronic
kidney disease and the direct costs of treatment. J Manag Care Pharm, 2007. 13(5): p. 397-411. [7]. Rodriguez, M., et al., The Use of Calcimimetics for the Treatment of Secondary Hyperparathyroidism: A
10 Year Evidence Review. Semin Dial, 2015. 28(5): p. 497-507. [8]. Shigematsu, T., et al., Long-term cinacalcet HCl treatment improved bone metabolism in Japanese
hemodialysis patients with secondary hyperparathyroidism. Am J Nephrol, 2009. 29(3): p. 230-6. [9]. Torres, P.A. and M. De Broe, Calcium-sensing receptor, calcimimetics, and cardiovascular calcifications
in chronic kidney disease. Kidney Int, 2012. 82(1): p. 19-25. [10]. Young, E.W., et al., Predictors and consequences of altered mineral metabolism: the Dialysis
Outcomes and Practice Patterns Study. Kidney Int, 2005. 67(3): p. 1179-87. Page 9/14 Page 9/14 [11]. Tentori, F., et al., Mortality risk for dialysis patients with different levels of serum calcium, phosphorus,
and PTH: the Dialysis Outcomes and Practice Patterns Study (DOPPS). Am J Kidney Dis, 2008. 52(3): p. 519-30. [12]. Fukagawa, M., et al., Abnormal mineral metabolism and mortality in hemodialysis patients with
secondary hyperparathyroidism: evidence from marginal structural models used to adjust for time-
dependent confounding. Am J Kidney Dis, 2014. 63(6): p. 979-87. [13]. Cozzolino, M., et al., Treatment of secondary hyperparathyroidism: the clinical utility of etelcalcetide. Ther Clin Risk Manag, 2017. 13: p. 679-689. [14]. References Fukagawa, M., et al., Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int, 1991. 39(5): p. 874-81. [30]. Fukagawa, M., et al., Regulation of parathyroid hormone synthesis in chronic renal failure in rats. Kidney Int, 1991. 39(5): p. 874-81. Figures References Nemeth, E.F., The search for calcium receptor antagonists (calcilytics). J Mol Endocrinol, 2002. 29(1):
p. 15-21. [15]. Nemeth, E.F., Misconceptions about calcimimetics. Ann N Y Acad Sci, 2006. 1068: p. 471-6. [16]. KDIGO clinical practice guideline for the diagnosis, evaluation, prevention, and treatment of Chronic
Kidney Disease-Mineral and Bone Disorder (CKD-MBD). Kidney Int Suppl, 2009(113): p. S1-130. [17]. Galassi, A., et al., Phosphate balance in ESRD: diet, dialysis and binders against the low evident
masked pool. J Nephrol, 2015. 28(4): p. 415-29. [18]. Mizobuchi, M., et al., Calcium-sensing receptor expression is regulated by glial cells missing-2 in
human parathyroid cells. J Bone Miner Res, 2009. 24(7): p. 1173-9. [19]. Brown, A.J., et al., Decreased calcium-sensing receptor expression in hyperplastic parathyroid glands
of uremic rats: role of dietary phosphate. Kidney Int, 1999. 55(4): p. 1284-92. [20]. Ritter, C.S., et al., Reversal of secondary hyperparathyroidism by phosphate restriction restores
parathyroid calcium-sensing receptor expression and function. J Bone Miner Res, 2002. 17(12): p. 2206-
13. [21]. Yano, S., et al., Association of decreased calcium-sensing receptor expression with proliferation of
parathyroid cells in secondary hyperparathyroidism. Kidney Int, 2000. 58(5): p. 1980-6. [22]. Ritter, C.S., et al., Parathyroid hyperplasia in uremic rats precedes down-regulation of the calcium
receptor. Kidney Int, 2001. 60(5): p. 1737-44. [23]. Canalejo, A., et al., Development of parathyroid gland hyperplasia without uremia: role of dietary
calcium and phosphate. Nephrol Dial Transplant, 2010. 25(4): p. 1087-97. [24]. Corbetta, S., et al., Calcium-sensing receptor expression and signalling in human parathyroid
adenomas and primary hyperplasia. Clin Endocrinol (Oxf), 2000. 52(3): p. 339-48. Page 10/14 Page 10/14 [25]. Dusso, A.S., et al., p21(WAF1) and transforming growth factor-alpha mediate dietary phosphate
regulation of parathyroid cell growth. Kidney Int, 2001. 59(3): p. 855-65. [26]. Sakaguchi, K., Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated
parathyroid cell growth. J Biol Chem, 1992. 267(34): p. 24554-62. [27]. Kanesaka, Y., et al., Endothelin receptor antagonist prevents parathyroid cell proliferation of low
calcium diet-induced hyperparathyroidism in rats. Endocrinology, 2001. 142(1): p. 407-13. [28]. Imanishi, Y., et al., Primary hyperparathyroidism caused by parathyroid-targeted overexpression of
cyclin D1 in transgenic mice. J Clin Invest, 2001. 107(9): p. 1093-102. [29]. Matsushita, H., et al., Proliferation of parathyroid cells negatively correlates with expression of
parathyroid hormone-related protein in secondary parathyroid hyperplasia. Kidney Int, 1999. 55(1): p. 130-
8. [30]. [30]. Fukagawa, M., et al., Regulation of parathyroid hormone synthesis in chronic renal failure in rats.
Kidney Int, 1991. 39(5): p. 874-81. Figure 1 CASR protein expression in different parathyroid tissues. Analysis of variance. P＜0.05. CASR protein expression in different parathyroid tissues. Analysis of variance. P＜0.05. CASR protein expression in different parathyroid tissues. Analysis of variance. P＜0.05. Page 11/14
CASR protein expression in different parathyroid tissues. Analysis of variance. P＜0.05. Page 11/14 Figure 2
CASR expression in secondary hyperparathyroidism tissue (++, × 20)
Figure 3
CASR expression in tissues of primary hyperparathyroidism (+, × 20) Figure 2 Figure 2 Figure 2 g
CASR expression in secondary hyperparathyroidism tissue (++, × 20)
Figure 3
CASR expression in tissues of primary hyperparathyroidism (+, × 20) CASR expression in secondary hyperparathyroidism tissue (++, × 20) Figure 3 CASR expression in tissues of primary hyperparathyroidism (+, × 20) Page 12/14 Page 12/14 Page 12/14 Figure 4
CASR expression in normal parathyroid glands (+++, × 20)
Figure 5
CASR expression in thyriod glands (0, × 20) Figure 4 CASR expression in normal parathyroid glands (+++, × 20)
Figure 5
CASR expression in thyriod glands (0, × 20) CASR expression in normal parathyroid glands (+++, × 20) CASR expression in normal parathyroid glands (+++, × 20) Figure 5
CASR expression in thyriod glands (0, × 20) Figure 5 CASR expression in thyriod glands (0, × 20) Page 13/14 Page 13/14 Figure 6
T test for SHPT and PHPT CASR gene expression (real time PCR 2-△ct), P＜0.05. Figure 6 T test for SHPT and PHPT CASR gene expression (real time PCR 2-△ct), P＜0.05. T test for SHPT and PHPT CASR gene expression (real time PCR 2-△ct), P＜0.05. T test for SHPT and PHPT CASR gene expression (real time PCR 2-△ct), P＜0.05. Page 14/14
|
https://openalex.org/W2908995163
|
https://europepmc.org/articles/pmc6359416?pdf=render
|
English
| null |
Extracellular Vesicle-Mediated Cell–Cell Communication in the Nervous System: Focus on Neurological Diseases
|
International journal of molecular sciences
| 2,019
|
cc-by
| 16,706
|
Received: 10 December 2018; Accepted: 17 January 2019; Published: 20 January 2019 Abstract: Extracellular vesicles (EVs), including exosomes, are membranous particles released by
cells into the extracellular space. They are involved in cell differentiation, tissue homeostasis, and
organ remodelling in virtually all tissues, including the central nervous system (CNS). They are
secreted by a range of cell types and via blood reaching other cells whose functioning they can modify
because they transport and deliver active molecules, such as proteins of various types and functions,
lipids, DNA, and miRNAs. Since they are relatively easy to isolate, exosomes can be characterized,
and their composition elucidated and manipulated by bioengineering techniques. Consequently,
exosomes appear as promising theranostics elements, applicable to accurately diagnosing pathological
conditions, and assessing prognosis and response to treatment in a variety of disorders. Likewise,
the characteristics and manageability of exosomes make them potential candidates for delivering
selected molecules, e.g., therapeutic drugs, to specific target tissues. All these possible applications are
pertinent to research in neurophysiology, as well as to the study of neurological disorders, including
CNS tumors, and autoimmune and neurodegenerative diseases. In this brief review, we discuss what
is known about the role and potential future applications of exosomes in the nervous system and its
diseases, focusing on cell–cell communication in physiology and pathology. Keywords: exosomes; extracellular vesicles; nervous system; central nervous system; cell–cell
interaction; biomarkers; theranostics tools; neurological diseases Extracellular Vesicle-Mediated Cell–Cell
Communication in the Nervous System: Focus on
Neurological Diseases Celeste Caruso Bavisotto 1,2,3
, Federica Scalia 1,2, Antonella Marino Gammazza 1,2,
Daniela Carlisi 4, Fabio Bucchieri 1, Everly Conway de Macario 5, Alberto J. L. Macario 2,5
,
Francesco Cappello 1,2,*
and Claudia Campanella 1 1
Department of Biomedicine, Neuroscience and Advanced Diagnostics (BIND), Section of Human Anatomy,
University of Palermo, 90127 Palermo, Italy; celestebavisotto@gmail.com (C.C.B.);
scalia.fede@gmail.com (F.S.); antonella.marino@hotmail.it (A.M.G.); fabiobuk@gmail.com (F.B.);
claudiettacam@hotmail.com (C.C.) 1
Department of Biomedicine, Neuroscience and Advanced Diagnostics (BIND), Section of Human Anatomy,
University of Palermo, 90127 Palermo, Italy; celestebavisotto@gmail.com (C.C.B.);
scalia.fede@gmail.com (F.S.); antonella.marino@hotmail.it (A.M.G.); fabiobuk@gmail.com (F.B.);
claudiettacam@hotmail.com (C.C.) 2
Euro-Mediterranean Institute of Science and Technology (IEMEST), 90136 Palermo, Italy;
ajlmacario@som.umaryland.edu 3
Institute of Biophysics, National Research Council, 90143 Palermo, Italy
4 4
Department of Biomedicine, Neuroscience and Advanced Diagnostics (BIND), Section of Biochemi
University of Palermo, 90127 Palermo, Italy; daniela.carlisi@unipa.it 5
Department of Microbiology and Immunology, School of Medicine, University of Maryland at
Baltimore-Institute of Marine and Environmental Technology (IMET), Baltimore, MD 21202, USA;
econwaydemacario@som.umaryland.edu *
Correspondence: francapp@hotmail.com; Tel.: +3909123867521 Received: 10 December 2018; Accepted: 17 January 2019; Published: 20 January 2019 International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences International Journal of
Molecular Sciences 1. Exosomes, Microvesicles for Cell–Cell Communication and Tissue Homeostasis Eukaryotic cells in multicellular organisms need to communicate with each other in order to
maintain tissue homeostasis and to respond to pathogens in the extracellular milieu. Generally, cells
exchange information through direct cell–cell contact or by secretion of soluble factors [1]. Mechanisms Int. J. Mol. Sci. 2019, 20, 434; doi:10.3390/ijms20020434 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms 2 of 23 Int. J. Mol. Sci. 2019, 20, 434 of intercellular interaction are known that involve the production and release of extracellular vesicles
(EVs). Cells interact and influence the extracellular environment and other cells in various ways,
for instance by releasing different types of EVs, which serve various functions depending on their
origin and molecular composition. EVs include a variety of nanoscale membranous vesicles that are
released by many cell types into the extracellular environment and can reach virtually all parts of the
body [2]. EVs carry molecules such as nucleic acids, proteins, and lipids to specific target cells and
can be classified according to their size, biogenesis, functions, and composition [3,4]. There are three
main types of EVs: (1) microvesicles (100–1000 nm in diameter); (2) apoptotic blebs (1000–5000 nm in
diameter); and exosomes (diameter 20–150 nm). The former two represent heterogeneous populations
of vesicles generated by outward budding of the plasma membrane. Exosomes instead are generated
by invagination of endosomal membranes and subsequent production of multivesicular bodies
(MVBs) [5,6]. Frequently, in the literature, the terms exosomes and EVs are used imprecisely, most
likely because a standardized, uniformed method for their isolation–characterization is not used
universally and, therefore, the results vary among laboratories. Nevertheless, because of the increasing
interest in EVs and because exosomes are currently the best characterized among them, in this review
we will focus on the latter. It was initially thought that exosomes could be a mechanism for shedding the cytoplasm in
maturing sheep reticulocytes [7]. Later, it was demonstrated that exosomes are active players in
intercellular communication [8–11], originate in endosomes and are secreted by all cell types, including
neurons, under physiological and pathological conditions [12]. Exosomes are present in body fluids
such as blood; urine; breast milk; saliva; and cerebrospinal, bronchoalveolar lavage, ascitic, and
amniotic fluids [11,13–21]. Exosomes are released into the extracellular space after the merging of late endosomes with the
cell membrane. Previously, early endosomes become part of multivesicular bodies (MVBs), which
undergo a maturation process characterized by a gradual change in protein composition of the vesicles
(intraluminal vesicles, ILVs). 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d The nervous system, composed by the central nervous system (CNS), and the peripheral nervous
system (PNS), is implicated in the communication with both the external and internal environment of
the organism by responding to chemical and physical stimuli [55]. 2. Nervous System Cells and Tissues: An Overview
The nervous system, composed by the central nervous system (CNS), and the peripheral
nervous system (PNS), is implicated in the communication with both the external and internal
i
t f th
i
b
di
t
h
i
l
d
h
i
l ti
li [55] The main cell types found in the nervous tissue are neurons, or nerve cells, that have the ability to
rapidly receive and transmit impulses to and from different parts of the body, and neuroglia, or glial
cells, which assist in the propagation of the nerve impulses and provide nutrients to the neurons
(Figure 1). Both neurons and neuroglia cells develop from the dorsal ectoderm of the early embryo but
different types of them can be distinguished, which are characteristic of the CNS or PNS (Figure 1). Overall, these cells are responsible for most of the functional features of nervous tissue [56]. Since
exosomes can be specific for the cell type that produce them, we briefly recapitulate the main features
of the cells that constitute the nervous tissue and that, when their homeostasis is affected, can be
implicated in the pathogenesis of nervous system diseases. environment of the organism by responding to chemical and physical stimuli [55]. The main cell types found in the nervous tissue are neurons, or nerve cells, that have the ability to
rapidly receive and transmit impulses to and from different parts of the body, and neuroglia, or glial
cells, which assist in the propagation of the nerve impulses and provide nutrients to the neurons (Figure
1). Both neurons and neuroglia cells develop from the dorsal ectoderm of the early embryo but different
types of them can be distinguished, which are characteristic of the CNS or PNS (Figure 1). Overall, these
cells are responsible for most of the functional features of nervous tissue [56]. Since exosomes can be
specific for the cell type that produce them, we briefly recapitulate the main features of the cells that
constitute the nervous tissue and that, when their homeostasis is affected, can be implicated in the
pathogenesis of nervous system diseases. Figure 1. 1. Exosomes, Microvesicles for Cell–Cell Communication and Tissue Homeostasis During this maturation process, the vesicles that have accumulated
in the MVBs can follow three different pathways: (1) merge with the lysosomes, which leads to the
degradation of their protein cargo (e.g., in the case of signalling receptors); (2) constitute a temporary
storage compartment; and (3) blend with the plasma membrane, releasing exosomes. MVBs merge
with the plasma membrane, resulting in exocytosis of the vesicles contained in them so that the
vesicles’ membrane maintains the same topological orientation as the plasma–cell membrane [1,22,23]. The endosomal sorting complexes required for the transport machinery (constituted of the proteins
ESCRT-0, -I, -II, -III) is involved in exosome biogenesis and loading [24]. ESCRT-1 assists in the sorting
of the ubiquitinated cargo proteins at the endosome membrane and the ESCRT-associated protein
ALIX (apoptosis-linked gene 2-interacting protein X) can regulate this function [24,25]. The content of exosomes reflects that of the cell of origin and, consequently, there is interest in
characterizing it to obtain information on the cell of origin and the functions of exosomes, and to assess
the potential of exosomes as drug delivery tools. The composition of exosomes depends on parental
cell conditions, and includes lipids; proteins; and nucleic acids, such as DNA, non-coding RNA, rRNA
(ribosomal RNA) and miRNAs (microRNAs) [26]. The lipid composition of exosomes is characteristic and includes cholesterol, phosphatidylcholine,
sphingolipid ceramide, and sphingomyelin that probably stabilize the exosomal bilayer membrane
and maintain its integrity in the extracellular milieu [27]. The sphingolipid ceramide plays a key role
in the budding of exosomes [28]. Various classes of proteins are found in exosomes, such as proteins involved in the vesicles’
trafficking, cell surface receptors, and proteins involved in endocytic pathways (GTPases; annexins;
flotillin; endosomal sorting complex required for transport, ESCRT, such as Alix; tumor susceptibility
gene 101, TSG101; integrin; and a number of tetraspanins such as CD9, CD53, CD63, CD81, and CD82,
depending on the cell of origin). Also, in exosomes are proteins with specific post-translational
modifications (PTMs) [29,30], and proteins that are important in long-distance communication, Int. J. Mol. Sci. 2019, 20, 434 3 of 23 such as cytokines [31], hormones [32], growth and transcription factors [33], and heat-shock proteins
(HSPs) [10,30,34,35]. The presence of mRNA [36] and miRNA [37–41] in exosomes indicates activity in the regulation
of gene expression in both recipient and donor cells, suggesting horizontal transfer of genetic
information [42]. Int. J. Mol. Sci. 1. Exosomes, Microvesicles for Cell–Cell Communication and Tissue Homeostasis 2018, 19, x FOR PEER REVIEW
3 of 23
The presence of mRNA [36] and miRNA [37–41] in exosomes indicates activity in the regulation
f
i
i
b th
i i
t
d d
ll
ti
h
i
t l t
f
f
ti Depending on the parental cells and their contents, exosomes may have many different functions. They are involved in cell-to-cell information transfer [43], immune response [44], inflammation [45],
coagulation [46], stem cell activation [47], and programmed cell death [48]. Exosomes can participate
in cellular responses against stress [49]. It has been shown that exposing B-cell lines to heat stress
results in a marked increase of HSPs in exosomes and in an increase in the quantity of exosomes
produced [10,11,30,49–51]. of gene expression in both recipient and donor cells, suggesting horizontal transfer of genetic
information [42]. Depending on the parental cells and their contents, exosomes may have many different
functions. They are involved in cell-to-cell information transfer [43], immune response [44],
inflammation [45], coagulation [46], stem cell activation [47], and programmed cell death [48]. Exosomes can participate in cellular responses against stress [49]. It has been shown that exposing
B-cell lines to heat stress results in a marked increase of HSPs in exosomes and in an increase in the Since exosomes can mediate transfer of molecules, it is very likely that they play a key role in
intercellular interactions and in the maintenance of tissue homeostasis [52,53]. For example, exosomes
play physiological roles in neuronal development, transmission of electrical impulse, and regeneration,
and, consequently, can play a pathogenic role in neurological disease [54]. quantity of exosomes produced [10,11,30,49–51]. Since exosomes can mediate transfer of molecules, it is very likely that they play a key role in
intercellular interactions and in the maintenance of tissue homeostasis [52,53]. For example,
exosomes play physiological roles in neuronal development, transmission of electrical impulse, and
ti
d
tl
l
th
i
l i
l
i
l di
[54] 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d Cells of the central and peripheral nervous systems. These cells have functions and locales
of residence distinctive of each of them but they all can secrete exosomes and receive exosomes from
the others, as depicted in Figure 2. BBB: blood–brain barrier. Figure 1. Cells of the central and peripheral nervous systems. These cells have functions and locales of
residence distinctive of each of them but they all can secrete exosomes and receive exosomes from the
others, as depicted in Figure 2. BBB: blood–brain barrier. Figure 1. Cells of the central and peripheral nervous systems. These cells have functions and locales
of residence distinctive of each of them but they all can secrete exosomes and receive exosomes from
the others, as depicted in Figure 2. BBB: blood–brain barrier. Figure 1. Cells of the central and peripheral nervous systems. These cells have functions and locales of
residence distinctive of each of them but they all can secrete exosomes and receive exosomes from the
others, as depicted in Figure 2. BBB: blood–brain barrier. Neurons are highly specialized cells that receive, process, and transmit information through
chemically-mediated electrical signals [56] (Figure 1). Neurons are highly specialized cells that receive, process, and transmit information through
chemically-mediated electrical signals [56] (Figure 1). Despite the fact that neurons can be specialized and differ in a variety of features, they all share
several characteristics. The key function of neurons is to communicate between them and with other
cell types When the nerve impulses travel along the axon in the form of an action potential the
Despite the fact that neurons can be specialized and differ in a variety of features, they all share
several characteristics. The key function of neurons is to communicate between them and with other Int. J. Mol. Sci. 2019, 20, 434 4 of 23 cell types. When the nerve impulses travel along the axon in the form of an action potential, the vesicles
at the axon terminal, which contain neurotransmitters or neuromodulators, release their content by
exocytosis. These signals between neurons are passed via specialized connections called synapses [57],
in which either the axon terminal or an en passant bouton (a type of terminal located along the
length of the axon) of one cell contacts another neuron’s dendrite, soma or less commonly, axon [57]. The chemical transmitters travel across the synaptic cleft to reach receptors on the postsynaptic cell. 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d According to the neuron doctrine founded by Ramón y Cajal and then supported by subsequent
investigators, this seems an appropriate description for most synapses in vertebrates and invertebrates,
but several studies and new technology applications, such as electron microscopy, have pointed out
the existence within CNS of new synapses, called mix synapses and synapses à distance, where the
axon and the dendrites appear to be exchanging their roles. It could lead to reform of the neuron
primary doctrine and render it more pliable [58]. In the last decade, in vitro studies demonstrated
that, depending on synaptic activity, neurons release exosomes that can be retaken by other neurons
suggesting a novel way for inter-neuronal communication [59]. The term glia derives from the ancient Greek word “glía” meaning “glue” in English, and may
suggest a passive type of cell; however, glial cells are active, providing support and nutrition to the
neurons, form myelin, and, by insulating axons, speed up electrical communication [60]. A major
distinction between glia and neurons is that glia do not participate directly in synaptic interactions
and electrical signaling. However, emerging evidence suggests that glia, particularly astrocytes, are
involved in the formation of synapses and in modulating synaptic function through bidirectional
communication with neurons, both during development and in adulthood [60]. For many years it has been argued that the number of glial cells in the brain was significantly
higher than neurons, but recent work has revealed that neurons and glia are almost equal in number
in the human cortex [61–63]. However, it is possible that the proportions of neurons and glial cells
vary in different brain areas [64]. Neuroglia of the CNS can be divided into macroglia and microglia (Figure 1). The macroglia
includes oligodendrocytes, astrocytes, and ependymoglial cells that originate from the ectoderm, while
the microglial cells derive from the yolk sac and they are found in the CNS during early embryonic
development [65]. Spinal cord and brain contain different subclasses of oligodendrocytes (OLGs) which derive from
multiple sources [66] (Figure 1). OLGs provide a lipid-based insulation and, thus, increase the speed at
which the action potential can travel in the axon. Within the oligodendrocyte linage, there exist the NG2-glia/oligodendrocyte cells. They are
characterized by the presence on their surface of chondroitin sulfate proteoglycan and are considered an
independent glial population, but their function in the adult brain is not yet fully established. 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d Despite their role
in CSF-barrier homeostasis regulation, there is not yet evidence for the exosomes’ secretion by the
ependymal cells. However, they can be isolated by CSF [13,79], thus suggesting that they pass this
barrier by still unknown mechanisms. Differently from the previously described cells of the nervous system, the microglial cells originate
from mesodermal hematopoietic cells that in mammals come from the yolk sac [80,81]. They serve
as innate immunity elements of the CNS independently of blood cells. With self-renewal ability they
act as unique tissue-resident macrophages involved in immune reactions and inflammatory diseases
(Figure 1) [82]. To define the microglial cells just as macrophages would be an oversimplification because,
in addition to their role in defending against bacterial and viral infections, they play a crucial role in the
maturation of neural circuits by their “synaptic pruning” function [83]; they also produce brain-derived
neurotrophic factor (BDNF) to survey mature neurons, mediate synapses, and remove myelin debris
by phagocytosis [83,84]. Microglial cells have a great morphological plasticity and with their highly motile processes
without moving their bodies constantly explore their environment. By screening the brain parenchyma
these cells rapidly search for pathogens, signs of injury, or homeostatic disturbances [85,86]. Finally,
the regulation of the neuronal plasticity by microglia may occur also by EVs releasing that have been
reported implicated in the increase of neuronal synaptic activity in vitro and in vivo [75]. The functions of the nervous system and immune system are often considered independent
from one another, however, this is a simplistic distinction, because in the regulation of the organism
homeostasis, they are in constant communication [87,88]. This relationship was demonstrated long ago
by the description of the association between peripheral neurons and mast cells, that are implicated
in neuroinflammation [89]. The communication between neurons and mast-cells occurs through to
several paracrine signals and also synapses, but the full understanding of this relation may open
important scenarios pertaining to the onset of neuroinflammatory diseases [90]. In the PNS there are two types of neuroglia: Schwann cells (SCs) that myelinate axons; and satellite
glial cells, that regulate nutrient and neurotransmitter levels in ganglia (Figure 1). SCs are recognized as the PNS counterparts of the oligodendrocytes in CNS, as they are involved
in the neuromuscular synapse formation and in wrapping myelin around neuronal axons to form
the myelin sheath. 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d NG2-glia
maintains the physiological and homeostatic conditions of the nervous tissue generating mature
myelinating oligodendrocytes; furthermore, it forms synapses with neurons of the hippocampus and
probably in other parts of the brain, too [67]. Notably, NG2-glial cells have the ability to receive signals
without creating or propagating action potentials [67]. The astrocytes are supportive glial cells in neural tissue with a star-like appearance because of
their elaborate cytoplasmic processes [68–70]. Astrocytes play a role in a variety of complex and essential functions in the healthy CNS,
such as the maintenance of water and ion homeostasis and blood–brain barrier (BBB) integrity, as
well as participation in tripartite synapses, all of which make astrocytes active actors in synaptic
context [60,71]. Furthermore, astrocytes can inhibit or enhance overall levels of neuronal activity by
releasing neurotransmitters. For many years astrocytes were classified into just two types, but now,
according to their structure and anatomic location, up to four major classes of GFAP+ astrocytes are
known to occur in the human brain: interlaminar astrocytes are located in layers I and II of the cortex;
protoplasmic astrocytes reside in layers III and IV; astrocytes in varicose projections in layers V and VI;
and fibrous astrocytes in white matter [72]. Int. J. Mol. Sci. 2019, 20, 434 5 of 23 In the functional regulation of the microenvironment, astrocytes and oligodendrocytes release
EVs, in order to facilitate the cell–cell communication and the activity of target cells [73–75]. In the functional regulation of the microenvironment, astrocytes and oligodendrocytes release
EVs, in order to facilitate the cell–cell communication and the activity of target cells [73–75]. The CNS macroglia cells are the ependymoglial cells derived from the neuroepithelium. They
populate the interface between the brain parenchyma and the cavity of the ventricles in the CNS, and
the central canal of the spinal cord. Macroglial cells appear with various shapes from cuboidal to
columnar with cilia and microvilli on the apical surfaces to enhance absorbance and circulation of
cerebral spinal fluid (CSF). p
Ependymoglial cells are of three types: ependymocytes, which make contact with the basal lamina
labyrinths (remnants of embryonic blood vessels) and with the ventricles where they contribute to the
CSF movement [76,77]; choroid plexus epithelial cells, which secrete CSF; tanycytes, highly specialized
ependymal cells that form a blood–CSF barrier and blood–CSF homeostasis [78]. The Possible Two-Way Journey of Exosomes Released by CNS Cells
2.1. The Possible Two-Way Journey of Exosomes Released by CNS Ce The roles of exosomes in the CNS may be as follows:
on the one hand, they can be
active components necessary for the development and protection of the CNS under physiological
conditions [12,102,103], whereas on the other hand, they may participate in pathogenesis by favoring
some neurodegenerative and neuroinflammatory phenomena, as suggested, for example, by the fact
that microglial exosomes are found in high concentrations in patients with Alzheimer’s disease (AD)
and exosomes produced by oligodendroglioma cells induce neuronal death [104]. The roles of exosomes in the CNS may be as follows: on the one hand, they can be active
components necessary for the development and protection of the CNS under physiological
conditions [12,102,103], whereas on the other hand, they may participate in pathogenesis by favoring
some neurodegenerative and neuroinflammatory phenomena, as suggested, for example, by the fact
that microglial exosomes are found in high concentrations in patients with Alzheimer’s disease (AD)
and exosomes produced by oligodendroglioma cells induce neuronal death [104]. Si
i
l
d b
h i
h
l h
d
h
i
f CNS i i
i i Since exosomes are involved both in healthy and pathogenic state of CNS, it is not surprising that
these vesicles are released by most of the CNS cells, including neurons, microglia, oligodendrocytes,
astrocytes, and neural embryonic stem cells, Figure 2 [105,106]. Questions of great interest are whether
these exosomes have the ability to cross the BBB and hematoliquor barrier; and whether they come
from the CNS to the periphery, or they come from the periphery to the SNC in normal physiological
and in pathological conditions. Since exosomes are involved both in healthy and pathogenic state of CNS, it is not surprising
that these vesicles are released by most of the CNS cells, including neurons, microglia,
oligodendrocytes, astrocytes, and neural embryonic stem cells, Figure 2 [105,106]. Questions of great
interest are whether these exosomes have the ability to cross the BBB and hematoliquor barrier; and
whether they come from the CNS to the periphery, or they come from the periphery to the SNC in
normal physiological and in pathological conditions. Figure 2. Schematic representation of the nervous tissue and exosome traffic. Some of the cells
presented in Figure 1 are here seen in the central nervous tissue along with a blood vessel. 2. Nervous System Cells and Tissues: An Overview
regeneration, and, consequently, can play a pathogenic
ll
d This SC activity promotes the efficient and energy low-cost propagation of axon
potentials via saltatory conduction by maintaining internodal—each myelin segment is flanked by
unmyelinated nodes of Ranvier–myelin sheath thickness and length relative to the diameter of the
corresponding axon [91]. SCs can perform many unique functions, including duplicating the roles of
the astrocytes and microglia as seen in the central nervous system (CNS) [92]. The terminal Schwann
cells (tSCs, also called non-myelinating SCs or perisynaptic SCs) have a role in “synapse elimination”
during development and, throughout adult life [93], effect the regeneration of injured peripheral
motor axons [94]. Schwann cells provide trophic support and transfer materials to damaged axons
via exosomes [95]. Furthermore, they maintain developing synapses, and participate in synaptic
pruning [96,97]. Int. J. Mol. Sci. 2019, 20, 434 6 of 23 Satellite glial cells (SGCs) have the same origin as Schwann cells. Sensory ganglia of the dorsal
roots of the spinal cord are composed of afferent neurons without a myelin sheath but lined by SGCs
and connective tissue cells. Satellite glial cells (SGCs) have the same origin as Schwann cells. Sensory ganglia of the dorsal
roots of the spinal cord are composed of afferent neurons without a myelin sheath but lined by SGCs
and connective tissue cells. SGCs share many features with astrocytes, like the expression of glutamine synthetase and
various neurotransmitter transporters. They cover axon terminals that make synaptic contacts on,
or near, the neuronal somata, wrap around dendrites that emerge from neuronal somata to control
the microenvironment and, similarly to astrocytes, influence synaptic transmission [98,99]. SGCs
can be considered a substitute of the lacking BBB in sensory ganglia and have been shown to have
phagocytic activity [98]. The role in microenvironment regulation and inflammation modulation by
SGCs exosomes remains unknown [100,101]. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW
6 of 23
microenvironment and, similarly to astrocytes, influence synaptic transmission [98,99]. SGCs can be
considered a substitute of the lacking BBB in sensory ganglia and have been shown to have
phagocytic activity [98]. The role in microenvironment regulation and inflammation modulation by
SGCs exosomes remains unknown [100,101]. The Possible Two-Way Journey of Exosomes Released by CNS Cells
2.1. The Possible Two-Way Journey of Exosomes Released by CNS Ce Also present
are epithelial cells lining the inside of the blood vessel, the blood–brain barrier (BBB) separating the
lumen of the vessel from the nervous tissue, and exosomes secreted by the four types of nervous cells
shown. Exosomes follow different routes, as indicated by double parallel arrows, from one cell to
another or through the BBB they gain the general circulation and reach distant targets. Conversely,
exosomes can traverse the BBB from inside the vessel into the nervous tissue and reach any of the
nervous cell types in it. Figure 2. Schematic representation of the nervous tissue and exosome traffic. Some of the cells
presented in Figure 1 are here seen in the central nervous tissue along with a blood vessel. Also present
are epithelial cells lining the inside of the blood vessel, the blood–brain barrier (BBB) separating the
lumen of the vessel from the nervous tissue, and exosomes secreted by the four types of nervous cells
shown. Exosomes follow different routes, as indicated by double parallel arrows, from one cell to
another or through the BBB they gain the general circulation and reach distant targets. Conversely,
exosomes can traverse the BBB from inside the vessel into the nervous tissue and reach any of the
nervous cell types in it. Figure 2. Schematic representation of the nervous tissue and exosome traffic. Some of the cells
presented in Figure 1 are here seen in the central nervous tissue along with a blood vessel. Also present
are epithelial cells lining the inside of the blood vessel, the blood–brain barrier (BBB) separating the
lumen of the vessel from the nervous tissue, and exosomes secreted by the four types of nervous cells
shown. Exosomes follow different routes, as indicated by double parallel arrows, from one cell to
another or through the BBB they gain the general circulation and reach distant targets. Conversely,
exosomes can traverse the BBB from inside the vessel into the nervous tissue and reach any of the
nervous cell types in it. Figure 2. Schematic representation of the nervous tissue and exosome traffic. Some of the cells
presented in Figure 1 are here seen in the central nervous tissue along with a blood vessel. Also present
are epithelial cells lining the inside of the blood vessel, the blood–brain barrier (BBB) separating the
lumen of the vessel from the nervous tissue, and exosomes secreted by the four types of nervous cells
shown. 3. Exosome-Mediated Cross-Talk between Cells in Neurogenesis and Neurohomeostasis The development and maintenance of neuronal circuits in the CNS requires a complex series
of events involving coordinated short- and long-distance communication between numerous cell
types. Neurons interact continuously with each other and with glial cells through electrical signals
and through chemical mediators. In chemical synapses, more common than electrical synapses in the
human CNS, the transmission of signals is carried out by chemical mediators, named neurotransmitters. The neurotransmitters are secreted by the pre-synaptic cells inside vesicles that reach the post-synaptic
cells, where multiple downstream events, both electric and molecular, are triggered by binding of
the neurotransmitter to specific receptors [115]. In view of these phenomena, it is not surprising that
neuronal cells may also release different types of EVs, such as exosomes that could have an impact
on synaptic activity, in neurogenesis, and in the overall regulation of neurological activities. In this
section, we will briefly describe known physiological functions of exosomes in CNS (Figure 2). CNS neurons secrete exosomes to control the complex and coordinated communication among
them and, with astrocytes and microglia, thus exosomes mediate a generalized cross-talk, in order to
regulate neuronal regeneration and synaptic functions in development and adult life [116,117]. g
g
y
p
p
To the best of our knowledge, the first report regarding exosomes produced by neural cells is
relatively recent. Glial cell lines overexpressing a prion protein (PrPsc) released exosomes as a way
to spread the PrPsc and, these exosomes bearing PrPsc were infectious, contributing to the spread
of prions throughout different areas of CNS and the whole organism [118]. Successively, it has been
shown that neurons may exploit the exosome pathway to maintain homeostasis and regulate cell–cell
interactions, for instance, as a way to discard unwanted proteins or degraded products. This hypothesis
has been proposed to explain how primary cortical neurones in culture release exosomes in a controlled
manner, while their composition is regulated by cell depolarisation [12]. The exosomes released are
captured by neighbouring cells and the exosomal cargoes elicit distinct downstream events [12]. Other
studies on exosome secretion from neurons have been conducted with embryonic neurons in culture. It was hypothesized that exosome release is a key mechanism during neurogenesis that seems to be
necessary for protein removal, and is a consequence of the fusion of late endosomes with lysosomes,
during the neurite elongation [119–121]. The Possible Two-Way Journey of Exosomes Released by CNS Cells
2.1. The Possible Two-Way Journey of Exosomes Released by CNS Ce Exosomes follow different routes, as indicated by double parallel arrows, from one cell to
another or through the BBB they gain the general circulation and reach distant targets. Conversely,
exosomes can traverse the BBB from inside the vessel into the nervous tissue and reach any of the
nervous cell types in it. Int. J. Mol. Sci. 2019, 20, 434 7 of 23 Cells from malignant gliomas, i.e., primary tumors that arise from neuroglial stem or progenitor
cells, produce and release in circulation exosomes with potential to induce malignant transformation
of normal cells [107]. It has been reported that with a immunomagnetic exosome-RNA (iMER) analysis
platform, it is possible to enrich glioblastoma (GBM)-derived exosomes from blood of patients, and
compare the exosomes’ GBM-derived mRNA profiles against those of their cells of origin [108]. In amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), FTD-ALS, tauopathies,
and Parkinson’s (PD) and Alzheimer’s (AD) diseases, exosomes migrate via blood and CSF carrying
misfolded proteins or pro-inflammatory molecules [109–111]. Modified rabies virus glycoprotein (RVG)-targeted EVs were used to transport siRNA across
BBB [112], that can specifically inhibit target genes in the brain [112]. Modified blood-borne
macrophages were used to carry antioxidant proteins called nanozymes [113]. It was demonstrated
that the therapeutic protein crossed the BBB and it was suggested that one of the mechanisms used by
macrophages to transfer nanozymes to target recipient cells was the release of exosomes [113]. Furthermore, it was demonstrated that the uptake of EVs by neurons in vitro (neuronal rat adrenal
pheochromocytoma cell line, PC12 cells) and in vivo neurons and microglial cells of mouse, is more
efficient than that of other traditional carriers, i.e., liposomes [114]. 3. Exosome-Mediated Cross-Talk between Cells in Neurogenesis and Neurohomeostasis In addition, exosomes released from neurons may contribute
to the local elimination of receptors at synapses undergoing plastic changes and escaping from
the vesicles retrograde transport through the axon [59,122]. During neuronal remodelling, exosomes
released by neurons could have a role in synapse elimination, stimulating microglial phagocytosis [123]. Furthermore, as the cytoplasmic calcium levels increase, MVBs’ fusion to the plasma membrane occurs
and is followed the secretion of exosomes. This seems to be a mechanism used by neurons to detect the 8 of 23 Int. J. Mol. Sci. 2019, 20, 434 strength of the excitatory synapses and adjust them, a mechanism that might be necessary to regulate
the functioning of synapses and maintain homeostasis during neuronal plastic changes [124]. In order
to regulate extracellular glutamate levels and modulate synaptic activation, neurons communicate with
astrocytes by secreting exosomes, which contain several regulatory molecules that are internalized by
astrocytes, thus eliciting a neuronal-dependent modification of the expression of glutamate transporters
(e.g., GLT1) [125]. Multiple interactions between glia-derived exosomes and neurons (Figure 2), also
suggest a role of these vesicles in neural circuit development and maintenance, by promoting neurite
outgrowth from hippocampal neurons and increased survival of cortical neurons [74]. Microglia-derived exosomes can modulate neuronal activity also via enhanced sphingolipid
metabolism [75]. Inflammatory microglia-derived exosomes transfer their miRNA cargo (miR-146a-5p)
to neurons determining the loss of excitatory synapses, suggesting a role during brain inflammation,
probably silencing key synaptic genes [126]. In CNS, oligodendrocyte progenitor cells secrete exosome-like vesicles carrying myelin proteolipid
protein (PLP), 2’3’-cyclic-nucleotide-phosphodiesterase (CNP), myelin basic protein (MBP), and myelin
oligodendrocyte glycoprotein (MOG) [127]. The oligodendrocyte-derived exosomes may contribute
to balanced production of myelin proteins and lipids and, therefore, these exosomes may be part
of a mechanism of formation and control of myelin membrane biogenesis [103,127]. In adult CNS,
during cell renewal and tissue regeneration, oligodendrocytes use the exosomal pathway to induce the
microglia toward degradation of oligodendroglial membrane by macropinocytosis, without immune
system activation [128]. The concomitant transfer of antigens from oligodendrocytes to microglia could
be implicated in the pathogenesis of autoimmune conditions of the CNS. The fact that exosomes can reach the circulation and the CSF makes these vesicles likely means of
long-distance communication and transport for bioactive molecules to be delivered to selected targets. 3. Exosome-Mediated Cross-Talk between Cells in Neurogenesis and Neurohomeostasis Because of their capability to cross the BBB [129,130], and because their content reflects faithfully that
of the cell of origin, circulating exosomes can reveal the status of the tissue from which they come and,
thereby, provide an accurate means for early, minimally invasive (peripheral blood drawing) diagnosis
of neurological diseases [22,41,131] (Figure 2). On the other side of the matter, peripheral organs can influence the functions of CNS through
exosomes [132,133]. The gut–brain axis is an example of an unconventional system of communication
between the intestinal mucosa and brain, different from peripheral nerves. The intestinal microbiota-derived EVs (named outer membrane vesicles, OMVs) can also enter the
systemic circulation and pass through the BBB, inducing neuroinflammation that could be implicated
in the pathogenesis of depressive disorders [134] and affect the BBB permeability [135]. The modality
by which exosomes cross the BBB still remains unclear; however, this characteristic makes exosomes
good candidates as biomarkers for diagnostics purposes, and for delivering therapeutic agents to
neural tissues. 4. Role of Exosomes in Nervous System Pathogenesis and Theranostics Progress in the medical sciences has been steady over the last few decades, encompassing the
discovery of etiological agents, elucidation of pathogenic mechanisms, and development of new
diagnostic techniques and therapeutic strategies. One of the major obstacles to improving patient
management has been the heterogeneity of any given disease, which varies from patient to patient. Thus, personalized medicine has emerged to develop means of diagnosis and treatment for the
management of each patient in accordance with its specific characteristics. Theranostics is one
advance in this direction that also aims at combining diagnostic and therapeutic capabilities in a single
agent [136]. Examples of theranostics agents are nanoparticles such as liposomes, polymers, micelles,
solid (lipid) nanoparticles, antibodies, and now also exosomes, that can be modified and improved
with drugs and imaging agents [137]. Nanoparticles have the ability to interact in a site-specific manner
with biomolecules present on the cell membrane surface or inside the cell, co-delivering therapeutic
and diagnostic/monitoring agents at the same time into diseased tissue. Appropriate targeting can be 9 of 23 Int. J. Mol. Sci. 2019, 20, 434 implemented using diverse strategies; for instance, to identify a cancer biomarker aberrantly expressed
on the cell surface [138]. Theranostics has manifold advantages: (1) it can be carried out before, after,
or during treatment; (2) the specific localization of the theranostics agents on a defined target reduces,
or may even eliminate, possible side effects and can also help identify patients with susceptibility to
side effects; (3) allows tumor homing: the nanometric size of the particles and the typical irregularity
of blood vessels with dilated fenestrations, allow the extravasation and accumulation of nanoparticles
into the tumor mass, improving the enhanced-permeability-and-retention (EPR) effect; and (4) it
allows the achievement of a more effective individualized therapy for various diseases [139,140]. Theranostics is viewed as a significant step forward in non-invasive or minimally invasive treatment
modalities with potential to accelerate drug development. However, theranostics has limitations in
what pertains, for example, to the limited quantities of the therapeutic agent that can be delivered to the
site where it is needed, the possibility of inducing immune reactions against the agent, manufacturing
difficulties during nanoparticle production, and the need of elimination of toxic metabolites that
might be generated during production and/or administration. 4. Role of Exosomes in Nervous System Pathogenesis and Theranostics In this regard, the biocompatibility,
biodegradability, and toxicity of the materials used to prepare the theranostics agents and the
pharmacokinetic and pharmacodynamic parameters of the compounds used have to be carefully
evaluated before clinical use. In summary, the balance between benefits and disadvantages in each
case must be critically assessed. As described earlier, exosomes derived from different nervous system cells contain specific
molecules or cell markers, e.g., oligodendrocyte-derived exosomes contain proteins of the myelin
sheath; neuronal exosomes contain cell-adhesion proteins and receptor subunits; microglial-derived
exosomes carry peptidases and cytokines. This suggests that exosomes have the ability to regulate
and maintain functional cell homeostasis during health and under disease conditions. But on the
other hand, exosomes can favor the disease mechanism rather than stop it, when they carry and
deliver pathogenic molecules from the cell of their origin. This type of pathogenic role of exosomes
has been observed in neuronal disorders with misfolded proteins (neurodegenerative, autoimmune,
neuroinflammatory conditions), as will be discussed later. 4.1. Overview of CNS Disorders Neurologic disorders are numerous and diverse and can be caused by a variety of etiologic agents
with many of the disorders being the consequence of the convergence of more than one etiopathogenic
factor. An important group of neurological disorders are inherited, i.e., a mutated gene, or group of
mutated genes are present in the genome of an individual which transmits it to its descendants. Some of
these mutations are now well characterized [141–143]. However, the pathogenetic mechanisms of many
of these genetic diseases are still poorly understood. Other disorders are caused by sporadic random
gene mutations and are not heritable. Genetic polymorphisms; old age; gender; poor education,
endocrine, immune and metabolic conditions; oxidative stress; inflammation; stroke; hypertension;
diabetes; smoking; head trauma; depression; infection; tumors; vitamin deficiencies; and exposure to
certain chemicals are considered risk factors that may contribute to the development of neurological
diseases, including AD, PD and ALS, in individuals that are probably genetically pre-disposed [144]. Some gene mutations, random or inherited, affect development and functioning of the nervous system,
leading to neuropathies, myopathies, epilepsies, ataxias, and degenerative disorders of the brain
and spinal cord (Table 1, Figure 3) [145]. In what pertains to the etiology of nervous system tumors,
there are still doubts and obscure situations: although genes associated with pathogenesis have been
identified, other risk factors and comorbidities seem to also play determinant roles [146]. This situation
is reflected in the classification of neurological disorders, which are encompassed in various large
groups and subgroups, as summarized in Table 1 and in Figure 3. Because of the multifactorial
mode of etiology, many neurological disorders can be assigned to more than one group or subgroup. Also, neurological disorders can be classified considering the location of the characteristic anatomic
pathology, symptoms and signs, outcome, and other parameters. 10 of 23
10 of 23 Int. J. Mol. Sci. 2019, 20, 434
Int. J. Mol. Sci. 2018, 19, x FOR Figure 3. Diagrammatic representation of the various groups encompassing the neurological
diseases presented in Table 1. It can be seen that according to their main etiopathogenic feature,
neurological diseases can be classified into distinct groups. However, there are various examples in
which a disease can be classified into more than one group because the etiopathogenic features are
mixed, or incompletely understood. 4.1. Overview of CNS Disorders Disease
Main Etiopathogenic Feature b
Genetic
Autoim-Mune
Inflam-Matory
Degener-Ative
Vascular
Tumoral
PGV a
Multiple Sclerosis
x
x
x
Alzheimer’s
x
x
x
x
Parkinson’s
x
x
x
Amyotrophic
Lateral Sclerosis
x
x
x
Ependymoma
x
x
Medulloblastoma
x
x
x
Diffuse intrinsic
pontine glioma
x
x
Glioblastoma
x
x
x
Malignant
peripheral nerve
sheath tumor
x
x
x
a PGV, possible genetic variants. b The symbol “x” in table cell indicates that the etiopathogenic feature mentioned
at the top of the column is present in the corresponding disease mentioned in the left-most column. astoma; MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple scl
s disease
Table 1. Major neurological diseases and their main etiopathogenic features. astoma; MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple sc
s disease
Table 1. Major neurological diseases and their main etiopathogenic features. medulloblastoma; MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple sclerosis; PD,
Parkinson’s disease. Table 1. Major neurological diseases and their main etiopathogenic features. Disease
Main etiopathogenic featureb
Genetic
Autoim-
mune
Inflam-m
atory
Degener-
ative
Vascular
Tumoral
PGVa
Multiple Sclerosis
x
x
x
Alzheimer’s
x
x
x
x
Parkinson’s
x
x
x
Amyotrophic Lateral
Sclerosis
x
x
x
Ependymoma
x
x
Medulloblastoma
x
x
x
Diffuse intrinsic
pontine glioma
x
x
Table 1. Major neurological diseases and their main etiopathogenic features. Disease
Main Etiopathogenic Feature b
Genetic
Autoim-Mune
Inflam-Matory
Degener-Ative
Vascular
Tumoral
PGV a
Multiple Sclerosis
x
x
x
Alzheimer’s
x
x
x
x
Parkinson’s
x
x
x
Amyotrophic
Lateral Sclerosis
x
x
x
Ependymoma
x
x
Medulloblastoma
x
x
x
Diffuse intrinsic
pontine glioma
x
x
Glioblastoma
x
x
x
Malignant
peripheral nerve
sheath tumor
x
x
x
a PGV, possible genetic variants. b The symbol “x” in table cell indicates that the etiopathogenic feature mentioned
at the top of the column is present in the corresponding disease mentioned in the left-most column. ffuse intrinsic
ontine glioma
x
x
a PGV, possible genetic variants. b The symbol “x” in table cell indicates that the etiopathogenic feature mentioned
at the top of the column is present in the corresponding disease mentioned in the left-most column. Diffuse intrinsic
pontine glioma
x
x
a PGV, possible genetic variants. b The symbol “x” in table cell indicates that the etiopathogenic feature mentioned
at the top of the column is present in the corresponding disease mentioned in the left-most column. 4.1. Overview of CNS Disorders Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic
lateral sclerosis; DIPG, diffuse intrinsic pontine gliomas; EP, ependymoma; GB, glioblastoma; MB,
d ll bl
S
l
h
l
h
h
S
l
l
l
Figure 3. Diagrammatic representation of the various groups encompassing the neurological diseases
presented in Table 1. It can be seen that according to their main etiopathogenic feature, neurological
diseases can be classified into distinct groups. However, there are various examples in which a
disease can be classified into more than one group because the etiopathogenic features are mixed, or
incompletely understood. Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis;
DIPG, diffuse intrinsic pontine gliomas; EP, ependymoma; GB, glioblastoma; MB, medulloblastoma;
MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple sclerosis; PD, Parkinson’s disease. Figure 3. Diagrammatic representation of the various groups encompassing the neurological
diseases presented in Table 1. It can be seen that according to their main etiopathogenic feature,
neurological diseases can be classified into distinct groups. However, there are various examples in
which a disease can be classified into more than one group because the etiopathogenic features are
mixed, or incompletely understood. Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic
lateral sclerosis; DIPG, diffuse intrinsic pontine gliomas; EP, ependymoma; GB, glioblastoma; MB,
Figure 3. Diagrammatic representation of the various groups encompassing the neurological diseases
presented in Table 1. It can be seen that according to their main etiopathogenic feature, neurological
diseases can be classified into distinct groups. However, there are various examples in which a
disease can be classified into more than one group because the etiopathogenic features are mixed, or
incompletely understood. Abbreviations: AD, Alzheimer’s disease; ALS, amyotrophic lateral sclerosis;
DIPG, diffuse intrinsic pontine gliomas; EP, ependymoma; GB, glioblastoma; MB, medulloblastoma;
MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple sclerosis; PD, Parkinson’s disease. medulloblastoma; MPNSTs, malignant peripheral nerve sheath tumors; MS, multiple sclerosis; PD,
Parkinson’s disease. Table 1. Major neurological diseases and their main etiopathogenic features. Disease
Main etiopathogenic featureb
Genetic
Autoim-
mune
Inflam-m
atory
Degener-
ative
Vascular
Tumoral
PGVa
Multiple Sclerosis
x
x
x
Alzheimer’s
x
x
x
x
Parkinson’s
x
x
x
Amyotrophic Lateral
Sclerosis
x
x
x
Ependymoma
x
x
Medulloblastoma
x
x
x
Diffuse intrinsic
pontine glioma
x
x
Table 1. Major neurological diseases and their main etiopathogenic features. 4.2. Exosomes in Neurological Disorders Studies on exosomes have contributed to increasing our current understanding of the pathogenesis
of neurodegenerative disease. Since exosomal proteins were found accumulated in amyloid plaques in
the brain of AD patients [151], the involvement of exosomes in AD pathogenesis deserves investigation. Secretion of exosomes may remove misfolded and/or aggregated proteins and transfer them to
neighboring cells and, thereby, perpetuate the disease process. In vitro and in vivo experiments
have confirmed that exosomes from neuronal cells contain precursors of amyloidogenic proteins
and enzymes for the maturation of precursors [152–155]. The role of exosomes is not yet clear, but
a possibility is that they could promote the spreading of beta-amyloid peptides and/or assist in the
removal of neurotoxic beta-amyloid from cells [152,153]. The likelihood of exosome involvement in AD pathogenesis was also suggested by the finding of
hyperphosphorylated tau protein in exosomes from neural tissue in culture and in human CSF [152]. Tau protein aberrantly accumulates in AD and, in this regard, microglial cells may participate in
spreading tau protein through various brain regions by releasing exosomes as carriers [152,153]. Hyperphosphorylated tau protein in exosomes from transgenic mice would indicate that PTMs could
enhance the abnormal process of tau formation and the spreading by exosomes [131,156]. In fact
protein phosphorylation could be a signal required for their release by exosomes [157]. Exosomes may
propagate tauopathies and, in AD, contribute to cognitive loss. Currently, it is possible to diagnose AD when the disease is already established, e.g., when
the patient has already developed dementia. The previous stages often remain asymptomatic,
and these are the stages in which the patient could benefit most from treatment. Therefore, some
sort of early diagnostic procedure is needed and, for this purpose, exosomes could be considered
potentially useful biomarkers. Exosomes could act as Aβ scavengers binding Aβ to their surface
and, subsequently, microglial cells would internalize “charged exosomes” and process them for
degradation [158,159]. Consequently, exosomes derived from human adipose tissue-stem cells have
been proposed for therapeutic degradation of Aβ plaques [160]. AD is associated also with chronic
inflammatory responses. Microglia and astrocytes release inflammatory cytokines, and free radicals
and oxidative stress molecules are present in the affected brain areas. As previously mentioned,
Aβ is packaged into exosomes and the spreading from cell to cell and the promotion of amyloid
plaque formation can initiate an inflammatory cascade [159]. 4.1. Overview of CNS Disorders Glioblastoma
x
x
x
Malignant peripheral
nerve sheath tumor
x
x
x
aPGV, possible genetic variants. bThe symbol “x” in table cell indicates that the etiopathogenic feature
mentioned at the top of the column is present in the corresponding disease mentioned in the
left-most column. The nervous system cells, Figure 1, can be the target of adaptive cellular and humoral immune
responses, causing autoimmunity-induced damage [147]. Autoimmunity disorders involving the
nervous system have been extensively investigated over the last few decades as in the case of multiple
sclerosis (MS), which is characterized by inflammation with anti-myelin specific antibodies causing
demyelination and neurodegeneration [148]. The nervous system cells, Figure 1, can be the target of adaptive cellular and humoral immune
responses, causing autoimmunity-induced damage [147]. Autoimmunity disorders involving the
nervous system have been extensively investigated over the last few decades as in the case of
Neuroinflammatory disorders often include cases also classified within other groups. Neuroinflammation occurs as a direct response of the glial cells against injury, microbial infection,
chemical substances, autoimmunity, or neurodegeneration of nervous tissue, but when the activation 11 of 23 Int. J. Mol. Sci. 2019, 20, 434 of microglial or macroglial cells becomes aberrant it can trigger acute inflammatory responses that
can progress toward chronicity and have serious pathogenic consequences. Chronic inflammation
is typically associated with some neurodegenerative diseases such as AD and PD. These and
other disorders, for instance MS and ALS, differ in pathophysiology and can cause memory and
cognitive impairments or affect a person’s ability to move, speak, and breathe. The outcome of a
neurodegeneration is the loss of structural and functional neuronal integrity. Since there are several
types of neurons and glial cells (Figure 1), their impairment causes a range of different symptoms
and signs. g
Neurovascular diseases, owing to defects of blood vessels supplying blood to CNS, can increase
the risk of stroke. These neurovascular deficits are involved in pathogenic mechanisms in various
neurodegenerative diseases, as for instance in AD [149]. Tumors are benign and malignant neoplasias of the CNS, PNS, autonomic nervous system,
cranial nerves, and meninges (Table 1 and Figure 3). Genomic abnormalities can lead to glioblastoma,
ependymomas, medulloblastomas, and diffuse intrinsic pontine gliomas. Malignant peripheral nerve
sheath tumors (MPNSTs) are rare Schwann cell-derived neoplasms that can occur in individuals with
autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) [150]. 4.2. Exosomes in Neurological Disorders Exosomes with the transactive response
binding protein-43 (TDP43) are markers of amyotrophic lateral sclerosis and frontotemporal lobar
degeneration [161]. Neuronal cells, but not astrocytes or microglia, release in vitro exosomes with the
TDP43 full-length protein or its C-terminal fragments, both of which have been found in the brain 12 of 23 Int. J. Mol. Sci. 2019, 20, 434 of ALS patients [161,162]. Similarly to the transportation of protein tau in AD via exosomes with
the propagation of tauopathy (discussed earlier), the release of TDP43 facilitates the progression of
proteinopathy, neuroinflammation, and neurodegeneration [163]. In PD, alfa-synuclein aggregation
is the pathological marker. This presynaptic neuronal protein has been shown to be secreted via
exosomes and transferred to other normal cells [163,164], largely neurons and astrocytes, in which it
had toxic effects causing death of the recipient cells [165,166]. Abnormalities in miRNA molecules are found in inflammatory cell populations or pathological
samples of autoimmune disease [167]. It was demonstrated that exosomes carrying miRNAs can
affect the recipient neural cells and dysregulate gene expression [165]. Almost 100 miRNAs have been
found dysregulated in various affected tissue including brain, blood, and CSF of multiple sclerosis
patients [168]. miRNA expression profiles in MS-derived exosomes compared to exosomes derived
from healthy donors showed an overabundance of certain miRNAs, which were able to reduce the
frequency of immune cells via inhibition of naïve-cell differentiation. Therefore, altered miRNA
expression may play a role in pathogenesis of multiple sclerosis [168]. Exosome carrying miR-29b
can affect neuronal function in HIV patients by suppressing the expression of the neuroprotective
protein platelet-derived growth factor (PDGF)-B expression [169]. Also, in another infectious
neurodegenerative disorder, prion disease, it has been demonstrated an alteration of exosomal miRNAs
and, it has also been shown that prion protein scrapie (PrPSc) in neuronal exosomes can be passed to
other cells via the exosomes and, thereby, infect neuronal and non-neuronal cells [118]. Tumor cells, derived from primary brain tumours or from metastases, use exosomes as packages
to spread proteins and other molecules associated with malignancy [41,170]. Exosomes with their cargo
would participate in the modulation of the tumor microenvironment, for instance by regulation of
gene expression in the target cells and the functioning of the immune system, creating a pro-metastatic
niche [171]. Tumor cell-derived exosomes can cross the BBB, which enhances tumor dissemination. 4.2. Exosomes in Neurological Disorders This capability of tumor-derived exosomes to influence their environment has been demonstrated
by showing that the exosomal microRNAs secreted by astrocytes target and inhibit the PTEN tumor
suppressor gene expression in brain tumor cells, leading to enhanced oncogenicity [172]. Several
findings confirmed the role of the brain tumor-derived exosomes in modulating immune functions
by facilitating the induction of immunosuppressed phenotypes that favour the immune escape by
means of their cargo of molecular mediators [158,159,173]. Moreover, glioblastoma-derived exosomes
increase angiogenesis, which promotes tumor growth [174–176] and may support tumor dissemination
also through the BBB [177]. Exosome-bearing tumorigenic mediators released by neuronal malignant
cells have been isolated from serum of glioblastoma patients [174,178]. The current diagnostic approaches for most neurological disorders are limited to evaluation of
clinical symptoms and radiologic signs. Consequently, the diagnosis can be tardive and treatment often
produces negligible benefits. Therefore, it is necessary to find biomarkers that can be measured with
minimally invasive procedures if progress in early diagnosis and reliable and timely assessment of
response to treatment are to be achieved. Within this context, exosomes appear as suitable biomarker
candidates as the key specimens of liquid biopsies. Efforts should be made to standardize assays with
high specificity and sensitivity that would extract as much clinically relevant information as possible
from exosomes. This approach is promising, considering that exosomes are a showcase of molecules
present in their cells of origin. 4.3. Exosomes as Potential Therapeutic Tools Some properties of exosomes make them, in principle, convenient for use as drug carriers for
delivery to the CNS. For example, exosomes can cross physiological barriers and can interact with
plasma-cell membranes, which may eventually lead to their penetration into target cells. Current
knowledge suggests that exosomes may have advantages in comparison with other drug delivery
agents such as liposomes, for example, in what concerns safety and selectivity, but more research is
needed to determine their practical value in clinics. Some of these issues are discussed below. Int. J. Mol. Sci. 2019, 20, 434 13 of 23 In the last few years, research efforts have been focused on the manipulation of the exosomes’
content and their targeting to the CNS pathological sites for treating specific pathologies. The potential
application of exosomes and EV in general, as therapeutic tools, has led to the development of new and
advantageous therapies, particularly for brain tumors. Illustrative examples pertain to exosomes from
bone marrow and mesenchymal stem cells (MSCs) [179] that were re-engineered to carry therapeutic
drugs or other therapeutic molecules to diseased brain regions [180–182]. In one of the first studies,
in a zebrafish model, endothelial cell-derived exosomes loaded with doxorubicin had the ability
to pass through the BBB and reach brain tissue [180]. In other models, it has been found how the
engineered exosomes enhanced the anti-tumor properties of immune cells [183] and could confer drug
sensitivity [184,185]. In an animal experimental model of stroke, it has been shown that the intravenous administration
of MSC-derived exosomes enhanced neurite remodelling, neurogenesis, and angiogenesis, leading
to functional recovery [186]. The effect of neuronal damage recovery of MSC-derived exosomes was
demonstrated also in a model of spinal cord injury, in which the beneficial effect was probably mediated
by the transfer of miRNA-133b [187]. Mouse models have also been used in exosome-based therapies
targeting AD. Exosomes were loaded by electroporation with exogenous siRNA and engineered to
expose a brain-specific peptide and were delivered through the BBB [114]. This approach resulted in
a significant and dose-dependent knockdown of the mRNA and protein for BACE1, a protease that
produces N-terminal cleavage of amyloid precursor proteins that lead to Aβ aggregation [112]. In what pertains to the exosome-based strategies for the treatment of PD, the engineering of
exosomes by electroporation with catalase can be mentioned [114]. 4.3. Exosomes as Potential Therapeutic Tools In a mouse model of neuronal
inflammation, intranasal administration of the engineered exosomes allowed them to interact with the
target neighbouring neurons and deliver the antioxidant activity of catalase into these cells [114]. In a brain ischemia mouse model, engineered exosomes loaded with curcumin reached the
target brain lesion after intravenous administration, supressing inflammation and apoptosis [188]. The efficiency of exosomes in passing through the BBB and in delivering a cargo protein was also
demonstrated in another in vivo model [189]. Exosomes from naïve macrophages interacted with
endothelial cells of microvessels in the BBB via native surface receptors. The possible toxicity of exosomal preparations and the side effects of their administration in
neuronal tissue are still to be explored further and various technical hurdles need to be overcome. However, exosomes have a great potential to be part of a versatile strategy to treat neurological
disorders for all the reasons discussed above, such as the requirement of minimally invasive techniques,
low immunogenicity, and ability to cross the BBB and reach the target pathological cells. References 1. Raposo, G.; Stoorvogel, W. Extracellular vesicles: Exosomes, microvesicles, and friends. J. Cell Biol. 2013, 200,
373–383. [CrossRef] 2. Lopez-Verrilli, M.A.; Court, F.A. Exosomes: Mediators of communication in eukaryotes. Biol. Res. 2013, 46,
5–11. [CrossRef] 3. Fais, S.; O’Driscoll, L.; Borras, F.E.; Buzas, E.; Camussi, G.; Cappello, F.; Carvalho, J.; Cordeiro da Silva, A.;
Del Portillo, H.; El Andaloussi, S.; et al. Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in
Nanomedicine. ACS Nano 2016, 10, 3886–3899. [CrossRef] [PubMed] 4. Lee, Y.; El Andaloussi, S.; Wood, M.J.A. Exosomes and microvesicles: Extracellular vesicles for genetic
information transfer and gene therapy. Hum. Mol. Genet. 2012, 21, 1–10. [CrossRef] [PubMed] 5. Stoorvogel, W.; Kleijmeer, M.J.; Geuze, H.J.; Raposo, G. The biogenesis and functions of exosomes. Traffic
2002, 3, 321–330. [CrossRef] [PubMed] 6. Gould, S.J.; Raposo, G. As we wait: Coping with an imperfect nomenclature for extracellular vesicles. J. Extracell. Vesicles 2013, 2, 20389. [CrossRef] [PubMed] 7. Pan, B.T.; Blostein, R.; Johnstone, R.M. Loss of the transferrin receptor during the maturation of sheep
reticulocytes in vitro. An immunological approach. Biochem. J. 1983, 210, 37–47. [CrossRef] [PubMed] 8. Cossetti, C.; Lugini, L.; Astrologo, L.; Saggio, I.; Fais, S.; Spadafora, C. Soma-to-germline transmission of
RNA in mice xenografted with human tumour cells: Possible transport by exosomes. PLoS ONE 2014, 9,
e101629. [CrossRef] [PubMed] 9. Caruso Bavisotto, C.; Marino Gammazza, A.; Rappa, F.; Fucarino, A.; Pitruzzella, A.; David, S.; Campanella, C. Exosomes: Can doctors still ignore their existence? EuroMediterr. Biomed. J. 2013, 8. [CrossRef] 10. Campanella, C.; Bucchieri, F.; Merendino, A.M.; Fucarino, A.; Burgio, G.; Corona, D.F.V.; Barbieri, G.;
David, S.; Farina, F.; Zummo, G.; et al. The odyssey of Hsp60 from tumor cells to other destinations includes
plasma membrane-associated stages and Golgi and exosomal protein-trafficking modalities. PLoS ONE 2012,
7, e42008. [CrossRef] 11. Campanella, C.; Rappa, F.; Sciumè, C.; Marino Gammazza, A.; Barone, R.; Bucchieri, F.; David, S.; Curcurù, G.;
Caruso Bavisotto, C.; Pitruzzella, A.; et al. Heat shock protein 60 levels in tissue and circulating exosomes
in human large bowel cancer before and after ablative surgery. Cancer 2015, 121, 3230–3239. [CrossRef]
[PubMed] 12. Fauré, J.; Lachenal, G.; Court, M.; Hirrlinger, J.; Chatellard-Causse, C.; Blot, B.; Grange, J.; Schoehn, G.;
Goldberg, Y.; Boyer, V.; et al. Exosomes are released by cultured cortical neurones. Mol. Cell. Neurosci. 2006,
31, 642–648. [CrossRef] [PubMed] 13. 5. Conclusions Incidence of CNS disorders is increasing worldwide, but no parallel progress in prevention and
treatment occurs [190]. Novel treatment strategies based on the use of exosomes may help correct
this deficiency and improve patient management. The reasons for the growing interest in exosomes
as theranostics tools for CNS disorders, can be attributed to their characteristics and may be listed
as follows: (1) the possibility of using exosomes as biomarkers, thus providing information about
the status of the CNS; (2) exosomes are able to transverse the BBB; (3) they can be collected and
administered with minimally invasive methods (e.g., peripheral blood and/or intranasal delivery);
(4) their content can be manipulated as needed; and (5) their membrane proteins allows their targeting
to precisely defined cell types, improving by engineering the specificity of any given treatment and,
thus, reducing the side effects. Despite the range of available information about exosomes as potential disease biomarkers
and the increasing number of clinical trials on exosome-based drug delivery strategies, in cancer,
for example [191,192], comparatively little is known about exosomes in the CNS. Therefore, much
remains to be done to standardize the use of exosomes as therapeutic tools in CNS diseases. 14 of 23 Int. J. Mol. Sci. 2019, 20, 434 14 of 23 Author Contributions: C.C.B. and F.C. collecting material, writing the manuscript and revision; F.S. writing the
manuscript; D.C., A.M.G. and C.C. revision the manuscript, final editing; F.B., E.C.d.M. and A.J.L.M. writing,
revising, editing the manuscript. Acknowledgments: Part of this work was funded by the Italian National Operational Programme (PON) for
Research and Competitiveness 2007–2013; grant awarded by the Italian Ministry of University and Research
to the project titled “Cyber Brain – Polo di innovazione” (Project code: PONa3_00210, European Regional
Development Fund). Part of this work was funded by the Italian National Operational Programme (PON)
«Imprese e Competitività» 2014-2020 FESR, grant awarded by the Italian Ministry of Economic Development to the
project titled «Gestione di un servizio integrato multicentrico di diagnostica e terapia personalizzata in oncologia»
(Project code: F/090012/01-02/X36). A.J.L.M, and E.C. de M. were partially supported by IMET. This work was
done under the agreement between IEMEST (Italy) and IMET (USA) (this is IMET contribution number 19-002). Conflicts of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References Yáñez-Mó, M.; Siljander, P.R.-M.; Andreu, Z.; Zavec, A.B.; Borràs, F.E.; Buzas, E.I.; Buzas, K.; Casal, E.;
Cappello, F.; Carvalho, J.; et al. Biological properties of extracellular vesicles and their physiological
functions. J. Extracell. Vesicles 2015, 4, 27066. [CrossRef] 14. Yang, C.; Guo, W.-B.; Zhang, W.-S.; Bian, J.; Yang, J.-K.; Zhou, Q.-Z.; Chen, M.-K.; Peng, W.; Qi, T.; Wang, C.-Y.;
et al. Comprehensive proteomics analysis of exosomes derived from human seminal plasma. Andrology 2017,
5, 1007–1015. [CrossRef] [PubMed] 15 of 23 Int. J. Mol. Sci. 2019, 20, 434 15. Vlassov, A.V.; Magdaleno, S.; Setterquist, R.; Conrad, R. Exosomes: Current knowledge of their composition,
biological functions, and diagnostic and therapeutic potentials. Biochim. Biophys. Acta-Gen. Subj. 2012, 1820,
940–948. [CrossRef] [PubMed] 6. Pisitkun, T.; Shen, R.-F.; Knepper, M.A. Identification and proteomic profiling of exosomes in human u
Proc. Natl. Acad. Sci. USA 2004, 101, 13368–13373. [CrossRef] [PubMed] 17. Admyre, C.; Johansson, S.M.; Qazi, K.R.; Filén, J.-J.; Lahesmaa, R.; Norman, M.; Neve, E.P.A.; Scheynius, A.;
Gabrielsson, S. Exosomes with immune modulatory features are present in human breast milk. J. Immunol. 2007, 179, 1969–1978. [CrossRef] [PubMed] 18. Ogawa, Y.; Miura, Y.; Harazono, A.; Kanai-Azuma, M.; Akimoto, Y.; Kawakami, H.; Yamaguchi, T.; Toda, T.;
Endo, T.; Tsubuki, M.; et al. Proteomic analysis of two types of exosomes in human whole saliva. Biol. Pharm. Bull. 2011, 34, 13–23. [CrossRef] 19. Keller, S.; Rupp, C.; Stoeck, A.; Runz, S.; Fogel, M.; Lugert, S.; Hager, H.-D.; Abdel-Bakky, M.S.; Gutwein, P.;
Altevogt, P. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int. 2007, 72,
1095–1102. [CrossRef] 20. Manek, R.; Moghieb, A.; Yang, Z.; Kumar, D.; Kobessiy, F.; Sarkis, G.A.; Raghavan, V.; Wang, K.K.W. Protein
Biomarkers and Neuroproteomics Characterization of Microvesicles/Exosomes from Human Cerebrospinal
Fluid Following Traumatic Brain Injury. Mol. Neurobiol. 2018, 55, 6112–6128. [CrossRef] 21. Vella, L.J.; Greenwood, D.L.V.; Cappai, R.; Scheerlinck, J.-P.Y.; Hill, A.F. Enrichment of prion protein in
exosomes derived from ovine cerebral spinal fluid. Vet. Immunol. Immunopathol. 2008, 124, 385–393. [CrossRef] [PubMed] 22. Van Niel, G.; Porto-Carreiro, I.; Simoes, S.; Raposo, G. Exosomes: A common pathway for a specialized
function. J. Biochem. 2006, 140, 13–21. [CrossRef] [PubMed] 23. Cappello, F.; Logozzi, M.; Campanella, C.; Caruso Bavisotto, C.; Marcilla, A.; Properzi, F.; Fais, S. Exosome
levels in human body fluids: A tumor marker by themselves? Eur. J. Pharm. Sci. 2017, 96. [CrossRef]
[PubMed] 24. References [CrossRef] 35. Caruso Bavisotto, C.; Cappello, F.; Macario, A.J.L.; Conway de Macario, E.; Logozzi, M.; Fais, S.;
Campanella, C. Exosomal HSP60: A potentially useful biomarker for diagnosis, assessing prognosis, and
monitoring response to treatment. Expert Rev. Mol. Diagn. 2017, 17. [CrossRef] [PubMed] 36. Ratajczak, J.; Miekus, K.; Kucia, M.; Zhang, J.; Reca, R.; Dvorak, P.; Ratajczak, M.Z. Embryonic stem
cell-derived microvesicles reprogram hematopoietic progenitors: Evidence for horizontal transfer of mRNA
and protein delivery. Leukemia 2006, 20, 847–856. [CrossRef] [PubMed] 37. Kosaka, N.; Iguchi, H.; Yoshioka, Y.; Takeshita, F.; Matsuki, Y.; Ochiya, T. Secretory mechanisms and
intercellular transfer of microRNAs in living cells. J. Biol. Chem. 2010, 285, 17442–17452. [CrossRef]
[PubMed] 38. Wei, H.; Xu, Y.; Xu, W.; Zhou, Q.; Chen, Q.; Yang, M.; Feng, F.; Liu, Y.; Zhu, X.; Yu, M.; et al. Serum Exosomal
miR-223 Serves as a Potential Diagnostic and Prognostic Biomarker for Dementia. Neuroscience 2018, 379,
167–176. [CrossRef] 39. Zheng, P.; Chen, L.; Yuan, X.; Luo, Q.; Liu, Y.; Xie, G.; Ma, Y.; Shen, L. Exosomal transfer of tumor-associated
macrophage-derived miR-21 confers cisplatin resistance in gastric cancer cells. J. Exp. Clin. Cancer Res. 2017,
36, 53. [CrossRef] 40. Qu, Y.; Zhang, Q.; Cai, X.; Li, F.; Ma, Z.; Xu, M.; Lu, L. Exosomes derived from miR-181-5p-modified
adipose-derived mesenchymal stem cells prevent liver fibrosis via autophagy activation. J. Cell. Mol. Med. 2017. [CrossRef] 41. Caruso Bavisotto, C.; Graziano, F.; Rappa, F.; Marino Gammazza, A.; Logozzi, M.; Fais, S.; Maugeri, R.;
Bucchieri, F.; Conway de Macario, E.; Macario, A.J.L.; et al. Exosomal Chaperones and miRNAs in
Gliomagenesis: State-of-Art and Theranostics Perspectives. Int. J. Mol. Sci. 2018, 19, 2626. [CrossRef]
[PubMed] 42. Fernández-Messina, L.; Gutiérrez-Vázquez, C.; Rivas-García, E.; Sánchez-Madrid, F.; de la Fuente, H. Immunomodulatory role of microRNAs transferred by extracellular vesicles. Biol. Cell 2015, 107, 61–77. [CrossRef] [PubMed] 43. Ostrowski, M.; Carmo, N.B.; Krumeich, S.; Fanget, I.; Raposo, G.; Savina, A.; Moita, C.F.; Schauer, K.;
Hume, A.N.; Freitas, R.P.; et al. Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 2010, 12, 19–30. [CrossRef] [PubMed] 44. Xie, Y.; Zhang, H.; Li, W.; Deng, Y.; Munegowda, M.A.; Chibbar, R.; Qureshi, M.; Xiang, J. Dendritic
cells recruit T cell exosomes via exosomal LFA-1 leading to inhibition of CD8+ CTL responses through
downregulation of peptide/MHC class I and Fas ligand-mediated cytotoxicity. J. Immunol. 2010, 185,
5268–5278. [CrossRef] [PubMed] 45. References Colombo, M.; Moita, C.; van Niel, G.; Kowal, J.; Vigneron, J.; Benaroch, P.; Manel, N.; Moita, L.F.; Théry, C.;
Raposo, G. Analysis of ESCRT functions in exosome biogenesis, composition and secretion highlights the
heterogeneity of extracellular vesicles. J. Cell Sci. 2013, 126, 5553–5565. [CrossRef] [PubMed] 25. Baietti, M.F.; Zhang, Z.; Mortier, E.; Melchior, A.; Degeest, G.; Geeraerts, A.; Ivarsson, Y.; Depoortere, F.;
Coomans, C.; Vermeiren, E.; et al. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes. Nat. Cell Biol. 2012, 14, 677–685. [CrossRef] [PubMed] 26. Théry, C.; Zitvogel, L.; Amigorena, S. Exosomes: Composition, biogenesis and function. Nat. Rev. Immunol. 2002, 2, 569–579. [CrossRef] [PubMed] 27. Möbius, W.; Ohno-Iwashita, Y.; van Donselaar, E.G.; Oorschot, V.M.J.; Shimada, Y.; Fujimoto, T.;
Heijnen, H.F.G.; Geuze, H.J.; Slot, J.W. Immunoelectron microscopic localization of cholesterol using
biotinylated and non-cytolytic perfringolysin O. J. Histochem. Cytochem. 2002, 50, 43–55. [CrossRef] 28. Trajkovic, K.; Hsu, C.; Chiantia, S.; Rajendran, L.; Wenzel, D.; Wieland, F.; Schwille, P.; Brügger, B.; Simons, M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes. Science 2008, 319, 1244–1247. [CrossRef] 29. Moreno-Gonzalo, O.; Villarroya-Beltri, C.; Sánchez-Madrid, F. Post-Translational Modifications of Exosomal
Proteins. Front. Immunol. 2014, 5, 383. [CrossRef] 30. Campanella, C.; D’Anneo, A.; Marino Gammazza, A.; Caruso Bavisotto, C.; Barone, R.; Emanuele, S.;
Lo Cascio, F.; Mocciaro, E.; Fais, S.; Conway de Macario, E.; et al. The histone deacetylase inhibitor SAHA
induces HSP60 nitration and its extracellular release by exosomal vesicles in human lung-derived carcinoma
cells. Oncotarget 2016, 7. [CrossRef] 31. Qu, Y.; Franchi, L.; Nunez, G.; Dubyak, G.R. Nonclassical IL-1 beta secretion stimulated by P2X7 receptors
is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J. Immunol. 2007, 179, 1913–1925. [CrossRef] [PubMed] 32. Phoonsawat, W.; Aoki-Yoshida, A.; Tsuruta, T.; Sonoyama, K. Adiponectin is partially associated with
exosomes in mouse serum. Biochem. Biophys. Res. Commun. 2014, 448, 261–266. [CrossRef] [PubMed] 33. Chen, L.; Chen, R.; Kemper, S.; Charrier, A.; Brigstock, D.R. Suppression of fibrogenic signaling in hepatic
stellate cells by Twist1-dependent microRNA-214 expression: Role of exosomes in horizontal transfer of
Twist1. Am. J. Physiol. Gastrointest. Liver Physiol. 2015, 309, G491–G499. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 434 16 of 23 16 of 23 34. Campanella, C.; Caruso Bavisotto, C.; Marino Gammazza, A.; Nikolic, D.; Rappa, F.; David, S.; Cappello, F.;
Bucchieri, F.; Fais, S. Exosomal Heat Shock Proteins as New Players in Tumour Cell-to-cell Communication. J. Circ. Biomark. 2014, 1. References Smooth and sparsely-spined stellate cells in the visual cortex of the rat: A study using a
combined golgi-electron microscope technique. J. Comp. Neurol. 1978, 181, 129–171. [CrossRef] [PubMed]
58. Palay, S.L.; Chan-Palay, V. General Morphology of Neurons and Neuroglia. In Comprehensive Physiology; John ,
;
,
p
y p
y
g
combined golgi-electron microscope technique. J. Comp. Neurol. 1978, 181, 129–171. [CrossRef] [PubMed]
58. Palay, S.L.; Chan-Palay, V. General Morphology of Neurons and Neuroglia. In Comprehensive Physiology; John
Wiley & Sons, Inc.: Hoboken, NJ, USA, 2011; pp. 5–37. 58. Palay, S.L.; Chan-Palay, V. General Morphology of Neurons and Neuroglia. In Comprehensive Physiology; John
Wiley & Sons, Inc.: Hoboken, NJ, USA, 2011; pp. 5–37. 59. Korkut, C.; Li, Y.; Koles, K.; Brewer, C.; Ashley, J.; Yoshihara, M.; Budnik, V. Regulation of postsynaptic
retrograde signaling by presynaptic exosome release. Neuron 2013, 77, 1039–1046. [CrossRef] [PubMed]
60. Allen, N.J.; Barres, B.A. Neuroscience: Glia—More than just brain glue. Nature 2009, 457, 675–677. [CrossRef]
[P bM d] 59. Korkut, C.; Li, Y.; Koles, K.; Brewer, C.; Ashley, J.; Yoshihara, M.; Budnik, V. Regulation of postsynaptic
retrograde signaling by presynaptic exosome release. Neuron 2013, 77, 1039–1046. [CrossRef] [PubMed] ,
;
,
;
,
;
,
;
y, J ;
,
;
,
g
p
y
p
retrograde signaling by presynaptic exosome release. Neuron 2013, 77, 1039–1046. [CrossRef] [PubMed]
60. Allen, N.J.; Barres, B.A. Neuroscience: Glia—More than just brain glue. Nature 2009, 457, 675–677. [CrossRef]
[PubMed] 60. Allen, N.J.; Barres, B.A. Neuroscience: Glia—More than just brain glue. Nature 2009, 457, 675–677. [CrossRef]
[PubMed] 1. Verkhratsky, A.; Nedergaard, M. Physiology of Astroglia. Physiol. Rev. 2018, 98, 239–389. [CrossRef] 61. Verkhratsky, A.; Nedergaard, M. Physiology of Astroglia. Physiol. Rev. 2018, 98, 239–389. [CrossRef]
62. Verkhratsky, A.; Zorec, R. Astroglial signalling in health and disease. Neurosci. Lett. 2018. [CrossRef] 62. Verkhratsky, A.; Zorec, R. Astroglial signalling in health and disease. Neurosci. Lett. 2018. [CrossRef] 63. Herculano-Houzel, S. Longevity and sexual maturity vary across species with number of cortical neurons,
and humans are no exception. J. Comp. Neurol. 2018. [CrossRef] 64. Azevedo, F.A.C.; Carvalho, L.R.B.; Grinberg, L.T.; Farfel, J.M.; Ferretti, R.E.L.; Leite, R.E.P.; Jacob Filho, W.;
Lent, R.; Herculano-Houzel, S. Equal numbers of neuronal and nonneuronal cells make the human brain an
isometrically scaled-up primate brain. J. Comp. Neurol. 2009, 513, 532–541. [CrossRef] [PubMed] 65. Janowska, J.; Gargas, J.; Ziemka-Nalecz, M.; Teresa, Z.; Buzanska, L.; Sypecka, J. References Van Niel, G.; Charrin, S.; Simoes, S.; Romao, M.; Rochin, L.; Saftig, P.; Marks, M.S.; Rubinstein, E.;
Raposo, G. The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during
melanogenesis. Dev. Cell 2011, 21, 708–721. [CrossRef] [PubMed] 46. Rana, S.; Yue, S.; Stadel, D.; Zöller, M. Toward tailored exosomes: The exosomal tetraspanin web contributes
to target cell selection. Int. J. Biochem. Cell Biol. 2012, 44, 1574–1584. [CrossRef] 7. Schorey, J.S.; Bhatnagar, S. Exosome function: From tumor immunology to pathogen biology. Traffic 200
871–881. [CrossRef] 48. Choi, D.-S.; Lee, J.-M.; Park, G.W.; Lim, H.-W.; Bang, J.Y.; Kim, Y.-K.; Kwon, K.-H.; Kwon, H.J.; Kim, K.P.;
Gho, Y.S. Proteomic analysis of microvesicles derived from human colorectal cancer cells. J. Proteome Res. 2007, 6, 4646–4655. [CrossRef] 49. Muralidharan-Chari, V.; Clancy, J.W.; Sedgwick, A.; D’Souza-Schorey, C. Microvesicles: Mediators of
extracellular communication during cancer progression. J. Cell Sci. 2010, 123, 1603–1611. [CrossRef] 50. Merendino, A.M.; Bucchieri, F.; Campanella, C.; Marcianò, V.; Ribbene, A.; David, S.; Zummo, G.; Burgio, G.;
Corona, D.F.V.; Conway de Macario, E.; et al. Hsp60 is actively secreted by human tumor cells. PLoS ONE
2010, 5, e9247. [CrossRef] 51. Lancaster, G.I.; Febbraio, M. A Exosome-dependent trafficking of HSP70: A novel secretory pathway for
cellular stress proteins. J. Biol. Chem. 2005, 280, 23349–23355. [CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 434 17 of 23 52. Yuana, Y.; Sturk, A.; Nieuwland, R. Extracellular vesicles in physiological and pathological conditions. Blood Rev. 2013, 27, 31–39. [CrossRef] [PubMed] 53. Frühbeis, C.; Fröhlich, D.; Kuo, W.P.; Krämer-Albers, E.-M. Extracellular vesicles as mediators of neuron-glia
communication. Front. Cell. Neurosci. 2013, 7, 182. [CrossRef] [PubMed] 54. Tasaki, M.; Ueda, M.; Ochiai, S.; Tanabe, Y.; Murata, S.; Misumi, Y.; Su, Y.; Sun, X.; Shinriki, S.; Jono, H.; et al. Transmission of circulating cell-free AA amyloid oligomers in exosomes vectors via a prion-like mechanism. Biochem. Biophys. Res. Commun. 2010, 400, 559–562. [CrossRef] [PubMed] 55. Mtui, E.; Gruener, G.; Dockery, P. Fitzgerald’s Clinical Neuroanatomy and Neuroscience; Elsevier Saunders:
Edinburgh, UK, 2006; ISBN 9780702067273. 56. Clarke, C.; Howard, R.; Rossor, M.; Shorvon, S. Neurology: A Queen Square Textbook; Wiley-Blackwell:
Chichester, UK, 2016; ISBN 9781118486177. 57. Peters, A.; Fairén, A. Smooth and sparsely-spined stellate cells in the visual cortex of the rat: A study using a
combined golgi-electron microscope technique. J. Comp. Neurol. 1978, 181, 129–171. [CrossRef] [PubMed] 57. Peters, A.; Fairén, A. References Directed glial differentiation
and transdifferentiation for neural tissue regeneration. Exp. Neurol. 2018. [CrossRef] [PubMed] 66. Bradl, M.; Lassmann, H. Oligodendrocytes: Biology and pathology. Acta Neuropathol. 2010, 119, 37–53. [CrossRef] [PubMed] 67. Jäkel, S.; Dimou, L. Glial Cells and Their Function in the Adult Brain: A Journey through the History of
Their Ablation. Front. Cell. Neurosci. 2017, 11, 24. [CrossRef] [PubMed] 68. Kriegstein, A.R.; Noctor, S.C. Patterns of neuronal migration in the embryonic cortex. Trends Neurosci. 2004,
27, 392–399. [CrossRef] 69. Hatton, G.I. Pituicytes, glia and control of terminal secretion. J. Exp. Biol. 1988, 139, 67–79. 70. Hoogland, T.M.; Kuhn, B. Recent developments in the understanding of astrocyte function in the cerebellum
in vivo. Cerebellum 2010, 9, 264–271. [CrossRef] 71. Newman, E.A. New roles for astrocytes: Regulation of synaptic transmission. Trends Neurosci. 2003, 26,
536–542. [CrossRef] 72. Vasile, F.; Dossi, E.; Rouach, N. Human astrocytes: Structure and functions in the healthy brain. Brain Struct. Funct. 2017, 222, 2017–2029. [CrossRef] 73. Frühbeis, C.; Fröhlich, D.; Kuo, W.P.; Amphornrat, J.; Thilemann, S.; Saab, A.S.; Kirchhoff, F.;
Möbius, W.; Goebbels, S.; Nave, K.-A.; et al. Neurotransmitter-triggered transfer of exosomes mediates
oligodendrocyte-neuron communication. PLoS Biol. 2013, 11, e1001604. [CrossRef] 74. Wang, S.; Cesca, F.; Loers, G.; Schweizer, M.; Buck, F.; Benfenati, F.; Schachner, M.; Kleene, R. Synapsin
I Is an Oligomannose-Carrying Glycoprotein, Acts as an Oligomannose-Binding Lectin, and Promotes
Neurite Outgrowth and Neuronal Survival When Released via Glia-Derived Exosomes. J. Neurosci. 2011, 31,
7275–7290. [CrossRef] [PubMed] 75. Antonucci, F.; Turola, E.; Riganti, L.; Caleo, M.; Gabrielli, M.; Perrotta, C.; Novellino, L.; Clementi, E.;
Giussani, P.; Viani, P.; et al. Microvesicles released from microglia stimulate synaptic activity via enhanced
sphingolipid metabolism. EMBO J. 2012, 31, 1231–1240. [CrossRef] [PubMed] 18 of 23 Int. J. Mol. Sci. 2019, 20, 434 76. Jiménez, A.J.; Domínguez-Pinos, M.-D.; Guerra, M.M.; Fernández-Llebrez, P.; Pérez-Fígares, J.-M. Structure
and function of the ependymal barrier and diseases associated with ependyma disruption. Tissue Barriers
2014, 2, e28426. [CrossRef] [PubMed] 77. Wolburg, H.; Paulus, W. Choroid plexus: Biology and pathology. Acta Neuropathol. 2010, 119, 75
[CrossRef] [PubMed] 78. Kaur, C.; Ling, E.-A. The circumventricular organs. Histol. Histopathol. 2017, 32, 879–892. [CrossRef]
[PubMed] 79. Gomes de Andrade, G.; Reck Cechinel, L.; Bertoldi, K.; Galvão, F.; Valdeci Worm, P.; Rodrigues Siqueira, I. The Aging Process Alters IL-1β and CD63 Levels Differently in Extracellular Vesicles Obtained from the
Plasma and Cerebrospinal Fluid. Neuroimmunomodulation 2018, 25, 18–22. [CrossRef] 80. References Barcia, C.; Guillemin, G.J.; Curtin, J.F.; Zirger, J.M. Editorial: Glial Cells: Managers of Neuro-Immunity. Front. Cell. Neurosci. 2016, 10, 60. [CrossRef] [PubMed] 81. Mammana, S.; Fagone, P.; Cavalli, E.; Basile, M.; Petralia, M.; Nicoletti, F.; Bramanti, P.; Mazzon, E. The Role of Macrophages in Neuroinflammatory and Neurodegenerative Pathways of Alzheimer’s Disease,
Amyotrophic Lateral Sclerosis, and Multiple Sclerosis: Pathogenetic Cellular Effectors and Potential
Therapeutic Targets. Int. J. Mol. Sci. 2018, 19, 831. [CrossRef] 82. Prinz, M.; Tay, T.L.; Wolf, Y.; Jung, S. Microglia: Unique and common features with other tissue macro
Acta Neuropathol. 2014, 128, 319–331. [CrossRef] 83. Shemer, A.; Jung, S. Differential roles of resident microglia and infiltrating monocytes in murine CNS
autoimmunity. Semin. Immunopathol. 2015, 37, 613–623. [CrossRef] 84. Garden, G.A.; Möller, T. Microglia Biology in Health and Disease. J. Neuroimmune Pharmacol. 2006, 1, 127–137. [CrossRef] [PubMed] 85. Shemer, A.; Erny, D.; Jung, S.; Prinz, M. Microglia Plasticity During Health and Disease: An Immunological
Perspective. Trends Immunol. 2015, 36, 614–624. [CrossRef] [PubMed] 86. Hickman, S.; Izzy, S.; Sen, P.; Morsett, L.; El Khoury, J. Microglia in neurodegeneration. Nat. Neurosci. 2018,
21, 1359–1369. [CrossRef] [PubMed] 87. Gamble, H.J.; Goldby, S. Mast cells in peripheral nerve trunks. Nature 1961, 189, 766–767. [CrossRef]
[PubMed] 88. Mattick, J.S. Challenging the dogma: The hidden layer of non-protein-coding RNAs in complex organisms. Bioessays 2003, 25, 930–939. [CrossRef] 89. Suzuki, R.; Furuno, T.; McKay, D.M.; Wolvers, D.; Teshima, R.; Nakanishi, M.; Bienenstock, J. Direct
neurite-mast cell communication in vitro occurs via the neuropeptide substance P. J. Immunol. 1999, 163,
2410–2415. [PubMed] 90. Forsythe, P. Mast Cells in Neuroimmune Interactions. Trends Neurosci. 2018. [CrossRef] 91. Tricaud, N. Myelinating Schwann Cell Polarity and Mechanically-Driven Myelin Sheath Elongation. Front. Cell. Neurosci. 2018, 11, 414. [CrossRef] 92. Saab, A.S.; Nave, K.-A. Myelin dynamics: Protecting and shaping neuronal functions. Curr. Opin. Neurobiol. 2017, 47, 104–112. [CrossRef] 93. il Lee, Y.; Thompson, W.J.; Harlow, M.L. Schwann cells participate in synapse elimination at the developing
neuromuscular junction. Curr. Opin. Neurobiol. 2017, 47, 176–181. [CrossRef] 4. Sugiura, Y.; Lin, W. Neuron-glia interactions: The roles of Schwann cells in neuromuscular synapse forma
and function. Biosci. Rep. 2011, 31, 295–302. [CrossRef] [PubMed] 95. Lopez-Verrilli, M.A.; Court, F.A. Transfer of vesicles from schwann cells to axons: A novel mechanism of
communication in the peripheral nervous system. Front. Physiol. 2012, 3, 205. [CrossRef] 96. Hyung, S.; Jung, K.; Cho, S.-R.; Jeon, N.L. References Chivet, M.; Hemming, F.; Pernet-Gallay, K.; Fraboulet, S.; Sadoul, R. Emerging role of neuronal exosomes in
the central nervous system. Front. Physiol. 2012, 3, 145. [CrossRef] [PubMed] 107. Liu, C.A.; Chang, C.Y.; Hsueh, K.W.; Su, H.L.; Chiou, T.W.; Lin, S.Z.; Harn, H.J. Migration/Invasion of
Malignant Gliomas and Implications for Therapeutic Treatment. Int. J. Mol. Sci. 2018, 19, 1115. [CrossRef]
[PubMed] 108. Shao, H.; Chung, J.; Lee, K.; Balaj, L.; Min, C.; Carter, B.S.; Hochberg, F.H.; Breakefield, X.O.; Lee, H.;
Weissleder, R. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat. Commun. 2015, 6, 6999. [CrossRef] [PubMed] 109. Quek, C.; Hill, A.F. The role of extracellular vesicles in neurodegenerative diseases. Biochem. Biophys. Res. Commun. 2017, 483, 1178–1186. [CrossRef] [PubMed] 110. Campanella, C.; Pace, A.; Caruso Bavisotto, C.; Marzullo, P.; Marino Gammazza, A.; Buscemi, S.; Palumbo
Piccionello, A. Heat shock proteins in Alzheimer’s disease: Role and targeting. Int. J. Mol. Sci. 2018, 19, 2603. [CrossRef] 111. Marino Gammazza, A.; Caruso Bavisotto, C.; Barone, R.; Conway de Macario, E.; Macario, A.J.L. Alzheimer’s
disease and molecular chaperones: Current knowledge and the future of chaperonotherapy. Curr. Pharm. Des. 2016, 22. [CrossRef] 112. Alvarez-Erviti, L.; Seow, Y.; Yin, H.; Betts, C.; Lakhal, S.; Wood, M.J.A. Delivery of siRNA to the mouse brain
by systemic injection of targeted exosomes. Nat. Biotechnol. 2011, 29, 341–345. [CrossRef] 113. Haney, M.J.; Zhao, Y.; Harrison, E.B.; Mahajan, V.; Ahmed, S.; He, Z.; Suresh, P.; Hingtgen, S.D.;
Klyachko, N.L.; Mosley, R.L.; et al. Specific transfection of inflamed brain by macrophages: A new therapeutic
strategy for neurodegenerative diseases. PLoS ONE 2013, 8, e61852. [CrossRef] 114. Haney, M.J.; Klyachko, N.L.; Zhao, Y.; Gupta, R.; Plotnikova, E.G.; He, Z.; Patel, T.; Piroyan, A.; Sokolsky, M.;
Kabanov, A.V.; et al. Exosomes as drug delivery vehicles for Parkinson’s disease therapy. J. Control. Release
2015, 207, 18–30. [CrossRef] 115. Bullmore, E.; Sporns, O. Complex brain networks: Graph theoretical analysis of structural and functional
systems. Nat. Rev. Neurosci. 2009, 10, 186–198. [CrossRef] [PubMed] 116. Gross, J.C.; Chaudhary, V.; Bartscherer, K.; Boutros, M. Active Wnt proteins are secreted on exosomes. Nat. Cell Biol. 2012, 14, 1036–1045. [CrossRef] 117. Sheldon, H.; Heikamp, E.; Turley, H.; Dragovic, R.; Thomas, P.; Oon, C.E.; Leek, R.; Edelmann, M.; Kessler, B.;
Sainson, R.C.A.; et al. New mechanism for Notch signaling to endothelium at a distance by Delta-like 4
incorporation into exosomes. Blood 2010, 116, 2385–2394. [CrossRef] 118. Février, B.; Raposo, G. References The Schwann Cell as an Active Synaptic Partner. ChemPhysChem
2018, 19, 1123–1127. [CrossRef] 97. Heredia, D.J.; Schubert, D.; Maligireddy, S.; Hennig, G.W.; Gould, T.W. A Novel Striated Muscle-Specific
Myosin-Blocking Drug for the Study of Neuromuscular Physiology. Front. Cell. Neurosci. 2016, 10, 276. [CrossRef] 98. Hanani, M. Satellite glial cells: More than just ‘rings around the neuron’. Neuron Glia Biol. 2010, 6, 1–2. [CrossRef] [PubMed] 99. Pannese, E. Biology and Pathology of Perineuronal Satellite Cells in Sensory Ganglia. In Advances in Anatomy,
Embryology, and Cell Biology; Springer: Berlin/Heidelberg, Germany, 2018; Volume 226, pp. 1–63. Int. J. Mol. Sci. 2019, 20, 434 19 of 23 19 of 23 100. Pietrangelo, T.; Di Filippo, E.S.; Locatelli, M.; Piacenza, F.; Farina, M.; Pavoni, E.; Di Donato, A.; Innosa, D.;
Provinciali, M.; Fulle, S. Extracellular Guanosine 5′-Triphosphate Induces Human Muscle Satellite Cells to
Release Exosomes Stuffed with Guanosine. Front. Pharmacol. 2018, 9, 152. [CrossRef] [PubMed] 101. Vinterhøj, H.S.H.; Stensballe, A.; Duroux, M.; Gazerani, P. Characterization of rat primary trigeminal satellite
glial cells and associated extracellular vesicles under normal and inflammatory conditions. J. Proteom. 2019,
190, 27–34. [CrossRef] 102. Marzesco, A.-M.; Janich, P.; Wilsch-Bräuninger, M.; Dubreuil, V.; Langenfeld, K.; Corbeil, D.; Huttner, W.B. Release of extracellular membrane particles carrying the stem cell marker prominin-1 (CD133) from neural
progenitors and other epithelial cells. J. Cell Sci. 2005, 118, 2849–2858. [CrossRef] [PubMed] 103. Bakhti,
M.;
Winter,
C.;
Simons,
M. Inhibition
of
myelin
membrane
sheath
formation
by
oligodendrocyte-derived exosome-like vesicles. J. Biol. Chem. 2011, 286, 787–796. [CrossRef] 104. Porro, C.; Trotta, T.; Panaro, M.A. Microvesicles in the brain: Biomarker, messenger or mediator? J. Neuroimmunol. 2015, 288, 70–78. [CrossRef] [PubMed] 105. Pegtel, D.M.; Peferoen, L.; Amor, S. Extracellular vesicles as modulators of cell-to-cell communication in the
healthy and diseased brain. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014, 369, 20130516. [CrossRef] [PubMed]
106. Chivet, M.; Hemming, F.; Pernet-Gallay, K.; Fraboulet, S.; Sadoul, R. Emerging role of neuronal exosomes in 105. Pegtel, D.M.; Peferoen, L.; Amor, S. Extracellular vesicles as modulators of cell-to-cell communication in the
healthy and diseased brain. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014, 369, 20130516. [CrossRef] [PubMed] healthy and diseased brain. Philos. Trans. R. Soc. Lond. B Biol. Sci. 2014, 369, 20130516. [CrossRef] [PubMed]
106. Chivet, M.; Hemming, F.; Pernet-Gallay, K.; Fraboulet, S.; Sadoul, R. Emerging role of neuronal exosomes in
the central nervous system. Front. Physiol. 2012, 3, 145. [CrossRef] [PubMed] 106. References Exosomes: Endosomal-derived vesicles shipping extracellular messages. Curr. Opin. Cell Biol. 2004, 16, 415–421. [CrossRef] [PubMed] 119. Arantes, R.M.E.; Andrews, N.W. A role for synaptotagmin VII-regulated exocytosis of lysosomes in neurite
outgrowth from primary sympathetic neurons. J. Neurosci. 2006, 26, 4630–4637. [CrossRef] 120. Putz, U.; Howitt, J.; Lackovic, J.; Foot, N.; Kumar, S.; Silke, J.; Tan, S.-S. Nedd4 family-interacting protein 1
(Ndfip1) is required for the exosomal secretion of Nedd4 family proteins. J. Biol. Chem. 2008, 283, 32621–32627. [CrossRef] [PubMed] (
p )
q
[CrossRef] [PubMed] Int. J. Mol. Sci. 2019, 20, 434 20 of 23 121. Ghidoni, R.; Paterlini, A.; Albertini, V.; Glionna, M.; Monti, E.; Schiaffonati, L.; Benussi, L.; Levy, E.; Binetti, G. Cystatin C is released in association with exosomes: A new tool of neuronal communication which is
unbalanced in Alzheimer’s disease. Neurobiol. Aging 2011, 32, 1435–1442. [CrossRef] 22. Regehr, W.G.; Carey, M.R.; Best, A.R. Activity-Dependent Regulation of Synapses by Retrograde Messen
Neuron 2009, 63, 154–170. [CrossRef] [PubMed] 123. Bahrini, I.; Song, J.; Diez, D.; Hanayama, R. Neuronal exosomes facilitate synaptic pruning by up-regulating
complement factors in microglia. Sci. Rep. 2015, 5, 7989. [CrossRef] 124. Lachenal, G.; Pernet-Gallay, K.; Chivet, M.; Hemming, F.J.; Belly, A.; Bodon, G.; Blot, B.; Haase, G.;
Goldberg, Y.; Sadoul, R. Release of exosomes from differentiated neurons and its regulation by synaptic
glutamatergic activity. Mol. Cell. Neurosci. 2011, 46, 409–418. [CrossRef] 125. Morel, L.; Regan, M.; Higashimori, H.; Ng, S.K.; Esau, C.; Vidensky, S.; Rothstein, J.; Yang, Y. Neuronal
exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1. J. Biol. Chem. 2013, 288, 7105–7116. [CrossRef] [PubMed] 126. Prada, I.; Gabrielli, M.; Turola, E.; Iorio, A.; D’Arrigo, G.; Parolisi, R.; De Luca, M.; Pacifici, M.; Bastoni, M.;
Lombardi, M.; et al. Glia-to-neuron transfer of miRNAs via extracellular vesicles: A new mechanism
underlying inflammation-induced synaptic alterations. Acta Neuropathol. 2018, 135, 529–550. [CrossRef]
[PubMed] 127. Krämer-Albers, E.-M.; Bretz, N.; Tenzer, S.; Winterstein, C.; Möbius, W.; Berger, H.; Nave, K.-A.; Schild, H.;
Trotter, J. Oligodendrocytes secrete exosomes containing major myelin and stress-protective proteins: Trophic
support for axons? Proteom. Clin. Appl. 2007, 1, 1446–1461. [CrossRef] [PubMed] 128. Fitzner, D.; Schnaars, M.; van Rossum, D.; Krishnamoorthy, G.; Dibaj, P.; Bakhti, M.; Regen, T.; Hanisch, U.-K.;
Simons, M. Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis. J. Cell Sci. 2011, 124, 447–458. [CrossRef] 129. References 2012, 2, a008888. [CrossRef] 143. Chen, S.; Sayana, P.; Zhang, X.; Le, W. Genetics of amyotrophic lateral sclerosis: An update. Mol. Neurodegener. 2013, 8, 28. [CrossRef] 144. Brown, R.C.; Lockwood, A.H.; Sonawane, B.R. Neurodegenerative diseases: An overview of environmental
risk factors. Environ. Health Perspect. 2005, 113, 1250–1256. [CrossRef] 145. Hima Bindu, A.; Aliya, S.; Jeevani, T. Genetic and Degenerative Neurological Disorders—An Emphasis on
Alzheimer’s, the Mystery. J. Genet. Syndr. Gene Ther. 2011, 2, 109. [CrossRef] 146. Cogen, P.H.; Daneshvar, L.; Metzger, A.K.; Edwards, M.S. Deletion mapping of the medulloblastoma locus
on chromosome 17p. Genomics 1990, 8, 279–285. [CrossRef] 47. Dalmau, J.; Geis, C.; Graus, F. Autoantibodies to Synaptic Receptors and Neuronal Cell Surface Protein
Autoimmune Diseases of the Central Nervous System. Physiol. Rev. 2017, 97, 839–887. [CrossRef] 147. Dalmau, J.; Geis, C.; Graus, F. Autoantibodies to Synaptic Receptors and Neuronal Cell Surface Proteins in
Autoimmune Diseases of the Central Nervous System. Physiol. Rev. 2017, 97, 839–887. [CrossRef]
148
Y
hii F Shi
h
Y A t i
N
l
i
l Di
Autoimmune Dis 2004 47 425 430 148. Yoshii, F.; Shinohara, Y. Autoimmune Neurological Diseases. Autoimmune Dis. 2004, 47, 425–430. 149. Kisler, K.; Nelson, A.R.; Montagne, A.; Zlokovic, B.V. Cerebral blood flow regulation and neurovascular
dysfunction in Alzheimer disease. Nat. Rev. Neurosci. 2017, 18, 419–434. [CrossRef] [PubMed] 150. Carroll, S.L. The Challenge of Cancer Genomics in Rare Nervous System Neoplasms: Malignant Peripheral
Nerve Sheath Tumors as a Paradigm for Cross-Species Comparative Oncogenomics. Am. J. Pathol. 2016, 186,
464–477. [CrossRef] 151. Rajendran, L.; Honsho, M.; Zahn, T.R.; Keller, P.; Geiger, K.D.; Verkade, P.; Simons, K. Alzheimer’s disease
beta-amyloid peptides are released in association with exosomes. Proc. Natl. Acad. Sci. USA 2006, 103,
11172–11177. [CrossRef] [PubMed] 152. Pluta, R.; Ułamek-Kozioł, M.; Januszewski, S.; Czuczwar, S.J. Exosomes as possible spread factor and
potential biomarkers in Alzheimer’s disease: Current concepts. Biomark. Med. 2018, 12. [CrossRef] 153. De Toro, J.; Herschlik, L.; Waldner, C.; Mongini, C. Emerging roles of exosomes in normal and path
conditions: New insights for diagnosis and therapeutic applications. Front. Immunol. 2015, 6, 203. [Cr 154. Reilly, P.; Winston, C.N.; Baron, K.R.; Trejo, M.; Rockenstein, E.M.; Akers, J.C.; Kfoury, N.; Diamond, M.;
Masliah, E.; Rissman, R.A.; et al. Novel human neuronal tau model exhibiting neurofibrillary tangles and
transcellular propagation. Neurobiol. Dis. 2017, 106, 222–234. [CrossRef] 155. Grey, M.; Dunning, C.J.; Gaspar, R.; Grey, C.; Brundin, P.; Sparr, E.; Linse, S. References Zhuang, X.; Xiang, X.; Grizzle, W.; Sun, D.; Zhang, S.; Axtell, R.C.; Ju, S.; Mu, J.; Zhang, L.; Steinman, L.; et al. Treatment of Brain Inflammatory Diseases by Delivering Exosome Encapsulated Anti-inflammatory Drugs
from the Nasal Region to the Brain. Mol. Ther. 2011, 19, 1769–1779. [CrossRef] [PubMed] 130. Chen, C.C.; Liu, L.; Ma, F.; Wong, C.W.; Guo, X.E.; Chacko, J.V.; Farhoodi, H.P.; Zhang, S.X.; Zimak, J.;
Ségaliny, A.; et al. Elucidation of Exosome Migration Across the Blood–Brain Barrier Model In Vitro. Cell. Mol. Bioeng. 2016, 9, 509–529. [CrossRef] 131. Fiandaca, M.S.; Kapogiannis, D.; Mapstone, M.; Boxer, A.; Eitan, E.; Schwartz, J.B.; Abner, E.L.; Petersen, R.C.;
Federoff, H.J.; Miller, B.L.; et al. Identification of preclinical Alzheimer’s disease by a profile of pathogenic
proteins in neurally derived blood exosomes: A case-control study. Alzheimer’s Dement. 2015, 11, 600–607.e1. [CrossRef] 132. Abbott, N.J. Astrocyte-endothelial interactions and blood-brain barrier permeability. J. Anat. 2002, 200,
629–638. [CrossRef] 133. Abbott, N.J. Inflammatory mediators and modulation of blood-brain barrier permeability. Cell. Mol. Neurobiol. 2000, 20, 131–147. [CrossRef] 134. Verstrepen, L.; Bekaert, T.; Chau, T.-L.; Tavernier, J.; Chariot, A.; Beyaert, R. TLR-4, IL-1R and TNF-R signaling
to NF-κB: Variations on a common theme. Cell. Mol. Life Sci. 2008, 65, 2964–2978. [CrossRef] 135. Wispelwey, B.; Hansen, E.J.; Scheld, W.M. Haemophilus influenzae outer membrane vesicle-induced
blood-brain barrier permeability during experimental meningitis. Infect. Immun. 1989, 57, 2559–2562. [PubMed] 136. Funkhouser, J. Reinventing Pharma: The Theranostic Revolution. Curr. Drug Discov. 2002, 2, 17–19. 137. Fan, Z.; Fu, P.P.; Yu, H.; Ray, P.C. Theranostic nanomedicine for cancer detection and treatment. J. Food
Drug Anal. 2014, 22, 3–17. [CrossRef] [PubMed] 138. Xie, J.; Lee, S.; Chen, X. Nanoparticle-based theranostic agents. Adv. Drug Deliv. Rev. 2010, 62, 1064–1079. [CrossRef] [PubMed] 139. Ahmed, N.; Fessi, H.; Elaissari, A. Theranostic applications of nanoparticles in cancer. Drug Discov. Today
2012, 17, 928–934. [CrossRef] [PubMed] 140. Lammers, T.; Aime, S.; Hennink, W.E.; Storm, G.; Kiessling, F. Theranostic Nanomedicine. Acc. Chem. Res. 2011, 44, 1029–1038. [CrossRef] 21 of 23 21 of 23 Int. J. Mol. Sci. 2019, 20, 434 141. Lanoiselée, H.M.; Nicolas, G.; Wallon, D.; Rovelet-Lecrux, A.; Lacour, M.; Rousseau, S.; Richard, A.C.;
Pasquier, F.; Rollin-Sillaire, A.; Martinaud, O.; et al. APP, PSEN1, and PSEN2 mutations in early-onset
Alzheimer disease: A genetic screening study of familial and sporadic cases. PLoS Med. 2017, 14, e1002270. [CrossRef] [PubMed] 142. Klein, C.; Westenberger, A. Genetics of Parkinson’s disease. Cold Spring Harb. Perspect. Med. References Acceleration of α-Synuclein
Aggregation by Exosomes. J. Biol. Chem. 2015, 290, 2969–2982. [CrossRef] [PubMed] 156. Saman, S.; Kim, W.; Raya, M.; Visnick, Y.; Miro, S.; Saman, S.; Jackson, B.; McKee, A.C.; Alvarez, V.E.;
Lee, N.C.Y.; et al. Exosome-associated Tau Is Secreted in Tauopathy Models and Is Selectively Phosphorylated
in Cerebrospinal Fluid in Early Alzheimer Disease. J. Biol. Chem. 2012, 287, 3842–3849. [CrossRef] [PubMed] 157. Wei, Y.; Wang, D.; Jin, F.; Bian, Z.; Li, L.; Liang, H.; Li, M.; Shi, L.; Pan, C.; Zhu, D.; et al. Pyruvate kinase
type M2 promotes tumour cell exosome release via phosphorylating synaptosome-associated protein 23. Nat. Commun. 2017, 8, 14041. [CrossRef] [PubMed] 158. Yuyama, K.; Sun, H.; Usuki, S.; Sakai, S.; Hanamatsu, H.; Mioka, T.; Kimura, N.; Okada, M.; Tahara, H.;
Furukawa, J.; et al. A potential function for neuronal exosomes: Sequestering intracerebral amyloid-β
peptide. FEBS Lett. 2015, 589, 84–88. [CrossRef] [PubMed] 159. Cai, Z.Y.; Xiao, M.; Quazi, S.; Ke, Z.Y. Exosomes: A novel therapeutic target for Alzheimer’s disease? Neural Regen. Res. 2018, 13, 930. [CrossRef] [PubMed] 160. Katsuda, T.; Oki, K.; Ochiya, T. Potential application of extracellular vesicles of human adipose tissue-derived
mesenchymal stem cells in Alzheimer’s disease therapeutics. Methods Mol. Biol. 2015, 1212, 171–181. [CrossRef] 161. Feiler, M.S.; Strobel, B.; Freischmidt, A.; Helferich, A.M.; Kappel, J.; Brewer, B.M.; Li, D.; Thal, D.R.; Walther, P.;
Ludolph, A.C.; et al. TDP-43 is intercellularly transmitted across axon terminals. J. Cell Biol. 2015, 211,
897–911. [CrossRef] 22 of 23 Int. J. Mol. Sci. 2019, 20, 434 162. Iguchi, Y.; Eid, L.; Parent, M.; Soucy, G.; Bareil, C.; Riku, Y.; Kawai, K.; Takagi, S.; Yoshida, M.;
Katsuno, M.; et al. Exosome secretion is a key pathway for clearance of pathological TDP-43. Brain
2016, 139, 3187–3201. [CrossRef] Levy, E. Exosomes in the Diseased Brain: First Insights from In vivo Studies. Front. Neurosci. 2017, 11, 142
[CrossRef] 164. Danzer, K.M.; Kranich, L.R.; Ruf, W.P.; Cagsal-Getkin, O.; Winslow, A.R.; Zhu, L.; Vanderburg, C.R.;
McLean, P.J. Exosomal cell-to-cell transmission of alpha synuclein oligomers. Mol. Neurodegener. 2012, 7, 42. [CrossRef] 165. Gupta, A.; Pulliam, L. Exosomes as mediators of neuroinflammation. J. Neuroinflamm. 2014, 11, 68. [CrossRef]
[PubMed] 166. Emmanouilidou, E.; Melachroinou, K.; Roumeliotis, T.; Garbis, S.D.; Ntzouni, M.; Margaritis, L.H.;
Stefanis, L.; Vekrellis, K. Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by
exosomes and impacts neuronal survival. J. Neurosci. 2010, 30, 6838–6851. [CrossRef] [PubMed] 167. References Momen-Heravi, F.; Saha, B.; Kodys, K.; Catalano, D.; Satishchandran, A.; Szabo, G. Increased number of
circulating exosomes and their microRNA cargos are potential novel biomarkers in alcoholic hepatitis. J. Transl. Med. 2015, 13, 261. [CrossRef] [PubMed] 168. Ebrahimkhani, S.; Vafaee, F.; Young, P.E.; Hur, S.S.J.; Hawke, S.; Devenney, E.; Beadnall, H.; Barnett, M.H.;
Suter, C.M.; Buckland, M.E. Exosomal microRNA signatures in multiple sclerosis reflect disease status. Sci. Rep. 2017, 7, 14293. [CrossRef] [PubMed] 169. Hu, G.; Yao, H.; Chaudhuri, A.D.; Duan, M.; Yelamanchili, S.V.; Wen, H.; Cheney, P.D.; Fox, H.S.;
Buch, S. Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal
dysfunction. Cell Death Dis. 2012, 3, e381. [CrossRef] [PubMed] 170. Graziano, F.; Caruso Bavisotto, C.; Marino Gammazza, A.; Rappa, F.; Conway de Macario, E.; Macario, A.J.L.;
Cappello, F.; Campanella, C.; Maugeri, R.; Iacopino, D. Chaperonology: The Third Eye on Brain Gliomas. Brain Sci. 2018, 8, 110. [CrossRef] [PubMed] 171. Schiera, G.; Di Liegro, C.M.; Di Liegro, I. Molecular Determinants of Malignant Brain Cancers: From
Intracellular Alterations to Invasion Mediated by Extracellular Vesicles. Int. J. Mol. Sci. 2017, 18, 2774. [CrossRef] 172. Zhang, L.; Zhang, S.; Yao, J.; Lowery, F.J.; Zhang, Q.; Huang, W.-C.; Li, P.; Li, M.; Wang, X.; Zhang, C.; et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature
2015, 527, 100–104. [CrossRef] 173. Szajnik, M.; Czystowska, M.; Szczepanski, M.J.; Mandapathil, M.; Whiteside, T.L. Tumor-derived
microvesicles induce, expand and up-regulate biological activities of human regulatory T cells (Treg). PLoS ONE 2010, 5, e11469. [CrossRef] 174. Skog, J.; Würdinger, T.; van Rijn, S.; Meijer, D.H.; Gainche, L.; Sena-Esteves, M.; Curry, W.T.; Carter, B.S.;
Krichevsky, A.M.; Breakefield, X.O. Glioblastoma microvesicles transport RNA and proteins that promote
tumour growth and provide diagnostic biomarkers. Nat. Cell Biol. 2008, 10, 1470–1476. [CrossRef] 175. Van der Vos, K.E.; Abels, E.R.; Zhang, X.; Lai, C.; Carrizosa, E.; Oakley, D.; Prabhakar, S.; Mardini, O.;
Crommentuijn, M.H.W.; Skog, J.; et al. Directly visualized glioblastoma-derived extracellular vesicles
transfer RNA to microglia/macrophages in the brain. Neuro-Oncology 2016, 18, 58–69. [CrossRef] [PubMed] 176. De Vrij, J.; Maas, S.L.N.; Kwappenberg, K.M.C.; Schnoor, R.; Kleijn, A.; Dekker, L.; Luider, T.M.; de Witte, L.D.;
Litjens, M.; van Strien, M.E.; et al. Glioblastoma-derived extracellular vesicles modify the phenotype of
monocytic cells. Int. J. Cancer 2015, 137, 1630–1642. [CrossRef] [PubMed] 177. Al-Nedawi, K.; Meehan, B.; Micallef, J.; Lhotak, V.; May, L.; Guha, A.; Rak, J. References Intercellular transfer of the
oncogenic receptor EGFRvIII by microvesicles derived from tumour cells. Nat. Cell Biol. 2008, 10, 619–624. [CrossRef] [PubMed] 178. Graner, M.W.; Alzate, O.; Dechkovskaia, A.M.; Keene, J.D.; Sampson, J.H.; Mitchell, D.A.; Bigner, D.D. Proteomic and immunologic analyses of brain tumor exosomes. FASEB J. 2009, 23, 1541–1557. [CrossRef]
[PubMed] 179. Katakowski, M.; Buller, B.; Zheng, X.; Lu, Y.; Rogers, T.; Osobamiro, O.; Shu, W.; Jiang, F.; Chopp, M. Exosomes from marrow stromal cells expressing miR-146b inhibit glioma growth. Cancer Lett. 2013, 335,
201–204. [CrossRef] [PubMed] 23 of 23 Int. J. Mol. Sci. 2019, 20, 434 23 of 23 180. Yang, T.; Martin, P.; Fogarty, B.; Brown, A.; Schurman, K.; Phipps, R.; Yin, V.P.; Lockman, P.; Bai, S. Exosome
delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio. Pharm. Res. 2015, 32, 2003–2014. [CrossRef] 81. Gomari, H.; Forouzandeh Moghadam, M.; Soleimani, M. Targeted cancer therapy using engineered exos
as a natural drug delivery vehicle. OncoTargets Ther. 2018, 11, 5753–5762. [CrossRef] 182. Jia, G.; Han, Y.; An, Y.; Ding, Y.; He, C.; Wang, X.; Tang, Q. NRP-1 targeted and cargo-loaded exosomes
facilitate simultaneous imaging and therapy of glioma in vitro and in vivo. Biomaterials 2018, 178, 302–316. [CrossRef] 183. Yao, Y.; Chen, L.; Wei, W.; Deng, X.; Ma, L.; Hao, S. Tumor cell-derived exosome-targeted dendritic cells
stimulate stronger CD8+ CTL responses and antitumor immunities. Biochem. Biophys. Res. Commun. 2013,
436, 60–65. [CrossRef] 184. Munoz, J.L.; Bliss, S.A.; Greco, S.J.; Ramkissoon, S.H.; Ligon, K.L.; Rameshwar, P. Delivery of Functional
Anti-miR-9 by Mesenchymal Stem Cell-derived Exosomes to Glioblastoma Multiforme Cells Conferred
Chemosensitivity. Mol. Ther. Nucleic Acids 2013, 2, e126. [CrossRef] 185. Zeng, A.; Wei, Z.; Yan, W.; Yin, J.; Huang, X.; Zhou, X.; Li, R.; Shen, F.; Wu, W.; Wang, X.; et al. Exosomal
transfer of miR-151a enhances chemosensitivity to temozolomide in drug-resistant glioblastoma. Cancer Lett. 2018, 436, 10–21. [CrossRef] 186. Xin, H.; Li, Y.; Cui, Y.; Yang, J.J.; Zhang, Z.G.; Chopp, M. Systemic administration of exosomes released from
mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J. Cereb. Blood Flow Metab. 2013, 33, 1711–1715. [CrossRef] [PubMed] 187. Yu, Y.M.; Gibbs, K.M.; Davila, J.; Campbell, N.; Sung, S.; Todorova, T.I.; Otsuka, S.; Sabaawy, H.E.; Hart, R.P.;
Schachner, M. MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult
zebrafish. Eur. J. Neurosci. 2011, 33, 1587–1597. [CrossRef] [PubMed] 188. References Tian, T.; Zhang, H.X.; He, C.P.; Fan, S.; Zhu, Y.L.; Qi, C.; Huang, N.P.; Xiao, Z.D.; Lu, Z.H.; Tannous, B.A.;
Gao, J. Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy. Biomaterials 2018, 150, 137–149. [CrossRef] [PubMed] 189. Yuan, D.; Zhao, Y.; Banks, W.A.; Bullock, K.M.; Haney, M.; Batrakova, E.; Kabanov, A.V. Macrophage
exosomes as natural nanocarriers for protein delivery to inflamed brain. Biomaterials 2017, 142, 1–12. [CrossRef] 190. Hebert, L.E.; Weuve, J.; Scherr, P.A.; Evans, D.A. Alzheimer disease in the United States (2010-2050) estimated
using the 2010 census. Neurology 2013, 80. [CrossRef] 191. Morse, M.A.; Garst, J.; Osada, T.; Khan, S.; Hobeika, A.; Clay, T.M.; Valente, N.; Shreeniwas, R.; Sutton, M.A.;
Delcayre, A.; et al. A phase I study of dexosome immunotherapy in patients with advanced non-small cell
lung cancer. J. Transl. Med. 2005, 3, 9. [CrossRef] 192. Besse, B.; Charrier, M.; Lapierre, V.; Dansin, E.; Lantz, O.; Planchard, D.; Le Chevalier, T.; Livartoski, A.;
Barlesi, F.; Laplanche, A.; et al. Dendritic cell-derived exosomes as maintenance immunotherapy after first
line chemotherapy in NSCLC. Oncoimmunology 2015, 5, e1071008. [CrossRef] © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
|
W3094519585.txt
|
https://www.mdpi.com/2223-7747/9/11/1417/pdf?version=1604315760
|
en
|
Strawberry FaNAC2 Enhances Tolerance to Abiotic Stress by Regulating Proline Metabolism
|
Plants
| 2,020
|
cc-by
| 9,047
|
plants
Article
Strawberry FaNAC2 Enhances Tolerance to Abiotic
Stress by Regulating Proline Metabolism
Jiahui Liang 1 , Jing Zheng 1 , Ze Wu 2
1
2
*
and Hongqing Wang 1, *
Department of Fruit Science, College of Horticulture, China Agricultural University, Beijing 100193, China;
bs20173170791@cau.edu.cn (J.L.); sy20193172524@cau.edu.cn (J.Z.)
Key Laboratory of Landscaping Agriculture, Ministry of Agriculture and Rural Affairs, College of
Horticulture, Nanjing Agricultural University, Nanjing 210095, China; wuze@njau.edu.cn
Correspondence: wanghq@cau.edu.cn; Tel.: +86-13683018901
Received: 23 September 2020; Accepted: 21 October 2020; Published: 23 October 2020
Abstract: The quality and yields of strawberry plants are seriously affected by abiotic stress every
year. NAC (NAM, ATAF, CUC) transcription factors are plant-specific, having various functions in
plant development and response to stress. In our study, FaNAC2 from strawberry (Fragaria × ananassa,
cultivar “Benihoppe”) was isolated and found to be a member of the ATAF sub-family, belonging to the
NAC family of transcription factors. FaNAC2 was strongly expressed in the shoot apical meristem and
older leaves of strawberries, and was induced by cold, high salinity, and drought stress. To investigate
how FaNAC2 functions in plant responses to abiotic stress, transgenic Nicotiana benthamiana plants
ectopically overexpressing FaNAC2 were generated. The transgenic plants grew better under salt
and cold stress, and, during simulated drought treatment, these transgenic lines not only grew
better, but also showed higher seed germination rates than wild-type plants. Gene expression
analysis revealed that key genes in proline biosynthesis pathways were up-regulated in FaNAC2
overexpression lines, while its catabolic pathway genes were down-regulated and proline was
accumulated more with the overexpression of FaNAC2 after stress treatments. Furthermore, the gene
expression of abscisic acid biosynthesis was also promoted. Our results demonstrate that FaNAC2
plays an important positive role in response to different abiotic stresses and may be further utilized to
improve the stress tolerance of strawberry plants.
Keywords: strawberry; ATAF; FaNAC2; abiotic stress
1. Introduction
Agricultural crops grow in a constantly changing environment and are often subjected to abiotic
stresses such as drought, heat, cold, and high salinity. These stresses are associated with increased
accumulation of certain deleterious chemicals like reactive oxygen species (ROS), which affect the
stability of cell membranes and the structure of proteins, finally leading to reduced crop yield and
even death [1,2]. To adapt to environmental stress and complete their life cycle, plants have evolved
a complex mechanism that tightly regulates gene expression through precise signaling. Until now,
many stress-induced proteins including transcription factors (TFs), osmotic stress-adaptive proteins
and key enzymes in abscisic acid (ABA) biosynthesis and signaling pathways have been reported [3–5].
The NAC [No apical meristem (NAM), Arabidopsis transcription activation factor (ATAF),
and Cup-shaped cotyledon (CUC)] superfamily is one of the largest groups of plant-specific TFs,
which not only play an important role in various stages of plant growth and development, but also
participate in responses to biotic and abiotic stress [6–10]. Many NAC genes have been identified
through their function in response to drought, cold, and high salinity stress [5]. Drought-induced
genes ANAC019, ANAC055, and RD26/ANAC072, when overexpressed in Arabidopsis, improved
Plants 2020, 9, 1417; doi:10.3390/plants9111417
www.mdpi.com/journal/plants
Plants 2020, 9, 1417
2 of 17
drought tolerance [11,12]. Overexpression of SlNAM1 from tomato could improve chilling tolerance of
transgenic tobacco [13]. Besides, it has also been shown that OsNAC6 (from rice), SNAC2 (from rice),
TaNAC4 (from wheat), TaNAC8 (from wheat), and CarNAC1 (from chickpea), function as transcriptional
activators in response to various abiotic stresses [9,14–17].
The ATAF TFs comprise a sub-family of NAC proteins. The first report of a stress-inducible
ATAF-like gene was StNAC from potato [18]. Elicited ATAF1 and ATAF2 are considered to function
as repressors of responsive genes under biotic and abiotic stress, because ataf1 and ataf2 mutants
showed high stress resistance [19–21]. However, overexpression of ATAF1 in Arabidopsis also
exhibited enhanced plant tolerance to drought [22]. Subsequently, more ATAF family members
from different species were explored in response to abiotic stress. OsNAC52 from rice belongs
to the ATAF sub-family, and functions as an important transcriptional activator in ABA-inducible
gene expression [23]. GmNAC2 plays a negative regulatory role in abiotic stress in Glycine max,
and participates in the ROS signaling pathway by regulating the expression of ROS removal genes [24].
Overexpression of SlNAC2 (from tomato) in Arabidopsis resulted in enhanced tolerance to salinity
stress [25]. In addition, SlNAC11 from tomato plays a stress-inducible TF role, depicting a positive
response to abiotic stress tolerance [26]. DgNAC1 from chrysanthemum worked as a positive regulator
in responses to salt stress, as its overexpression in transgenic chrysanthemum showed lower levels
of MDA (malondialdehyde) and reactive oxygen species (H2 O2 and O2− ), greater activities of SOD
(superoxide dismutase), POD (peroxidase) and CAT (catalase), as well as more proline content than
wild-type (WT) under salt stress [27]. In addition, it was reported that CsATAF1 (from cucumber) was
a crucial activator of the drought stress response via an ABA-dependent pathway, and inhibited ROS
accumulation [28].
Strawberries are one of the most economically valuable crops in the world, which often suffers
from water deficit, high salinity, flooding, and extreme temperature, leading to yield reduction [29–31].
Although many studies on responses of strawberry plants to abiotic stress have been reported, few data
have showed NAC family members participating in abiotic stress resistance. Zhang et al. [31] showed
that there are five FvNAC genes significantly contributing to various abiotic and biotic stress responses
in woodland strawberry (Fragaria vesca), but their regulatory mechanisms are largely unknown.
In our study, FaNAC2 was isolated from the cultivar strawberry (Fragaria × ananassa, cultivar
“Benihoppe”); it was highly expressed in shoot apical meristem and old leaves of strawberry plants,
and showed especially high expression in the guard cells of leaves. FaNAC2 was induced under abiotic
stress treatment, and many cis-acting elements that are responsive to abiotic stress were predicted in
the FaNAC2 promoter. We overexpressed FaNAC2 in Nicotiana benthamiana (N. benthamiana), and found
that the transgenic plants showed higher drought, cold and salt tolerance. These results indicated that
FaNAC2 might play a positive role in plant responses to abiotic stress.
2. Results
2.1. FaNAC2 Encodes an ATAF Protein that Belongs to the NAC Family
FaNAC2 encodes a protein of 289 amino acids, and is a member of a plant-specific NAC family
of transcription factors. We used the cDNA sequence of FaNAC2 as a query to perform a BLAST
search against 122 NAC family members of Arabidopsis, using MEGA 7.0 software. It was found
that FaNAC2 has the closest relationship with AT1G01720, which encodes an ATAF sub-group protein,
and is also named AtNAC2 (Figure 1A). A multiple sequence alignment of ATAF homologues from
Arabidopsis, rose, Suaeda liaotungensis, soybean, cucumber and strawberry was performed. As shown in
Figure 1B, high sequence similarities between FaNAC2 and other plant ATAF proteins were found in
the N-terminus, which contained several distinguishable conserved domains, and five sub-domains.
These results indicated that FaNAC2 encoded an ATAF1 protein and was a typical member of the NAC
transcription factors.
Plants 2020, 9, x FOR PEER REVIEW
3 of 17
domains. These results indicated that FaNAC2 encoded an ATAF1 protein and was a typical member
3 of 17
of the NAC transcription factors.
Plants 2020, 9, 1417
Figure
Figure 1.1. Phylogenetic
Phylogenetic analysis
analysis and
and amino
amino acid
acidsequence
sequencealignment
alignmentof
ofFaNAC2.
FaNAC2. (A)
(A) Phylogenetic
Phylogenetic
relationship
relationshipbetween
betweenFaNAC2
FaNAC2(red
(redboxed)
boxed)from
from strawberry
strawberry and
and NAC
NAC family
family members
members in
in Arabidopsis.
Arabidopsis.
MEGA
MEGA 7.0
7.0 software
software was
was used
used totoconstruct
constructthe
theNeighbor-Joining
Neighbor-Joiningtree.
tree. The
The nearest
nearest NAC
NAC gene
gene isis
AT1G01720
AT1G01720(AtATAF1
(AtATAF1or
orAtNAC2).
AtNAC2).(B)
(B)Protein
Proteinsequence
sequencealignment
alignment of
ofATAFs.
ATAFs.The
Theblack
blackunderlines
underlines
indicate
indicatethe
theconserved
conservedN-terminal
N-terminaldomain
domainof
ofNAC
NACfamily.
family.A–E
A–E represent
representfive
fiveconserved
conservedsub-domains.
sub-domains.
Accession
Numbers:
Rosa
hybrid
cultivar,
RhATAF1
(AXT99858.1);
Arabidopsis
Accession Numbers: Rosa hybrid cultivar, RhATAF1 (AXT99858.1); Arabidopsis thaliana,
thaliana, AtATAF1
AtATAF1
(AT1G01720),
(AT1G01720), AtATAF2
AtATAF2(AT5G08790);
(AT5G08790);Suaeda
Suaedaliaotungensis
liaotungensisK.,
K., SlNAC2
SlNAC2 (JX860282.1);
(JX860282.1); Glycine
Glycine max,
max,
GmATAF1-like/GmNAC2
GmATAF1-like/GmNAC2(AAX85979.1);
(AAX85979.1);Cucumis
Cucumissativus
sativusL.,
L.,CsATAF1
CsATAF1(Csa4M361820.1).
(Csa4M361820.1).
2.2. Expression Pattern of FaNAC2
2.2. Expression Pattern of FaNAC2
To explore the function of FaNAC2, we first analyzed its spatial and temporal expression patterns
To explore the function of FaNAC2, we first analyzed its spatial and temporal expression
in strawberry plants. The qRT-PCR (quantitative RT-PCR) results showed that FaNAC2 was expressed
patterns in strawberry plants. The qRT-PCR (quantitative RT-PCR) results showed that FaNAC2 was
at higher levels in shoot apical meristem, old leaves and flowers, compared to roots and fruits
expressed at higher levels in shoot apical meristem, old leaves and flowers, compared to roots and
(Figure 2A). For different stages of leaves, the expression of FaNAC2 in older leaves was more than
fruits (Figure 2A). For different stages of leaves, the expression of FaNAC2 in older leaves was more
that in younger leaves and mature leaves, indicating that the accumulation of FaNAC2 might increase
than that in younger leaves and mature leaves, indicating that the accumulation of FaNAC2 might
with the senescence of leaves. Amongst the different sizes of flower buds, FaNAC2 had a high level of
increase with the senescence of leaves. Amongst the different sizes of flower buds, FaNAC2 had a
expression in the late petals and early stages of pistils (Figure 2A).
high level of expression in the late petals and early stages of pistils (Figure 2A).
Plants 2020, 9, 1417
Plants 2020, 9, x FOR PEER REVIEW
4 of 17
4 of 17
Figure
2. Expression
pattern
(A) qRT-PCR
qRT-PCRanalysis
analysis
FaNAC2
in different
tissues
of
Figure
2. Expression
patternofofFaNAC2.
FaNAC2. (A)
of of
FaNAC2
in different
tissues
of
strawberry.
ROOT:
roots;SAM:
SAM: shoot
shoot apical
meristem;
YL: YL:
young
leavesleaves
(the second
ML: leaf);
strawberry.
ROOT:
roots;
apical
meristem;
young
(the folded
secondleaf);
folded
mature leaves
third
or or
fourth
fullyfully
expanded
leaf); OL:
old
leaves
seventh
SPE: leaf);
ML: mature
leaves(the
(the
third
fourth
expanded
leaf);
OL:
old(below
leavesthe
(below
theleaf);
seventh
flower
buds
(length
<0.5 cm);
middle
flower
buds
(length
0.5–0.8
SPE: petals
petalsfrom
fromsmall
small
flower
buds
(length
< 0.5MPE:
cm);petals
MPE:from
petals
from
middle
flower
buds
(length
cm);
LPE:
petals
from
large
opened
flower;
SST:
stamen
from
small
flower
buds;
MST:
stamen
from
0.5–0.8 cm); LPE: petals from large opened flower; SST: stamen from small flower buds; MST: stamen
middle flower buds; LST: stamen from large opened flower; MPI: pistils from middle flower buds;
from middle flower buds; LST: stamen from large opened flower; MPI: pistils from middle flower
LPI: pistils from large flower buds; LSE: sepals from large opened flower; SRE: receptacles from small
buds; LPI: pistils from large flower buds; LSE: sepals from large opened flower; SRE: receptacles
flower buds; MRE: receptacles from middle flower buds; LRE: receptacles from large opened flower;
from small flower buds; MRE: receptacles from middle flower buds; LRE: receptacles from large
SGF: small green fruits; MGF: middle green fruits; LGF: large green fruits; RF: red fruits. The FaACTIN
opened
SGF:
greenreference
fruits; MGF:
middle the
green
fruits; LGF:
green
fruits; RF:
geneflower;
was used
as small
an internal
to normalize
expression
data. large
Data are
presented
as red
fruits.
The FaACTIN
gene wasrepeats.
used as
an are
internal
normalize
the expression
averages
of three biological
Bars
meansreference
(±S.D.) of to
three
independent
experiments.data.
(B,C) Data
are presented
as averages
of threeofbiological
repeats.
Bars are means
(±S.D.) of three
independent
β-glucuronidase
(GUS) analysis
5 DAG (days
after germination)
ProFaNAC2–GUS
N. benthamiana
experiments.
β-glucuronidase
(GUS)GUS
analysis
of 5ofDAG
(days
after
germination)
(Nicotiana(B,C)
benthamiana)
seedlings. (D,E)
analysis
true leaf
of 13
DAG
seedling andProFaNAC2–GUS
its enlarged
view. The red
arrow points
to guard cells
in (E). (F,G)
analysis
of stigma
35 DAG
N. benthamiana
(Nicotiana
benthamiana)
seedlings.
(D,E)GUS
GUS
analysis
of trueand
leafanther
of 13of
DAG
seedling
ProFaNAC2–GUS
lines
after
on soil. to guard cells in (E). (F,G) GUS analysis of stigma and
and its
enlarged view.
The
redplanting
arrow points
anther of 35 DAG ProFaNAC2–GUS lines after planting on soil.
To better understand the expression pattern of FaNAC2, the transgenic N. benthamiana contained
a β-glucuronidase
(GUS) the
reporter
gene which,
under
the controlthe
of transgenic
FaNAC2 promoter,
was produced
To
better understand
expression
pattern
of FaNAC2,
N. benthamiana
contained
and detected. The results revealed that the GUS signals were detected in cotyledons and true leaves
a β-glucuronidase (GUS) reporter gene which, under the control of FaNAC2 promoter, was produced
and detected. The results revealed that the GUS signals were detected in cotyledons and true leaves
of young plants (Figure 2B,C), and it showed strong expression in the guard cells of true leaves
Plants 2020, 9, 1417
5 of 17
(Figure 2D,E), suggesting that FaNAC2 might have a function in controlling stoma. In addition,
GUS expression was also be seen in the stigmas and anthers of the flowers (Figure 2F,G).
2.3. FaNAC2 Is Induced by Cold, Salinity, and Drought Treatment
Since many cis-elements related to abiotic stress, such as ABRE (response to abscisic acid),
LTR (response to cold) and MBS (response to drought), were found on the promoter sequence
of FaNAC2 (Table 1), we hypothesized that FaNAC2 would also respond to abiotic stress. Thus,
we performed stress treatments, including 200 mM NaCl, 20% polyethylene glycol (PEG) 6000,
and 4 ◦ C, to detect the expression changes of FaNAC2 using 3 MAC (months after cutting node bud
from runners) tissue cultured seedlings of strawberry for qRT-PCR assay. The results revealed that its
expression was strongly up-regulated in both SAM (shoot apical meristem) and leaves in response to
abiotic stress (Figure 3A,B). During cold and salt treatment, the expression of FaNAC2 in the SAM
was induced at 3 h and then gradually decreased compared to the initial expression levels, while the
expression of FaNAC2 from the leaves was a little slower, being induced only at 6 h following stress.
Under the condition of simulated drought treatment, FaNAC2 showed a great difference in expression
pattern in different tissues; although FaNAC2 was induced at 9 h, the expression of FaNAC2 in the SAM
was gradually decreased from 12 h after induction, while FaNAC2 expression in the leaves increased at
all the times tested, which indicated that FaNAC2 might participate in drought stress response, mainly
in the leaves.
Table 1. Predicted cis-elements in the promoter of FaNAC2.
Cis-elements
Sequence
Number
Character
ABRE
ARE
Box-4
CGTCA-motif
G-box
GCN4-motif
LTR
MBS
RY-element
TCA-element
TGA-element
ACGTG
AAACCA
ATTAAT
CGTCA
CACGTG
TGAGTCA
CCGAAA
CAACTG
CATGCATG
TCAGAAGAGG
AACGAC
4 (+)
1 (+)
1 (+)
3 (+)
7 (+)
1 (+)
1 (+)
1 (+)
1 (+)
2 (+)
2 (+)
Response to abscisic acid
Response to anaerobic process
Response to light
Response to Jasmonic Acid
Response to light reaction
Involved in endosperm expression
Response to cold
Response to drought
Specific seeds’ regulation
Response to salicylic acid
Response to auxin
To confirm the expression pattern, different stress treatments were performed at 7 DAG (days
after germination) of ProFaNAC2–GUS N. benthamiana, and the results showed that the GUS expression
became stronger under cold, simulated drought and salt stress, indicating that FaNAC2 could be
induced by different abiotic stresses (Figure 3C). Taking all these results together, it could be inferred
that FaNAC2 might function in different tissues to cope with different stress conditions.
2.4. Overexpression of FaNAC2 Improves Stress Tolerance in Transgenic N. benthamiana
To further investigate how FaNAC2 plays roles in abiotic stress resistance, FaNAC2 was ectopically
transformed into N. benthamiana under the control of a CaMV (Cauliflower Mosaic Virus)–35S promoter,
and ten positive lines were obtained through screening using kanamycin and RT-PCR analysis.
By observing the growth potential of germination of 13 DAG 35S::FaNAC2 and wild-type lines under
salt treatments, we found that 35S::FaNAC2 lines exhibited better growth under salt stress; for example,
the leaf areas of transgenic plants were larger than WT following salt treatments (Figure 4A,B),
indicating that overexpression of FaNAC2 promoted the salt tolerance of plants.
Plants 2020, 9, 1417
Plants 2020, 9, x FOR PEER REVIEW
6 of 17
6 of 17
Figure 3. FaNAC2 is induced by cold, drought, and salinity stress. (A) The expression level of FaNAC2
Figure(shoot
3. FaNAC2
is induced
by cold,
drought, and
salinity
stress. (A)
The expression
level
of FaNAC2
in the SAM
apical
meristem)
of strawberry
under
different
abiotic
stresses. (B)
The
expression
in
the
SAM
(shoot
apical
meristem)
of
strawberry
under
different
abiotic
stresses.
(B)
The
level of FaNAC2 in the leaves of strawberry under different abiotic stresses. (C) Differentexpression
abiotic stress
level of FaNAC2 in the leaves of strawberry under different abiotic stresses. (C) Different abiotic stress
treatments
to 7 DAG (days after germination) ProFaNAC2–GUS seedlings. Salinity treatment used
treatments to 7 DAG (days after germination) ProFaNAC2–GUS seedlings. Salinity treatment used
200 mM NaCl. Three independent experiments were performed and error bars indicate standard
200 mM NaCl. Three independent experiments were performed and error bars indicate standard
deviation (Student’s t–test; * p < 0.05; ** p < 0.01). The scale bar represents 1 cm.
deviation (Student’s t–test; * p < 0.05; ** p < 0.01). The scale bar represents 1 cm.
ectopically transformed into N. benthamiana under the control of a CaMV (Cauliflower Mosaic Virus)–
35S promoter, and ten positive lines were obtained through screening using kanamycin and RT-PCR
analysis. By observing the growth potential of germination of 13 DAG 35S::FaNAC2 and wild-type
lines under salt treatments, we found that 35S::FaNAC2 lines exhibited better growth under salt stress;
for example, the leaf areas of transgenic plants were larger than WT following salt treatments (Figure
Plants 2020, 9, 1417
7 of 17
4A,B), indicating that overexpression of FaNAC2 promoted the salt tolerance of plants.
Figure
4. Comparison
of resistance
to salinity
35S::FaNAC2and
andwild-type
wild-type (WT)
Figure
4. Comparison
of resistance
to salinity
ofof
35S::FaNAC2
(WT)lines.
lines.(A)
(A)Analysis
Analysis of
of
13
DAG
seedlings
of
35S::FaNAC2
and
WT
seeds
under
salt
and
control
treatments.
OX:
13 DAG seedlings of 35S::FaNAC2 and WT seeds under salt and control treatments. OX: overexpression.
overexpression.
(B)
Leaf
area
of
35S::FaNAC2
and
WT
13
DAG
seedlings
under
salt
and
control
(B) Leaf area of 35S::FaNAC2 and WT 13 DAG seedlings under salt and control treatments. (C–E) Enzyme
treatments. (C–E) Enzyme activity of several enzymes related to plant resistance to salt. CAT: Catalase;
activity
of several enzymes related to plant resistance to salt. CAT: Catalase; POD: Peroxidase; SOD:
Superoxide dismutase. The scale bar represents 1 cm. Bars are means (±S.D.) of three independent
experiments (Student’s t–test; * p < 0.05; ** p < 0.01).
As the plants get stronger, the 40 DAG 35S::FaNAC2 lines and WT plants were irrigated with
300 mM NaCl for one week, with normal watering as a control. By comparing the enzyme activity of
antioxidant enzymes including catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD)
from two lines under different conditions, it was found that CAT enzyme activity in 35S::FaNAC2 lines
was significantly higher than that in WT lines under control conditions, but there was no significant
difference after salt treatment (Figure 4C). POD activity of 35S::FaNAC2 plants was higher than that of
WT lines under both control and salt treatment, while there was no significant change in SOD enzyme
activity (Figure 4D,E). These results suggested that FaNAC2 might promote plant salt tolerance by
partially affecting the activity of some antioxidant enzymes.
Plants 2020, 9, 1417
8 of 17
Through different treatments for seed germination, we found that there was no difference in seed
germination rates between 35S::FaNAC2 and WT lines under normal conditions. However, the seeds
of 35S::FaNAC2 lines had a 10% higher germination rates than that of WT plants under the treatment
of simulated drought (10% PEG 6000; Figure 5A,B). Subsequently, we conducted drought treatment on
40 DAG 35S::FaNAC2 and WT plants; 35S::FaNAC2 plants had a higher recovery rate after rehydration
(Figure 5C). Comparing the WLR (water loss rate) from leaves of the two lines, it was found that
the dryness of 35S::FaNAC2 leaves was lower, suggesting that they retained leaf water more easily
(Figure 5D). Besides, a key gene for ABA biosynthesis, NbNCED1 (9-cis-epoxycarotenoid dioxygenase 1),
was also detected in both the normal and drought treatment conditions, and NbNCED1 expression in
35S::FaNAC2 lines was significantly higher than that of WT (Figure 5E). Taking all these results together,
our data suggested that FaNAC2 might exist as a positive regulator of drought stress tolerance.
Figure 5. Comparison the rehydration rate of 35S::FaNAC2 and WT lines under drought stress.
(A,B) Germination rates of 35S::FaNAC2 and WT seeds under water and simulated drought conditions.
(C) Phenotypes of 40 DAG 35S::FaNAC2 and WT lines under control and rehydration conditions.
(D) Water loss rate (WLR) of leaves from 35S::FaNAC2 and WT lines. (E) The qRT-PCR analysis of
NbNCED1 expression, related to ABA biosynthesis pathways. The scale bar represents 1 cm. Bars are
means (± S.D.) of three independent experiments (Student’s t-test; * p < 0.05; ** p < 0.01).
In order to understand the function of FaNAC2 in cold stress, 35S::FaNAC2 lines and WT plants
were subjected to cold treatment, which was performed as follows: 4 ◦ C for 2 h, 0 ◦ C for 1 h, −5 ◦ C
for 1 h and 4 ◦ C for 1 h. The results showed that 35S::FaNAC2 lines were more cold-resistant than
WT plants, showing less damage (Figure 6A). Meanwhile, a key gene, NbNPK1 (Nicotiana protein
kinase 1), which was involved in cold resistance signal transmission in plants, was found to show
increased expression in 35S::FaNAC2 lines compared to WT plants under control treatment, while it
was significantly higher than that in WT plants under cold treatment (Figure 6B). All of these results
demonstrated that FaNAC2 might play a positive role in response to abiotic stress.
2.5. FaNAC2 Promotes Plant Abiotic Stress Tolerance via Regulating Proline Metabolism
To further investigate how FaNAC2 regulates plant stress tolerance, we detected the expression
changes of key genes in proline biosynthesis, NbP5CS1 (Pyrroline-5 carboxylate synthetase 1),
and catabolism, NbP5CDH (P5C dehydrogenase) and NbproDH2 (Proline dehydrogenase 2) from
35S::FaNAC2 and WT plants (Figure 7). Compared with WT, NbP5CS1 expression from 35S::FaNAC2
lines was increased either in control or salt stress conditions (Figure 7A,B). NbproDH2 expression
Plants 2020, 9, x FOR PEER REVIEW
Plants 2020, 9, 1417
9 of 17
9 of 17
plants, showing less damage (Figure 6A). Meanwhile, a key gene, NbNPK1 (Nicotiana protein kinase 1),
which was involved in cold resistance signal transmission in plants, was found to show increased
wasexpression
also higher
in transgenic lines than WT in control conditions, while its expression significantly
in 35S::FaNAC2 lines compared to WT plants under control treatment, while it was
decreased
after
salt
treatment.
expression
of another
gene in(Figure
the proline
catabolism
significantly higher
than thatThe
in WT
plants under
cold key
treatment
6B). All
of these pathway,
results
NbP5CDH,
in
35S::FaNAC2
lines
was
also
down-regulated
after
salt
treatment
(Figure
7B).
demonstrated that FaNAC2 might play a positive role in response to abiotic stress.
Figure
6. Comparison
of 35S::FaNAC2
and
WT under
plantscold
under
cold
(A) Phenotypes
of
Figure
6. Comparison
of 35S::FaNAC2
and WT
plants
stress.
(A)stress.
Phenotypes
of 35S::FaNAC2
35S::FaNAC2
lines
and
WT
under
control
and
cold
conditions.
(B)
Expression
of
NbNPK1
related
to
lines and WT under control and cold conditions. (B) Expression of NbNPK1 related to plant resistance
plantpathways
resistance in
to 35S::FaNAC2
cold pathwayslines
in 35S::FaNAC2
linescold
andtreatment.
WT under cold
treatment.
The scale bar
to cold
and WT under
The scale
bar represents
1 cm.
represents
are
means
(±S.D.) of three
independent
experiments
(Student’s
t-test; ** p 10
< 0.01).
Bars
are 2020,
means
(±S.D.)
three
independent
experiments
(Student’s
t-test;
** p < 0.01).
Plants
9,1 xcm.
FORBars
PEERof
REVIEW
of 17
2.5. FaNAC2 Promotes Plant Abiotic Stress Tolerance via Regulating Proline Metabolism
To further investigate how FaNAC2 regulates plant stress tolerance, we detected the expression
changes of key genes in proline biosynthesis, NbP5CS1 (Pyrroline-5 carboxylate synthetase 1), and
catabolism, NbP5CDH (P5C dehydrogenase) and NbproDH2 (Proline dehydrogenase 2) from 35S::FaNAC2
and WT plants (Figure 7). Compared with WT, NbP5CS1 expression from 35S::FaNAC2 lines was
increased either in control or salt stress conditions (Figure 7A,B). NbproDH2 expression was also
higher in transgenic lines than WT in control conditions, while its expression significantly decreased
after salt treatment. The expression of another key gene in the proline catabolism pathway, NbP5CDH,
in 35S::FaNAC2 lines was also down-regulated after salt treatment (Figure 7B).
Figure 7. The qRT-PCR analysis of several genes related to proline matabolism in 35S::FaNAC2 lines
Figure 7. The qRT-PCR analysis of several genes related to proline matabolism in 35S::FaNAC2 lines
and WTand
under
abiotic
stress
treatments.
genes
in 35S::FaNAC2
lines and
WT different
under different
abiotic
stress
treatments.(A)
(A) Expression
Expression ofof
genes
in 35S::FaNAC2
lines and
WT under
conditions.
(B) (B)
Expression
in35S::FaNAC2
35S::FaNAC2
lines
WT under
salt stress.
WTcontrol
under control
conditions.
Expressionofofgenes
genes in
lines
and and
WT under
salt stress.
(C) Expression
of genes
in 35S::FaNAC2
lines
and
WT
underdrought
drought stress.
Expression
of genes
(C) Expression
of genes
in 35S::FaNAC2
lines
and
WT
under
stress.(D)
(D)
Expression
of genes in
in 35S::FaNAC2
lines
WT under
stress.
Bars
aremeans
means (±S.D.)
of of
three
biological
replicantes
35S::FaNAC2
lines and
WTand
under
cold cold
stress.
Bars
are
(±S.D.)
three
biological
replicantes
experiments (Student’s t-test; * p < 0.05; ** p < 0.01).
experiments (Student’s t-test; * p < 0.05; ** p < 0.01).
Following drought treatment, the expression of NbP5CS1 in 35S::FaNAC2 was significantly
higher than that of WT, while the expression of NbP5CDH and NbproDH2 was decreased compared
with that of WT (Figure 7C).
Similar to the two previous stress treatments, further analysis revealed that the expression of
NbP5CS1 was up-regulated in 35S::FaNAC2 lines under both control and cold treatment. The
NbproDH2 expression of 35S::FaNAC2 lines was more than five times as high as that in WT plants
Plants 2020, 9, 1417
10 of 17
Following drought treatment, the expression of NbP5CS1 in 35S::FaNAC2 was significantly higher
than that of WT, while the expression of NbP5CDH and NbproDH2 was decreased compared with that
of WT (Figure 7C).
Similar to the two previous stress treatments, further analysis revealed that the expression of
NbP5CS1 was up-regulated in 35S::FaNAC2 lines under both control and cold treatment. The NbproDH2
expression of 35S::FaNAC2 lines was more than five times as high as that in WT plants under the control
treatment, whereas it was twice as high as that in WT plants under the cold stress, suggesting that
NbproDH2 of 35S::FaNAC2 was decreased during the cold resistance compared to control condition.
Meanwhile, NbP5CDH expression was relatively high in the control treatment, but lower in the WT
plants after cold treatment (Figure 7D).
In addition, proline levels were detected under control and abiotic stress. It was found that
proline content in 35S::FaNAC2 lines was slightly higher than WT plants in the control environment.
With different abiotic stress treatments on the plants, proline content in 35S::FaNAC2 strains was
Plants 2020, 9, x FOR PEER REVIEW
11 of 17
significantly higher than WT strains (Figure 8). Due to both the proline biosynthesis gene and
the proline catabolism gene being up-regulated under normal conditions, it was speculated that
might promote proline metabolism to sustain proline hemostasis for normal growth of transgenic
FaNAC2 might promote proline metabolism to sustain proline hemostasis for normal growth of
plants, but FaNAC2 could improve proline levels, in response to different abiotic stresses, for
transgenic plants, but FaNAC2 could improve proline levels, in response to different abiotic stresses,
increased tolerance.
for increased tolerance.
Figure 8. The proline content in 35S::FaNAC2 lines and WT leaves under different abiotic stress
Figure
8. TheThe
proline
content
in 35S::FaNAC2
lines
and WT
leaves underBars
different
abiotic
stressof
treatments.
fifth leaf
was collected
for proline
content
determination.
are means
(±S.D.)
treatments.
The
fifth
leaf
was
collected
for
proline
content
determination.
Bars
are
means
(±S.D.)
of
three independent experiments (Student’s t-test; * p < 0.05; ** p < 0.01).
three independent experiments (Student’s t-test; * p < 0.05; ** p < 0.01).
Taking all of these results into account, we concluded that, in general, FaNAC2 might be involved
Taking
all stress
of these
resultsbyinto
account,proline
we concluded
that,and
in general,
FaNAC2 might be
in plant
abiotic
tolerance
regulating
accumulation
catabolism.
involved in plant abiotic stress tolerance by regulating proline accumulation and catabolism.
3. Discussion
3. Discussion
3.1. The Expression Pattern of FaNAC2
3.1. The
Expression
Patternmembers
of FaNAC2
Many
NAC family
have been reported to be involved in plant growth and development
processes,
as SAM
(shoot
apical meristem)
establishment,
root development,
senescence
Many such
NAC
family
members
have been
reported tolateral
be involved
in plant leaf
growth
and
and
cell
wall
formation
[32–35].
Thus,
we
speculated
that
FaNAC2
may
also
be
involved
in
many
development processes, such as SAM (shoot apical meristem) establishment, lateral plant
root
developmental
processes.
Our
that FaNAC2
might
be involved
in leaf
senescence,
as it
development,
leaf
senescence
anddata
cell show
wall formation
[32–35].
Thus,
we speculated
that
FaNAC2 may
expressed
highly
in
old
leaves
(Figure
2A).
These
results
are
similar
to
the
function
of
other
members
also be involved in many plant developmental processes. Our data show that FaNAC2 might be
of the NAC
family.
Overexpression
of OsNAC2
has
shown
to promote
leaf senescence
ABA
involved
in leaf
senescence,
as it expressed
highly
in been
old leaves
(Figure
2A). These
results arevia
similar
whilemembers
ABA biosynthesis
was
also activated
in the 35S::FaNAC2
lines
tobiosynthesis
the function[36],
of other
of the NAC
family.
Overexpression
of OsNAC2 N.
hasbenthamiana
been shown
to
promote leaf senescence via ABA biosynthesis [36], while ABA biosynthesis was also activated in the
35S::FaNAC2 N. benthamiana lines as our data shown (Figure 5), thus, whether FaNAC2 promotes leaf
senescence via ABA synthesis needs further investigation.
GUS analysis showed high levels of FaNAC2 promoter activity in guard cells of transgenic N.
benthamiana leaves (Figure 2D,E). Considering these results, it is possible that there is a function for
Plants 2020, 9, 1417
11 of 17
as our data shown (Figure 5), thus, whether FaNAC2 promotes leaf senescence via ABA synthesis
needs further investigation.
GUS analysis showed high levels of FaNAC2 promoter activity in guard cells of transgenic
N. benthamiana leaves (Figure 2D,E). Considering these results, it is possible that there is a function for
FaNAC2 in plant development. Stomata can affect transpiration and photosynthesis by regulating their
closure with the changing environment, via sensing ABA signals under adverse conditions [37–40].
Thus, FaNAC2 may regulate transpiration and leaf water retention by adjusting guard cells. Besides,
although the theory that root-sourced ABA can act as a signal to regulate stomatal aperture gained
widespread acceptance, ABA biosynthetic mutants showed that stomatal aperture is predominantly
regulated by leaf-sourced ABA [41–44]. In our results (Figure 5E), FaNAC2 could promote the key gene
in ABA synthesis pathways in the leaf, thus we speculated that FaNAC2 may regulate stomatal closure
by participating in ABA biosynthesis in leaves, further improving the drought resistance of plants.
3.2. FaNAC2 Functions as a Positive Regulator in Response to Abiotic Stress
Abiotic stress is an important factor that threatens the yield and quality of strawberry. To alleviate
the damage of abiotic stress, plants usually initiate complex adaptation via genetic mechanisms
including regulation of gene expression and increased concentration of osmolytes [5,27]. There are
several reports revealing that the ATAF sub-group of TFs belonging to the NAC family play important
roles in response to abiotic stress; however, their function is still under debate. In Arabidopsis,
ATAF1 was reported to negatively regulate stress-responsive gene expression during drought stress,
because ataf1 mutants displayed higher recovery rates than WT under drought [20]. Overexpression of
GmNAC2 reduces abiotic stress tolerance in Glycine max, which also functions as a negative regulator
by participating in ROS signaling pathways [24]. It has been reported that AtATAF1 overexpression in
transgenic lines enhanced drought tolerance [22]. Subsequently, OsNAC52 from rice, SlNAC2 from
tomato, DgNAC1 from chrysanthemum and CsATAF1 from cucumber were reported to function as
positive regulators in response to abiotic stress [23,25,27,28].
In our study, GUS analysis showed that the promoter activity of strawberry FaNAC2 was induced
by drought, salt and cold stress (Figure 3C), and FaNAC2 expression levels exhibited the same trend
that was verified by qRT-PCR (Figure 3A,B) in strawberry. Besides, ectopic overexpression of FaNAC2
in N. benthamiana plants showed higher tolerance to salinity, drought and cold stress (Figures 4–6).
Members of the ATAF sub-family in dicotyledons have conserved domains and can be identified by
some conserved regions that respond positively to stress. Overexpressed transgenic lines of ANAC019,
ANAC055 and ANAC072 improved the drought resistance of plants, and the conserved cis-elements
CATGT and CACG, for their binding, were identified [12]. However, the central function of FaNAC2 in
response to stress in strawberry is still unknown, and further studies on the abiotic stress pathway
involving FaNAC2 are needed.
Proline accumulation has been reported to occur after biotic and abiotic stress [45–47]. It varies
across different species under stress and can be more than 100 times higher than that under control
conditions [48]. In our study, both the synthesis and catabolism pathways of proline were induced
in 35S::FaNAC2 lines (Figure 7A), suggesting that the overexpression of FaNAC2 can promote the
metabolism of proline. However, in a stress environment, the expression levels of NbP5CS1 in
35S::FaNAC2 were still higher than that in WT, and both NbproDH2 and NbP5CDH were decreased
compared to control condition (Figure 7B–D). Further, the proline content of 35S::FaNAC2 was higher
than WT under the abiotic stress condition (Figure 8). We speculate that FaNAC2 might promote
the accumulation of proline under adverse conditions by activating proline synthesis and inhibiting
proline degradation, so as to promote the stress tolerance of plants.
Taken together, FaNAC2 from strawberry might play a positive role in response to abiotic stress
by regulating proline metabolism. Although the role of FaNAC2 in stress tolerance needs to be further
validated in strawberries, we demonstrate that it can serve as a candidate gene to enhance stress
tolerance, as long as the spatial–temporal expression is controlled.
Plants 2020, 9, 1417
12 of 17
4. Materials and Methods
4.1. Plant Materials, Growth Conditions
The strawberry cultivar “Benihoppe” (Fragaria × ananassa Duch.) was used in this study and
maintained in a plant culture room (23 ± 1 ◦ C, relative humidity of 40%, 16 h/8 h light/dark cycles).
The tissue cultured seedlings were initiated from node bud of runners, which were collected from
actively growing plants and disinfected with 70% ethanol (30 s) and 1% NaClO (10 min).
Most of N. benthamiana seeds were germinated on MS solid medium with 20% sucrose and
grown for 13 days. Then, the seedlings were transplanted to the soil and grown in the culture room.
For N. benthamiana seeds treated by simulate drought, the seeds were simply spread flat on a filter
paper soaked in water or 10% PEG 6000.
4.2. Gene Isolation and Sequence Alignment
Total RNA samples were extracted from collected shoot apicals and leaves using an E.Z.N.A
Total RNA Kit (Omega., Norcross, Georgia, USA). HIScript II Reverse Transcriptase (Vazyme, Nanjing,
China) was used for cDNA synthesis. The primers of FaNAC2 were designed according to the
GDR Database (Genome Database for Rosaceae) [49]. The CDS (coding sequence) of FaNAC2 was
obtained from the cDNA of “Benihoppe”. Phylogenetic analysis was performed using MEGA
version 7 (http://www.megasoftware.net/) [50]. Alignments of the FaNAC2 full-length amino acid
sequence with ATAF homologues from other species were performed using BioEdit software (http:
//www.mbio.ncsu.edu/BioEdit/bioedit.html) and ClustalW for multiple sequence alignments (http:
//www.ch.embnet.org/software/ClustalW.html).
4.3. Promoter Isolation, Prediction of Cis-Elements and GUS Activity Assay
Genomic DNA was extracted from strawberry “Benihoppe” using a TIANquick Midi Purification
Kit (TianGen., Beijing, China). The primers for the promoter of FaNAC2 were designed using
the sequence from the GDR database and the sequence of the promoter was obtained using the
DNA of “Benihoppe” strawberry as template, then the fragment was proofread and sequenced.
FaNAC2 promoter was cloned into the pCAMBIA1391 vector using the TrelisfTM SoSoo cloning
Kit (TsingKe, Beijing, China) to generate the reporter construct pCAMBIA1391–ProFaNAC2–GUS.
The primers used are listed in Table S1. The construct was stably transformed into N. benthamiana by
Agrobacterium-mediated transformation, as described below. Prediction of cis-elements was performed
using the Plantcare online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
For GUS analysis, samples were incubated with GUS staining buffer (including 2 M ferri/
ferrocyanide, 0.1% Triton X-100, 0.1 M sodium phosphate buffer, 0.5 mg·mL−1 X-gluc, pH 7) at 37 ◦ C
for 9 h, then stained samples were decolorized using 75% ethanol.
4.4. Gene Expression Analysis
qRT-PCR was employed to detect the expression of different genes. The qRT-PCR reactions (20 µL
volume containing 500 ng cDNA as template) were run using SYBR Premix ExTaq (TAKARA., Beijing,
China) as enzyme and ABI QuantStudio™ 6 Flex PCR System (ABI., New York, NY, USA). The 2−∆∆CT
method was used for qRT-PCR analysis. FaACTIN and NbACTIN were used as internal controls
for gene expression. The qRT-PCR primers of NbNCED1/NbNPK1/Nb5CS1/NbproDH2/NbP5CDH for
qRT-PCR were designed according to the N. benthamiana database [51]. The template for analyzing the
expression of these genes was cDNA of the fifth tobacco leaves from different treatments. PCR was
performed in triplicate using RNA samples extracted from three independent plants. Each reaction
was performed using three biological replicates and verified by melting curve analysis. The primers
are listed in Table S1.
Plants 2020, 9, 1417
13 of 17
4.5. Stable Transformation of N. benthamiana
For overexpression of FaNAC2 in N. benthamiana, the vector pCAMBIA2300 with kanamycin
resistance was used for stable transformation. The FaNAC2 ORF was inserted into pCAMBIA2300 using
the TrelisfTM SoSoo cloning Kit (TsingKe) and driven by a CaMV 35S promoter. The 35S::FaHAN was
introduced into Agrobacterium tumefaciens strain GV3101 and then transformed into N. benthamiana leaf
dishes by the method of Agrobacterium-mediated transformation [52]. The obtained kanamycin-resistant
plants were screened again by RT-PCR. The PCR mix was from TsingKe and the PCR primers used for
vector construction are listed in Table S1.
4.6. Abiotic Stress Treatment
The 3-months-old “Benihoppe” strawberry tissue cultured seedlings, which were obtained from
the node bud of runners, were used for stress treatment. Three simulated stress conditions of 200 mM
NaCl, 20% PEG 6000 and 4 ◦ C were set, and MS liquid medium was used as a control to treat for 0 h, 3 h,
6 h, 12 h and 36 h. The qRT-PCR was performed in biological triplicate using RNA samples extracted
from three independent plants. For GUS staining, the abiotic stress treatment for 7 DAG (days after
germination) N. benthamiana seedlings was 200 mM NaCl, 15% PEG 6000 and 4 ◦ C; the treatment
lasted 12 h.
Regarding abiotic stress treatment of 35S::FaNAC2 transgenic N. benthamiana lines and WT
plants, simulated drought treatment for seeds was used with 10% PEG 6000; drought treatment of
40 DAG seedlings was conducted by stopping the watering for 20 d, until the leaves were all wilted,
then re-watering for 5 d to observe the recovery of plants. Cold treatment was performed to put the
plants under normal indoor conditions after 4 ◦ C for 2 h, 0 ◦ C for 1 h, −5 ◦ C for 1 h and 4 ◦ C for 1 h,
and the status of the plants was observed. Salt treatment for seedings of 35S::FaNAC2 and wild-type
lines were MS solid medium with 10 mM NaCl and 100 mM NaCl, respectively. Considering that
40 DAG seedlings were stronger, salt treatment was enhanced to 300 mM NaCl. These experiments
were repeated three times and each line had three biological replicates.
4.7. Determination of SOD, POD and CAT Enzyme Activities
Approximately 0.15 g of the fifth expanded leaf from each line (including 35S::FaNAC2 and WT
lines) was homogenized in 5 mL pre-cooled Phosphate buffered saline, at 4000 rpm for 10 min at
4 ◦ C, then 2 mL of supernatant was drained and placed on ice for determination of different enzyme
activities, according to measurements as follows.
CAT (Catalase) activity was measured spectrophotometrically at 240 nm [53]. The reaction mixture
contained 100 mM sodium phosphate buffer (pH 7.0), 30 mM H2 O2 and 100 µL of crude extract in a
total volume of 3 mL. The absorbance was read quickly every 1 min, for a total of 4 min.
The activity of POD (peroxidase) was determined at 420 nm using a spectrophotometer, with
callus lignin as substrate. The reaction mixture contained 100 mM sodium phosphate buffer (pH 6.0),
5 mM hydrogen peroxide, 5 mM guaiacol and 100 µL crude extract, with a total volume of 3 mL,
at room temperature (±25 ◦ C) [54].
The activity of SOD (superoxide dismutase) was determined by inhibiting the photoreduction of
SOD to NBT (nitro-blue tetrazolium) [55]. The reaction mixture contained a final volume of 3 mL of
50 mM sodium phosphate buffer (pH 7.6), 0.1 mM Ethylene Diamine Tetraacetic Acid (EDTA)-Na2 ,
50 mM sodium carbonate, 12 mM L-methionine, 50 mM NBT, 10 µM riboflavin and 100 µL of crude
extract. Then, the reaction mixture was exposed to white light for 30 min for the SOD reaction.
After incubation, the absorbance was recorded at 560 nm with a spectrophotometer.
4.8. Determination of WLR (Water Loss Rate) in N. benthamiana Leaves
The fifth leaf was collected from half-month-old tobacco plants which were grown in the soil and
dried naturally on filter paper. The temperature was 25 ◦ C, the relative humidity was 38%, and weight
Plants 2020, 9, 1417
14 of 17
measurements were taken at 0 h, 0.5 h, 1 h, 1.5 h, 2 h, 3 h, and 6 h. This experiment was repeated three
times to calculate the weight and rate of water loss in each period.
4.9. Proline Measurement
To determine free proline level, 0.1 g of fourth expanded leaf samples from each line (including
35S::FaNAC2 and WT lines) was homogenized in 3% (w/v) sulphosalycylic acid and then homogenate
filtered through filter paper [56]. The mixture was heated at 100 ◦ C for 30 min in a water bath after
the addition of acid ninhydrin and glacial acetic acid. The reaction was then stopped by ice bath.
The mixture was extracted with toluene and the absorbance of the fraction with toluene aspired from
the liquid phase was read at 520 nm. Proline concentration was determined using a calibration curve.
4.10. Statistical Analysis
Microsoft Excel 2019 (Microsoft Corp., Redmond, Washington, USA) and GraphPad (GraphPad
Software Inc., San Diego, California, USA) were used for analyzing the experimental data. Data for
p-values were analyzed by Student’s t test at a significance level of 0.05 or 0.01. Comparisons between
multiple samples were determined using Tukey’s multiple comparisons test.
Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/9/11/1417/s1,
Table S1: Primers used in this study.
Author Contributions: J.L. and J.Z. designed the experiments and performed the experiments; J.L., J.Z. and H.W.
drafted the manuscript; J.L., H.W. and Z.W. revised the manuscript. All authors have read and agreed to the
published version of the manuscript.
Funding: This work was supported by the National Key R&D Program of China (2019YFD001800).
Acknowledgments: We thank Mingfang Yi (China Agricultural University, Beijing, China) for the generous gift of
pCAMBIA2300 and pCAMBIA1391 vectors.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Choudhury, S.; Panda, P.; Sahoo, L.; Panda, S.K. Reactive oxygen species signaling in plants under abiotic
stress. Plant Signal. Behav. 2013, 8, e23681. [CrossRef] [PubMed]
Choudhury, F.K.; Rivero, R.M.; Blumwald, E.; Mittler, R. Reactive oxygen species, abiotic stress and stress
combination. Plant J. 2017, 90, 856–867. [CrossRef]
Zhu, J.K. Salt and drought stress signal transduction in plants. Ann. Rev. Plant Biol. 2002, 53, 247–273.
[CrossRef]
Zhu, J.K. Abiotic Stress Signaling and Responses in Plants. Cell 2016, 167, 313–324. [CrossRef] [PubMed]
Min, X.; Jin, X.; Zhang, Z.; Wei, X.; Ndayambaza, B.; Wang, Y.; Liu, W. Genome-Wide Identification of NAC
Transcription Factor Family and Functional Analysis of the Abiotic Stress-Responsive Genes in Medicago
sativa L. J. Plant Growth Regul. 2019, 1–14. [CrossRef]
Takada, S.; Hibara, K.; Ishida, T.; Tasaka, M. The CUP-SHAPED COTYLEDON1 gene of Arabidopsis regulates
shoot apical meristem formation. Development 2001, 128, 1127–1135.
Mallory, A.C.; Dugas, D.V.; Bartel, D.P.; Bartel, B. MicroRNA Regulation of NAC-Domain Targets Is Required
for Proper Formation and Separation of Adjacent Embryonic, Vegetative, and Floral Organs. Curr. Biol. 2004,
14, 1035–1046. [CrossRef]
Olsen, A.N.; Ernst, H.A.; Leggio, L.L.; Skriver, K. NAC transcription factors: Structurally distinct, functionally
diverse. Trends Plant Sci. 2005, 10, 79–87. [CrossRef]
Nakashima, K.; Tran, L.S.P.; Van Nguyen, D.; Fujita, M.; Maruyama, K.; Todaka, D.; Ito, Y.; Hayashi, N.;
Shinozaki, K.; Yamaguchi-Shinozaki, K. Functional analysis of a NAC-type transcription factor OsNAC6
involved in abiotic and biotic stress-responsive gene expression in rice. Plant J. 2007, 51, 617–630. [CrossRef]
Shao, H.; Wang, H.; Tang, X. NAC transcription factors in plant multiple abiotic stress responses: Progress
and prospects. Front. Plant Sci. 2015, 6, 902. [CrossRef]
Plants 2020, 9, 1417
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
15 of 17
Fujita, M.; Fujita, Y.; Maruyama, K.; Seki, M.; Hiratsu, K.; Ohme-Takagi, M.; Tran, L.P.; Yamaguchi-Shinozaki, K.;
Shinozaki, K. A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent
stress-signaling pathway. Plant J. 2004, 39, 863–876. [CrossRef]
Tran, L.S.P.; Nakashima, K.; Sakuma, Y.; Simpson, S.D.; Fujita, Y.; Maruyama, K.; Fujita, M.; Seki, M.;
Shinozaki, K.; Yamaguchi-Shinozaki, K. Isolation and functional analysis of Arabidopsis stress-inducible
NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration
stress 1 promoter. Plant. Cell 2004, 16, 2481–2498. [CrossRef] [PubMed]
Li, X.D.; Zhuang, K.Y.; Liu, Z.M.; Yang, D.Y.; Ma, N.N.; Meng, Q.W. Overexpression of a novel NAC-type
tomato transcription factor, SlNAM1, enhances the chilling stress tolerance of transgenic tobacco. J. Plant.
Physiol. 2016, 204, 54–65. [CrossRef] [PubMed]
Hu, H.; You, J.; Fang, Y.; Zhu, X.; Qi, Z.; Xiong, L. Characterization of transcription factor gene SNAC2
conferring cold and salt tolerance in rice. Plant. Mol. Biol. 2008, 67, 169–181. [CrossRef] [PubMed]
Peng, H.; Yu, X.; Cheng, H.; Shi, Q.; Zhang, H.; Li, J.; Ma, H. Cloning and characterization of a novel NAC
family gene CarNAC1 from chickpea (Cicer arietinum L.). Mol. Biotechnol. 2010, 44, 30. [CrossRef]
Xia, N.; Zhang, G.; Liu, X.Y.; Deng, L.; Cai, G.L.; Zhang, Y.; Wang, X.J.; Zhao, J.; Huang, L.L.; Kang, Z.S.
Characterization of a novel wheat NAC transcription factor gene involved in defense response against stripe
rust pathogen infection and abiotic stresses. Mol. Biol. Rep. 2010, 37, 3703–3712. [CrossRef]
Xia, N.; Zhang, G.; Sun, Y.F.; Zhu, L.; Xu, L.S.; Chen, X.M.; Liu, B.; Yu, Y.T.; Wang, X.J.; Huang, L.L.; et al.
TaNAC8, a novel NAC transcription factor gene in wheat, responds to stripe rust pathogen infection and
abiotic stresses. Physiol. Mol. Plant. Pathol. 2010, 74, 394–402. [CrossRef]
Collinge, M.; Boller, T. Differential induction of two potato genes, Stprx2 and StNAC, in response to infection
by Phytophthora infestans and to wounding. Plant. Mol. Biol. 2001, 46, 521–529. [CrossRef]
Delessert, C.; Kazan, K.; Wilson, I.W.; Straeten, D.V.D.; Manners, J.; Dennis, E.S.; Dolferus, R. The transcription
factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. Plant. J. 2005, 43,
745–757. [CrossRef]
Lu, P.L.; Chen, N.Z.; An, R.; Su, Z.; Qi, B.S.; Ren, F.; Chen, J.; Wang, X.C. A novel drought-inducible gene,
ATAF1, encodes a NAC family protein that negatively regulates the expression of stress-responsive genes in
Arabidopsis. Plant. Mol. Biol. 2007, 63, 289–305. [CrossRef] [PubMed]
Mauch-Mani, B.; Flors, V. The ATAF1 transcription factor: At the convergence point of ABA-dependent plant
defense against biotic and abiotic stresses. Cell Res. 2009, 19, 1322–1323. [CrossRef] [PubMed]
Wu, Y.; Deng, Z.; Lai, J.; Zhang, Y.; Yang, C.; Yin, B.; Zhao, Q.; Zhang, L.; Li, Y.; Yang, C.; et al. Dual function
of Arabidopsis ATAF1 in abiotic and biotic stress responses. Cell Res. 2009, 19, 1279–1290. [CrossRef]
Gao, F.; Xiong, A.; Peng, R.; Jin, X.; Xu, J.; Zhu, B.; Chen, J.; Yao, Q. OsNAC52, a rice NAC transcription
factor, potentially responds to ABA and confers drought tolerance in transgenic plants. Plant. Cell Tissue
Organ. Cult. 2010, 100, 255–262. [CrossRef]
Jin, H.; Huang, F.; Cheng, H.; Song, H.; Yu, D. Overexpression of the GmNAC2 gene, an NAC transcription
factor, reduces abiotic stress tolerance in tobacco. Plant. Mol. Biol. Rep. 2013, 31, 435–442. [CrossRef]
Yang, X.; Hu, Y.X.; Li, X.L.; Yu, X.D.; Li, Q.L. Molecular characterization and function analysis of SlNAC2 in
Suaeda liaotungensis K. Gene 2014, 543, 190–197. [CrossRef] [PubMed]
Wang, L.; Hu, Z.; Zhu, M.; Zhu, Z.; Hu, J.; Qanmber, G.; Chen, G. The abiotic stress-responsive NAC
transcription factor SlNAC11 is involved in drought and salt response in tomato (Solanum lycopersicum L.).
Plant. Cell Tissue Organ. Culture 2017, 129, 161–174. [CrossRef]
Wang, K.; Zhong, M.; Wu, Y.; Bai, Z.; Liang, Q.; Liu, Q.; Pan, Y.; Zhang, L.; Jiang, B.; Jia, Y.; et al. Overexpression
of a chrysanthemum transcription factor gene DgNAC1 improves the salinity tolerance in chrysanthemum.
Plant. Cell Rep. 2017, 36, 571–581. [CrossRef]
Wang, J.; Zhang, L.; Cao, Y.; Qi, C.; Li, S.; Liu, L.; Wang, G.; Mao, A.; Ren, S.; Guo, Y. CsATAF1 positively
regulates drought stress tolerance by an ABA-dependent pathway and by promoting ROS scavenging in
cucumber. Plant. Cell Physiol. 2018, 59, 930–945. [CrossRef]
Brown, J.; Voth, V. Salt damage to strawberries: Types of water, irrigation system, and soil condition found to
influence salt accumulation in strawberry plantings. Calif. Agric. 1955, 9, 11–12.
Razavi, F.; Pollet, B.; Steppe, K.; Van, L.M.C. Chlorophyll fluorescence as a tool for evaluation of drought
stress in strawberry. Photosynthetica 2008, 46, 631–633. [CrossRef]
Plants 2020, 9, 1417
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
16 of 17
Zhang, H.; Kang, H.; Su, C.; Qi, Y.; Liu, X.; Pu, J. Genome-wide identification and expression profile analysis
of the NAC transcription factor family during abiotic and biotic stress in woodland strawberry. PLoS ONE
2018, 13, e0197892. [CrossRef] [PubMed]
Souer, E.; van Houwelingen, A.; Kloos, D.; Mol, J.; Koes, R. The no apical meristem gene of Petunia is required
for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries. Cell
1996, 85, 159–170. [CrossRef]
He, X.J.; Mu, R.L.; Cao, W.H.; Zhang, Z.G.; Zhang, J.S.; Chen, S.Y. AtNAC2, a transcription factor downstream
of ethylene and auxin signaling pathways, is involved in salt stress response and lateral root development.
Plant. J. 2005, 44, 903–916. [CrossRef] [PubMed]
Mitsuda, N.; Seki, M.; Shinozaki, K.; Ohme-Takagi, M. The NAC transcription factors NST1 and NST2 of
Arabidopsis regulate secondary wall thickenings and are required for anther dehiscence. Plant. Cell 2005, 17,
2993–3006. [CrossRef]
Uauy, C.; Distelfeld, A.; Fahima, T.; Blechl, A.; Dubcovsky, J. A NAC gene regulating senescence improves
grain protein, zinc, and iron content in wheat. Science 2006, 314, 1298–1301. [CrossRef]
Mao, C.; Lu, S.; Lv, B.; Zhang, B.; Shen, J.; He, J.; Luo, L.; Xi, D.; Chen, X.; Ming, F. A rice NAC transcription
factor promotes leaf senescence via ABA biosynthesis. Plant. Physiol. 2017, 174, 1747–1763. [CrossRef]
Hetherington, A.M.; Woodward, F.I. The role of stomata in sensing and driving environmental change.
Nature 2003, 424, 901–908. [CrossRef] [PubMed]
Roelfsema, M.R.G.; Levchenko, V.; Hedrich, R. ABA depolarizes guard cells in intact plants, through a
transient activation of R-and S-type anion channels. Plant. J. 2004, 37, 578–588. [CrossRef]
Zhang, Y.; Xu, W.; Li zhong Deng, X.; Wu, W.; Xue, Y. F-Box Protein DOR Functions as a Novel Inhibitory
Factor for Abscisic Acid-Induced Stomatal Closure under Drought Stress in Arabidopsis. Plant. Physiol.
2008, 148, 4. [CrossRef]
Lawson, T.; Blatt, M.R. Stomatal size, speed, and responsiveness impact on photosynthesis and water use
efficiency. Plant. Physiol. 2014, 164, 1556–1570. [CrossRef]
Holbrook, N.M.; Shashidhar, V.R.; James, R.A.; Munns, R. Stomatal control in tomato with ABA-deficient
roots: Response of grafted plants to soil drying. J. Exp. Bot. 2002, 53, 1503–1514. [PubMed]
Christmann, A.; Weiler, E.W.; Steudle, E.; Grill, E. A hydraulic signal in root-to-shoot signalling of water
shortage. Plant. J. 2007, 52, 167–174. [CrossRef] [PubMed]
Mc Adam, S.A.; Sussmilch, F.C.; Brodribb, T.J. Stomatal responses to vapour pressure deficit are regulated by
high speed gene expression in angiosperms. Plant. Cell Environ. 2016, 39, 485–491. [CrossRef]
Zhang, F.; Sussmilch, F.; Nichols, D.S.; Cardoso, A.A.; Brodribb, T.J.; McAdam, S.A.M. Leaves, not roots or
floral tissue, are the main site of rapid, external pressure-induced ABA biosynthesis in angiosperms. J. Exp.
Bot. 2018, 69, 5. [CrossRef] [PubMed]
Saradhi, P.P.; AliaArora, S.; Prasad, K. Proline accumulates in plants exposed to UV radiation and protects
them against UV-induced peroxidation. Biochem. Biophys. Res. Commun. 1995, 209, 1–5. [CrossRef]
Hare, P.D.; Cress, W.A. Metabolic implications of stress-induced proline accumulation in plants. Plant.
Growth Regul. 1997, 21, 79–102. [CrossRef]
Siripornadulsil, S.; Traina, S.; Verma, D.P.S.; Sayre, R.T. Molecular mechanisms of proline-mediated tolerance
to toxic heavy metals in transgenic microalgae. Plant. Cell 2002, 14, 2837–2847. [CrossRef]
Verbruggen, N.; Hermans, C. Proline accumulation in plants: A review. Amino acids 2008, 35, 753–759.
[CrossRef]
Jung, S.; Ficklin, S.P.; Lee, T.; Cheng, C.H.; Blenda, A.; Zheng, P.; Yu, J.; Bombarely, A.; Cho, I.; Ru, S.; et al.
The Genome Database for Rosaceae (GDR): Year 10 update. Nucleic Acids Res. 2014, 42, D1237–D1244.
[CrossRef]
Kumar, S.; Stecher, G.; Tamura, K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger
datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [CrossRef]
Fernandez-Pozo, N.; Menda, N.; Edwards, J.D.; Saha, S.; Tecle, I.Y.; Strickler, S.R.; Bombarely, A.;
Fisher-York, T.; Pujar, A.; Foerster, H.; et al. The Sol Genomics Network (SGN)—From genotype to
phenotype to breeding. Nucleic Acids Res. 2015, 43, D1036–D1041. [CrossRef] [PubMed]
Wydro, M.; Kozubek, E.; Lehmann, P. Optimization of transient Agrobacterium-mediated gene expression
system in leaves of Nicotiana benthamiana. Acta Biochim. Pol. 2006, 53, 289–298. [CrossRef] [PubMed]
Plants 2020, 9, 1417
53.
54.
55.
56.
17 of 17
Bergmeyer, H.U.; Graßl, M. Methods of Enzymatic Analysis, 3rd ed.; Verlag Chemie: Weinheim, Germany, 1983;
pp. 267–268.
Onsa, G.H.; bin Saari, N.; Selamat, J.; Bakar, J. Purification and characterization of membrane-bound
peroxidases from Metroxylon sagu. Food Chem. 2004, 85, 365–376. [CrossRef]
Oktay, M.; Küfreviolu, I.; Kocaçalişkan, I.; Şaklrolu, H. Polyphenoloxidase from Amasya apple. J. Food Sci.
1995, 60, 494–496. [CrossRef]
Bates, L.S.; Waldren, R.P.; Teare, I.D. Rapid determination of free proline for water-stress studies. Plant. Soil
1973, 39, 205–207. [CrossRef]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional
affiliations.
© 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/).
|
|
https://openalex.org/W3200577068
|
https://www.scielo.br/j/ref/a/SVpXh9FZz8tPG5YM7m8LNjp/?lang=pt&format=pdf
|
Portuguese
| null |
Mulheres de sucesso no campo científico: uma análise de redes sociais
|
Revista Estudos Feministas
| 2,021
|
cc-by
| 10,307
|
Successful Women in The Scientifi c Field: An Analysis of Social Networks Abstract: The article presents results from research that examined how women considered successful
in science and technology narrate their successful trajectory. The empirical material consists of reports
published on the social network Facebook obtained from 2015 to 2018. As theoretical contributions,
studies of the thinking of Michel Foucault were used. Analysis has shown that the trajectories of
successful women are marked by individual coping with the difficulties of consolidating a career. This
confrontation is linked to the mechanisms of neoliberal rationality present in our society that positions
the individual as solely responsible for their education and professional qualification, weakening the
relevance of the collective and the actions of the state as ways of guaranteeing and sustaining gender
equity in the scientific areas. Keywords: Women; Scientific field; Social networks; Michel Foucault. Artigos Artigos Mulheres de sucesso no campo científi co:
uma análise de redes sociais
Polliane Trevisan Nunes1
0000-0003-3919-3455
Fernanda Wanderer1
0000-0002- 8198-7104
1Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil.
90040-060. ppgedu@ufrgs.br Resumo: Neste artigo, apresentamos resultados de uma pesquisa que examinou como as mulheres
consideradas bem-sucedidas nas áreas da ciência e tecnologia narram a sua trajetória de sucesso. O material empírico constitui-se de reportagens veiculadas na rede social Facebook, obtidas
no período de 2015 a 2018. Como aportes teóricos, foram utilizados estudos do pensamento de
Michel Foucault. A análise mostrou que as trajetórias das mulheres de sucesso são marcadas pelo
enfrentamento individual das dificuldades para consolidar uma carreira. Esse enfrentamento está
vinculado aos mecanismos da racionalidade neoliberal presentes em nossa sociedade que posiciona
o indivíduo como o único responsável por sua educação e qualificação profissional, esmaecendo
a relevância dos processos coletivos e as ações do Estado como formas de garantir e sustentar a
equidade de gênero nas áreas científicas. q
g
Palavras-chave: Mulheres; campo científico; redes sociais; Michel Foucault. Keywords: Women; Scientific field; Social networks; Michel Foucault. Introdução No artigo, apresentamos resultados de uma pesquisa que objetivou analisar as formas
pelas quais as redes sociais produzem a relação entre as mulheres e o campo científico. Em
especial, o estudo consistiu em examinar como as mulheres consideradas bem-sucedidas nas
áreas da ciência e tecnologia narram a sua trajetória de sucesso. Para isso, foram escrutinadas
diversas reportagens sobre o tema que provêm de fontes compartilhadas em redes sociais no
período de 2015 a 2018. Na contemporaneidade, proliferam debates e reflexões sobre as relações de gênero em
nossa sociedade, os quais se relacionam tanto às desigualdades entre homens e mulheres ainda
presentes em diversos setores, quanto aos avanços já alcançados em prol da equidade de
gênero. Muitas dessas conquistas emergem dos movimentos feministas que, desde a década de
1960, mobilizaram não só a geração de ações afirmativas, quanto a produção de teorizações Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 1 ,
p
,
( )
DOI: 10.1590/1806-9584-2021v29n268120 POLLIANE TREVISAN NUNES E FERNANDA WANDERER e investigações relatando as histórias das mulheres e suas condições de trabalho, educação,
saúde e participação política (Guacira Lopes LOURO, 2014). e investigações relatando as histórias das mulheres e suas condições de trabalho, educação,
saúde e participação política (Guacira Lopes LOURO, 2014). Apesar de conquistas, em algumas áreas, ainda se fazem presentes desigualdades,
como no campo científico. Tatiane Furukawa Liberato e Thales Haddas Novaes de Andrade
(2018) destacam que na área da ciência e tecnologia a participação das mulheres é ainda
menor que a masculina, sendo, às vezes, limitada e relegada a papéis marginais. Apoiando-se
em pesquisas e dados estatísticos, os autores apontam que estudos têm sido realizados para
refletir sobre as questões de gênero que marcam a área científica, como o menor número de
mulheres no campo, a desigual oportunidade de acesso e permanência na carreira, bem como
o desempenho inferior de mulheres em relação aos homens, o qual se manifesta, por exemplo,
em menores taxas de publicação científica. Em efeito, essas constatações já foram observadas e examinadas por estudiosas como
Valerie Walkerdine (2007), Louro (2014) e Londa Schiebinger (2008; 2001), as quais destacam que
há questões de ordem epistemológica que dificultaram – e ainda dificultam – o entendimento
de que a ciência é um espaço e uma atividade para mulheres. Schiebinger (2001, p. Introdução 26) afirma
que a maior contribuição do feminismo foi questionar a neutralidade do gênero na ciência,
“revelando que valores geralmente atribuídos às mulheres foram excluídos da ciência e que
desigualdades de gênero foram construídas na produção e estrutura do conhecimento”. Em
sua argumentação, a autora afirma que o processo de formalização da prática científica em
laboratórios e universidades restringiu o acesso das mulheres à ciência, uma vez que o acesso
à universidade, em geral, não era permitido a elas. Assim, destaca que o poder da ciência
ocidental “é celebrado por produzir conhecimento objetivo e universal, transcendendo as
restrições culturais. Entretanto, no que diz respeito ao gênero, à raça e a muito mais, a ciência
não é um valor neutro” (SCHIENINGER, 2008, p. 274). Pesquisas realizadas por Maria Rosa Lombardi (2004), Raimunda de Nazaré Fernandes
Corrêa (2011), Maria Celia Macedo Araújo Melo (2013), Adriana Zomer de Moraes (2016),
Fabiane Ferreira da Silva (2012) e Liberato e Andrade (2018) problematizaram as relações entre
as mulheres e as áreas de ciência e tecnologia. Mesmo desenvolvidas em diferentes tempos e
espaços, os resultados apontam para a presença de mulheres em todas as chamadas áreas
exatas, mas com distribuição desigual tanto na esfera acadêmica quanto no mercado de
trabalho. Isso indica que as construções discursivas sobre o campo científico e suas subdivisões
são mais difíceis de serem modificadas ao longo do tempo, uma vez que fazem parte de um
contexto social ainda bastante desigual. Porém, como apresentam Liberato e Andrade (2018),
apesar de muitos obstáculos, cada vez mais mulheres cientistas tornam-se reconhecidas e
valorizadas em suas áreas de atuação. Recentemente, algumas histórias das trajetórias de
mulheres com sucesso no campo científico passaram a circular na mídia, em especial nas redes
sociais. A pesquisa que realizamos emerge com o propósito de examinar como as mulheres
consideradas bem-sucedidas nas áreas científicas narram a sua trajetória de sucesso. Apoiamos-nos nas produções de Jorge Larrosa (2008) que discutem as formas como as pessoas
são constituídas por suas experiências, no interior de tramas discursivas, como as histórias que
relatamos sobre a nossa trajetória acadêmica e profissional. O autor afirma que a pessoa
humana “se fabrica no interior de certos aparatos (pedagógicos, terapêuticos) de subjetivação”
(LARROSA, 2008, p. 37). Poderíamos dizer que a mídia, ao criar e colocar em circulação
diferentes enunciações, é um destes aparatos que faz parte da constituição das subjetividades
das mulheres nas áreas de ciência e tecnologia. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS em congruência com a própria teorização, preferimos chamar de ‘produção’ de informação –
e de estratégias de descrição e análise (MEYER; PARAÍSO, 2012, p. 16). Além disso, as pesquisas realizadas com base nesse registro teórico, como a que
realizamos, são constituídas levando-se em consideração a noção de que, ao final do processo
de investigação, não se chegará a um resultado definitivo nem a uma verdade única (MEYER;
PARAÍSO, 2012). Isso decorre do entendimento de que “a verdade é uma invenção, uma
criação” (PARAÍSO, 2012, p. 27), ou seja, é resultado de relações de poder que estabelecem, em
cada momento histórico, o que é considerado verdadeiro. As investigações que se inscrevem no
pensamento pós-estruturalista estão mais interessadas em entender como os fenômenos sociais
se organizam, buscando “descrever e problematizar processos por meio dos quais significados
e saberes específicos são produzidos, no contexto de determinadas redes de poder, com certas
consequências para determinados indivíduos e/ou grupos” (MEYER, 2012, p. 51), do que em
explicar o que é, de fato, o objeto de estudo em questão. A parte empírica deste estudo envolveu a análise de enunciações a respeito das trajetórias
de sucesso de mulheres no campo científico obtidas nas redes sociais, em especial o Facebook,
a “rede mais popular e mais disseminada” que se impõe como uma “fonte privilegiada de
informação aos estudiosos” (Lúcia AMANTE, 2014, p. 28). Neste contexto, Marcilene Forechi
(2018) afirma que as redes sociais digitais disseminaram-se na sociedade, principalmente nos
últimos cinco anos, tornando-se relevante para o compartilhamento de notícias, para difusão
de ideias e denúncias, bem como mobilização e organização de movimentos sociais, tais como
os feministas. Para obter o material empírico, seguimos as publicações de fan pages, ou páginas, sobre
a temática “mulheres de sucesso no campo científico”. Selecionamos páginas brasileiras que, de
forma recorrente, publicavam reportagens com esse tema, abordando os seguintes conteúdos:
matérias de reconhecimento de descobertas científicas feitas por mulheres e divulgação de
casos de mulheres bem-sucedidas nas suas carreiras ou que tiveram recentemente algum
destaque acadêmico. Considerando esses critérios, as páginas escolhidas para acompanhar
regularmente foram: 1) ELAS nas Exatas: consiste em uma parceria entre o Fundo ELAS, Instituto Unibanco,
Fundação Carlos Chagas e ONU Mulheres para reverter uma certa tendência de as mulheres
escolherem as ciências humanas como área de atuação profissional. MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS 2) Meninas Olímpicas: projeto que busca o empoderamento de meninas para que elas
sejam protagonistas por meio da participação em olimpíadas científicas. 3) Parent in Science: grupo que surgiu com o intuito de discutir sobre a maternidade (e
paternidade) dentro do universo da ciência do Brasil. 4) Mulheres na Ciência: espaço para mulheres cientistas contarem suas histórias e
discutirem sua posição no mundo científico. O material empírico examinado nesse artigo emerge das fan pages acompanhadas que
funcionavam como agregadoras de notícias sobre o tema e, ao mesmo tempo, propagadoras,
compartilhando conteúdos produzidos por outras pessoas ou sites. Para examinarmos essas
reportagens, acompanhamos as discussões de Michel Foucault (2004) e Rosa Maria Bueno
Fischer (2001) sobre a análise do discurso. Como aprendemos com o filósofo, os discursos
não são expressões espontâneas dos indivíduos, mas constituídos conforme as regras de um
determinado tempo que, por sua vez, dependem das relações de poder e saber que vão dar
sentido e possibilidade àquilo que é expresso (FISCHER, 2001). Tendo esse entendimento, não
buscamos verificar a legitimidade ou a veracidade das informações compartilhadas pelas
páginas; consideramos a sua própria existência em circulação. Ou seja, o objetivo não foi buscar
significados ocultos ou uma suposta verdade a ser revelada nas entrelinhas das reportagens,
mas, sim, traçar um percurso de análise com o que se encontra na superfície, sem buscar uma
relação de causa e efeito. Ao final do processo de seleção do material, foram obtidas em torno de 20 notícias,
divulgadas por meio de links, nessas páginas. A estratégia analítica utilizada envolveu três
etapas. A primeira consistiu em selecionar todas as reportagens ou publicações encontradas
nas páginas sobre mulheres bem-sucedidas na área científica. Na sequência, estivemos atentas
para encontrar as recorrências e dispersões que se tornaram mais evidentes no material. E,
por último, organizamos os excertos em séries para visualizar alguns dos sentidos presentes nas
matérias selecionadas. O resultado desse exercício será descrito na próxima seção. Introdução Assim, na esteira de Larrosa (2008), narrar-se é uma forma de constituir-se enquanto
sujeito, uma vez que esse processo não está inicialmente determinado, mas vai se gestando
ao longo da vida. Nas palavras do autor: “a ideia do que é uma pessoa, ou um eu, ou um
sujeito, é histórica e culturalmente contingente” (LARROSA, 2008, p. 40). Tomar as trajetórias de
mulheres como objeto de análise pressupõe não as entender enquanto matérias jornalísticas
neutras, mas fazendo parte de um discurso que constitui determinados sujeitos. Conforme Larrosa
(2008), os discursos constituem os sujeitos que contam sobre si em diversos contextos (práticas
pedagógicas, práticas médicas, práticas religiosas etc.). Em termos metodológicos, acompanhamos as reflexões de Dagmar Meyer e Marlucy
Paraíso (2012) a respeito das pesquisas em Educação amparadas em uma perspectiva pós-
estruturalista de inspiração foucaultiana. Nesses trabalhos, segundo elas, não há uma única
metodologia a ser seguida, mas o entendimento de que os caminhos investigativos podem
ser definidos e construídos ao longo do processo, com base nos problemas que emergem do
campo empírico. As autoras entendem uma metodologia como um certo modo de perguntar, de interrogar, de formular questões e de construir problemas
de pesquisa que é articulado a um conjunto de procedimentos de coleta de informações – que, Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 2 Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 A sociedade espera que uma mulher tenha sonhos pequenos, mas eu decidi ser
astronauta Mylena Peixoto nasceu em Campos dos Goytacazes, no interior do Rio de Janeiro, tem 18
anos e visitou a sede da Nasa, no Texas (EUA) pela segunda vez em setembro. Na primeira
experiência, em 2016, Mylena foi para os Estados Unidos depois de descobrir cinco asteroides
ao participar de um programa internacional que mobilizou escolas públicas da sua cidade. […] Você é jovem e ainda está no início da carreira, mas já teve grandes experiências que
muitos cientistas só alcançam com mais tempo de estudo. Como você vê isso? Eu não diria
que sou uma pessoa sortuda, porque eu batalhei muito para chegar aqui e com a ajuda de
muitas pessoas. Mas eu me sinto privilegiada por ter tido a oportunidade de realizar um sonho
tão grande. Hoje, meus sonhos ganharam o mundo! […] me vejo representada nas histórias das
vencedoras que se esforçaram tanto para alcançar reconhecimento (MULHERES NA CIÊNCIA,
2017, online). Chama atenção, em todas as reportagens selecionadas, o fato de que as mulheres
podem realizar grandes feitos acadêmicos, como ganhar diversas medalhas ou conhecer a
National Aeronautics and Space Administration (NASA), nos Estados Unidos. A primeira questão
que se coloca, então, é: por que essas conquistas precisam ser divulgadas? Uma explicação
possível é que, culturalmente, as realizações acadêmicas nas áreas científicas não são
naturalmente associadas às mulheres. Isso se deve, principalmente, pelas características
relacionadas ao fazer científico e à cultura científica (SCHIEBINGER, 2008) que se constituíram
historicamente vinculadas ao masculino. Ao mesmo tempo em que essa divulgação ocorre,
também merece destaque o fato de que essas ações não estão estabelecidas como o padrão
da presença feminina no campo científico, mas como exceção, reforçando o argumento de
que há desigualdades de gênero na constituição do campo científico. Assim, há um movimento de divulgar as realizações das mulheres em contraposição a
décadas de silenciamento, onde esses feitos eram ignorados ou apropriados por outros atores,
em geral masculinos (SCHIEBINGER, 2001). Isso corrobora a ideia de Luciana Luzzardi (2017, p. 27), ao observar que “quando uma mulher se destaca e tem visibilidade em áreas de trabalho
dominadas por homens, há um esforço para entender e justificar tal êxito”. Mulheres de sucesso no campo científi co As matérias selecionadas para análise abordam a inserção de mulheres no campo
científico, com destaque para as histórias produzidas pelas próprias mulheres. Importa destacar
que as notícias referem-se a trajetórias tanto de jovens pesquisadoras, quanto daquelas com
a carreira já consolidada. Em geral, apresentam elementos positivos dessas trajetórias, como Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 3 POLLIANE TREVISAN NUNES E FERNANDA WANDERER a superação das dificuldades e o mérito acadêmico das entrevistadas. Os excertos abaixo
evidenciam duas dessas narrativas: a superação das dificuldades e o mérito acadêmico das entrevistadas. Os excertos abaixo
evidenciam duas dessas narrativas: Atleta de cálculos, gaúcha de 14 anos se prepara para mundial
A gaúcha Mariana, 14, coleciona 11 medalhas em ciências exatas, incluindo uma de ouro
na Obmep (Olimpíada Brasileira de Matemática das Escolas Públicas). Encontrou seu próprio
método de estudo e chegou a acompanhar aulas em uma universidade federal como ouvinte. […] Quer mostrar que as meninas são tão capazes de ter sucesso em matemática quanto os
garotos. […] Comecei do zero e descobri que sabia mais que alguns colegas que cursaram a
sexta série. Eu temia não saber os conteúdos, mas fui percebendo que eu sabia. Eu não gosto
de apenas ficar lendo. Gosto de ver vídeos e fazer resumos. Mas o método que eu descobri que
funciona comigo é fazer provas simuladas. Eu adoro simuladas! Para estudar história, porém,
prefiro ter aulas. É muito mais abrangente. […] Eu desenvolvi o raciocínio lógico e conseguia
simplificar os problemas na minha cabeça para resolvê-los. Depois da primeira medalha,
comecei a descobrir sozinha outras olimpíadas (Paula SPERB, 2016, online). A sociedade espera que uma mulher tenha sonhos pequenos, mas eu decidi ser
astronauta A sociedade espera que uma mulher tenha sonhos pequenos, mas eu decidi ser
t
t Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120
4 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Assim, se as profissões historicamente ocupadas por homens são as mais valorizadas, as
enunciações presentes nas reportagens apontam para uma tentativa de mostrar que as mulheres
também podem (e devem) estar incluídas. A divulgação de possibilidades para as mulheres,
que até então não são vistas como naturais, poderia ser parte de uma lógica inclusiva. Lopes
(2009) afirma que, ao vivermos em uma sociedade guiada por uma racionalidade neoliberal: Todos devem estar incluídos, mas em diferentes níveis de participação, nas relações que se
estabelecem entre Estado/população, públicos/comunidades e mercado. Não se admite que
alguém perca tudo ou fique sem jogar. Para tanto, as condições principais de participação
são três: primeiro, ser educado em direção a entrar no jogo; segundo, permanecer no jogo
(permanecer incluído); terceiro, desejar permanecer no jogo (LOPES, 2009, p. 155). As três condições de participação citadas acima estão conectadas e se fazem presentes
nas histórias das mulheres examinadas neste artigo. A formação para ter condições de entrar no
jogo passa por mecanismos que, segundo a autora, são mais educadores do que pedagógicos:
“eles simplesmente educam a partir daquilo que mobilizam nos indivíduos” (LOPES, 2009, p. 156). No caso das estudantes descritas acima, essa mobilização se observa na expectativa de
fazer parte do universo da ciência ativado por experiências não necessariamente escolares. Mylena, que visitou a Nasa, em um dado momento de sua narrativa, afirmou: “Quando estive
lá, tive a certeza de que era aquilo que eu queria. Foi quando vi a minha paixão pela Ciência
concretizada”. Já para Mariana, o fato de participar das Olimpíadas Brasileiras de Matemática
das Escolas Públicas evidencia a entrada nesse jogo, ou seja, o vínculo e a participação em
atividades relacionadas às carreiras científicas. Por condições de permanecer no jogo, Lopes (2009) entende as diversas políticas de
inclusão. Embora nenhuma política de equidade de gênero apareça de forma explícita no
material selecionado para este trabalho, é possível estabelecer um paralelo com o fato de que
as estudantes tiveram seu desempenho acima da média identificado por professores e isso, de
alguma forma, modificou suas trajetórias. Mylena, por exemplo, foi selecionada para participar
do projeto internacional de Astronomia pelo seu professor de Física, fato que possibilitou suas
visitas ao centro espacial nos Estados Unidos: “Como eu vinha me destacando no colégio, um
professor de Física começou a observar meu desempenho e interesse pela Ciência, então
me selecionou para participar do programa internacional ‘Caça aos Asteroides’”. MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS A estudante
Mariana foi incentivada por seus professores a participar das competições e a avançar na
escola: “No quinto ano, a professora percebeu que eu tinha um ótimo desempenho e rapidez
para aprender. Ela propôs, então, que eu pulasse o sexto ano”. Estas ações de professores a partir do desempenho escolar das estudantes podem
significar que, talvez, sem esse impulso, as alunas não teriam traçado o mesmo caminho de
destaques acadêmicos. Acerca da inclusão, Alfredo Veiga-Neto e Lopes (2007) afirmam que
a “igualdade de acesso não garante a inclusão e, na mesma medida, não afasta a sombra
da exclusão” (p. 958). Desta forma, não basta estar na escola para ter condições iguais de
competir no contexto acadêmico das ciências exatas. Conforme mostram as histórias de Mylena
e Mariana, a atuação dos professores e a forma como a escola incentiva (ou não) certas
atividades podem ser determinantes da futura inserção das estudantes no campo científico e
tecnológico. No que se refere à intenção de permanecer competindo, Lopes (2009, p. 156) afirma
que “é o desejo que faz com que ninguém fique de fora; é ele que mobiliza os jogadores
a quererem que seus pares continuem jogando”. Isso implica enfrentar as dificuldades que
possam surgir, como no caso de Mylena, que: “precisou movimentar a cidade para conseguir
apoio financeiro e realizar as duas viagens” e que pretende seguir a carreira científica mesmo
sabendo dos problemas que poderá encontrar: “Eu acredito na educação e estou disposta a
lutar por incentivos à ciência no Brasil, pois só ela é capaz de transformar vidas”. O desejo por
competir também se expressa na trajetória de Mariana que, depois de conquistar a primeira
premiação, seguiu competindo já por conta própria, sem o auxílio explícito dos professores:
“Depois da primeira medalha, comecei a descobrir sozinha outras olimpíadas”. O segundo conjunto de reportagens que compõem esta seção analítica aborda a
outra face do que foi apresentado acima: são os casos das mulheres que já têm uma carreira
consolidada na área de ciência e tecnologia, tendo ingressado nesse meio nas décadas de
1970 e 1980. As mulheres narraram suas trajetórias levando em conta as dificuldades pelas
quais passaram desde a graduação e ao longo da carreira, mas com ênfase na superação dos
obstáculos de diversas ordens. A sociedade espera que uma mulher tenha sonhos pequenos, mas eu decidi ser
astronauta Embora as matérias
aqui apresentadas não abordem carreiras consolidadas, pois tratam de jovens mulheres
inserindo-se em áreas de conhecimento onde a presença masculina é que predomina, é possível
ver uma excepcionalidade ao descrever as realizações dessas mulheres, bem como uma busca
por uma justificativa ao contar suas histórias de vida e hábitos escolares e acadêmicos, onde o
esforço aparece como naturalizado. A análise que empreendemos das reportagens sobre as histórias de sucesso de mulheres
nas áreas da ciência e tecnologia evidenciou ressonâncias do imperativo da inclusão (Maura
Corcini LOPES, 2009), mostrando que todos podem ter seu espaço nas carreiras científicas,
inclusive as mulheres. Mas, que inclusão seria essa? Aqui, acompanhamos a discussão de
Lopes (2009, p. 167) ao problematizar a inclusão a partir de uma perspectiva foucaultiana de
governamentalidade: Garantir para cada indivíduo uma condição econômica, escolar e de saúde pressupõe estar
fazendo investimentos para que a situação presente de pobreza, de falta de educação básica
e de ampla miserabilidade humana talvez se modifique em curto e médio prazo. A promessa
da mudança de status dentro de relações de consumo – uma promessa que chega até
aqueles que vivem em condição de pobreza absoluta –, articulada ao desejo de mudança
de condição de vida, são fontes que mantêm o Estado na parceria com o mercado e que
mantêm a inclusão como um imperativo do próprio neoliberalismo (LOPES, 2009, p. 167). No caso das histórias das mulheres aqui examinadas, diríamos que se trata apenas de
uma ressonância desse princípio no qual todos devem ter condições para estar inseridos na
sociedade contemporânea e naquilo que ela valorize, como a área científica e tecnológica. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120
4 4 Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 POLLIANE TREVISAN NUNES E FERNANDA WANDERER POLLIANE TREVISAN NUNES E FERNANDA WANDERER como generificado significa, nesta perspectiva, entendê-lo como não sendo um espaço neutro
em relação às diferenças de gênero, mas, sim, considerar que os discursos que estabelecem
essas diferenças também constituem e reverberam as relações de trabalho. Carpenedo (2011)
destaca que, historicamente, a inclusão das mulheres no mercado de trabalho não se deu
acompanhada necessariamente de uma mudança qualitativa nas relações de trabalho. Assim,
embora o número de mulheres presentes em carreiras de ciência e tecnologia tenha crescido,
é importante considerar a forma como essa inserção ocorreu. As reportagens selecionadas
abaixo evidenciam essa questão: como generificado significa, nesta perspectiva, entendê-lo como não sendo um espaço neutro
em relação às diferenças de gênero, mas, sim, considerar que os discursos que estabelecem
essas diferenças também constituem e reverberam as relações de trabalho. Carpenedo (2011)
destaca que, historicamente, a inclusão das mulheres no mercado de trabalho não se deu
acompanhada necessariamente de uma mudança qualitativa nas relações de trabalho. Assim,
embora o número de mulheres presentes em carreiras de ciência e tecnologia tenha crescido,
é importante considerar a forma como essa inserção ocorreu. As reportagens selecionadas
abaixo evidenciam essa questão: Primeira professora negra no ITA, Sônia Guimarães cobra igualdade para mulheres:
“conservadorismo já não é mais capaz de nos parar” co se
ado s
o já
ão é
a s capa de
os pa a
Sônia Guimarães foi a primeira mulher negra professora no Instituto Tecnológico de Aeronáutica
(ITA) de São José dos Campos. Ela entrou para a sala de aula do ITA quando as mulheres
ainda não eram aceitas no vestibular da instituição militar mais tradicional do país. As roupas
coloridas e a risada alta contrastam com os corredores silenciosos dos laboratórios e com as
fardas azuis dos militares da instituição. Professora de física há 26 anos no ITA, ela também é
pesquisadora na área – onde a presença feminina é ainda menor. […] Era a primeira negra
da instituição, que tinha um número pequeno de docentes mulheres. […] “Eu sei dos números
que eu represento e quero que outras mulheres olhem para mim e vejam que é possível. Eu
combato todos os dias um cenário que contrasta de mim só por estar aqui, mas eu quero mais
que isso” (Poliana CASEMIRO, 2018, online). POLLIANE TREVISAN NUNES E FERNANDA WANDERER O próximo caso é a história de uma professora da área de exatas que enfrentou grandes
dificuldades financeiras antes de ter uma carreira consolidada como pesquisadora na área da
Química: MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Ao olhar para as carreiras de mulheres na área científica e tecnológica, é preciso
considerar o que afirma Manoela Carpenedo (2011) acerca das relações de poder que
constituem as sociedades e, dentro delas, as relações de trabalho. A autora pontua que “o
mercado de trabalho pode ser considerado por si só generificado, caracterizado, sobretudo,
pela norma do trabalhador masculino” (CARPENEDO, 2011, p. 109). Tomar o mercado de trabalho Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 5 PhD em Química por Harvard, brasileira faz pesquisa de ponta com alunos no ensino
médio A fala doce, baixinha e de sotaque carregado já dá a pista. Aquela mulher de aparência
frágil, de não muito mais do que um metro e meio, tem o dom de contornar obstáculos. De
família pobre de Franca, no interior de São Paulo, a professora de Química Joana D’Arc Felix
de Souza, de 53 anos, estudou em apostilas emprestadas e, muitas vezes, dormiu com fome
quando morava em Campinas, onde fez graduação, doutorado e mestrado na Unicamp. De
lá, bateu asas para os Estados Unidos, onde concluiu seu pós-doutorado na Universidade de
Harvard, uma das mais prestigiadas do mundo. […] – Cheguei a passar fome, mas decidi
vencer pelos estudos. Meu pai dizia: para atingir seus objetivos, tem que passar pelo sacrifício. Quem não nasce em berço de ouro tem que arregaçar as mangas. Se você desistir, nunca vai
chegar lá. E ela chegou (Flávia JUNQUEIRA, 2017, online). O que se evidencia nessa matéria é a forma como narra a professora: alguém capaz
de superar os obstáculos com tranquilidade, sem pensar em desistir ou rebelar-se contra eles. A
docilidade faz com que Joana consiga superar as dificuldades em embates, como fica expresso
já no início da matéria, que conta a história trazendo elementos afetivos: “A fala doce, baixinha
e de sotaque carregado já dá a pista. Aquela mulher de aparência frágil, de não muito mais
do que um metro e meio, tem o dom de contornar obstáculos”. Aqui é possível estabelecer um
contraponto com o caso anterior, de Sônia, que ri alto e contrasta com o silêncio, enquanto
Joana fala baixo e contorna as dificuldades, configurando assim formas diferentes de inserção
de mulheres no campo científico. A notícia a seguir aborda a vida e a carreira de uma mulher que assumiu a direção de
uma escola de engenharia brasileira, possibilitando trazer para a discussão o que poderia ser
considerado um ponto alto destas trajetórias acadêmicas: MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Liedi afirma que sua chegada ao cargo de diretora é importante para as demais alunas
e engenheiras porque, no seu tempo de estudante, não havia para quem reportar qualquer
problema relativo a gênero: “Eram todos homens. A mulher se calava, engolia em seco. E, se
reagisse, era chamada de histérica, o que é pesadíssimo”. Segundo Liedi, é simbólico que
haja uma mulher dirigindo a Poli-USP, mostrando, com isso, que há espaço para mulheres em
uma instituição “conservadora e tradicional”, onde, a princípio, esse espaço pode ser difícil de
conseguir. Neste sentido, uma mulher ocupando o cargo de direção seria uma possibilidade de
ter um espaço de escuta mais receptivo às demandas sobre questões de gênero nesse âmbito. Na trajetória de Liedi também está presente a questão da conciliação entre a vida
pessoal e a carreira acadêmica, dinâmica esta que, muitas vezes, é difícil de organizar. Em seu
relato, vemos que ela teve que se dividir entre o trabalho e a formação acadêmica e o cuidado
dos filhos. Acerca da inserção das mulheres no mercado de trabalho, Carpenedo (2011) lembra
que essa inserção não é acompanhada muitas vezes pela diminuição das responsabilidades
femininas dentro da esfera privada no que diz respeito ao trabalho reprodutivo, visto que a
figura da mulher como a principal responsável pelos cuidados e reprodução da vida ainda
continua fortemente consolidada (CARPENEDO, 2011, p. 15). As três reportagens apresentadas anteriormente sobre as mulheres bem-sucedidas nas
áreas da ciência e tecnologia evidenciam suas formas de permanecer na carreira e de superar
as dificuldades para construir uma carreira, formas essas vinculadas ao esforço individual e à
autonomia para lidar com a competitividade. Pode-se entender que os obstáculos de ordem
estrutural e cultural, como o preconceito de gênero e a falta de recursos financeiros, foram
enfrentados de forma individual e não coletiva ou institucional, conforme se observa nos excertos
a seguir, extraídos das reportagens analisadas: “Apesar das ‘ofensas costumeiras’, como ela
descreve o machismo na Universidade, Liedi não duvidava da sua capacidade”; “De família
pobre (…) estudou em apostilas emprestadas e, muitas vezes, dormiu com fome quando morava
em Campinas, onde fez graduação, doutorado e mestrado na Unicamp”; “A partir do momento
que a gente não acredita no preconceito, conquistamos nossos objetivos”; “Temos que nos
expor, dar a cara à tapa. Se você se dispõe a ser diretora de uma instituição tradicionalmente
masculina, tem que encarar”. Mulher assume direção da Poli-USP pela primeira vez em 124 anos. Liedi Bernucci, 59,
chefi ará uma das maiores escolas de engenharia do país Mulher assume direção da Poli USP pela primeira vez em 124 anos. Liedi Bernucci, 59,
chefi ará uma das maiores escolas de engenharia do país
Ao chegar à sala de aula da Escola Politécnica da USP, em 1977, a estudante Liedi Bernucci,
então com 19 anos, ouviu de um professor: “Mulher não deveria entrar na engenharia, porque
o que elas querem é casar e acabam roubando a vaga de um homem”. É verdade que Liedi se
casou, tempos depois, e também “roubou a vaga” de um homem, por assim dizer: tornou-se a
primeira mulher a assumir a diretoria da Poli, uma das principais escolas de engenharia do país,
após 124 anos de chefia masculina. O episódio em sala de aula poderia ter feito a estudante
desistir, mas Liedi seguiu o conselho da mãe: “A melhor resposta é seguir em frente”. Com 59
anos, ela foi eleita nesta quarta-feira (7) para o cargo máximo administrativo da Poli, uma
instituição com 452 docentes e mais de 8.000 estudantes. Apesar das “ofensas costumeiras”,
como ela descreve o machismo na universidade, Liedi não duvidava da sua capacidade. Era
boa aluna, com notas altas, e isso bastava. “Sou engenheira e objetiva, acredito nos números. Eles falam. Se na comparação eu estava melhor, não tinha como falar que eu era burra.” (…)
“A vida era conturbada, com dois filhos pequenos, mais trabalho e estudo. Era raro mulher com
filho pequeno trabalhar naquela época. Mas, olhando para trás, isso me fortaleceu. Eu tinha
que organizar o meu tempo, me dividir em mil tarefas, e achar que ia dar certo. Me fez ser
otimista.” (Marina ESTARQUE, 2018, online). Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 6 POLLIANE TREVISAN NUNES E FERNANDA WANDERER POLLIANE TREVISAN NUNES E FERNANDA WANDERER é possível afirmar que o discurso da superação se articula com o discurso da competitividade. Liedi, por exemplo, ao enfrentar problemas com um professor, ainda na graduação, em um
episódio que “poderia ter feito a estudante desistir”, diz que “a melhor resposta é seguir em
frente”, ou seja, seguir no jogo. Na história de Joana, a superação fica mais evidente, e pode-se
afirmar, a partir de sua fala, que as dificuldades não constituem uma razão forte o suficiente para
deixar de competir e conquistar um espaço no campo científico, uma vez que ela, “estudando
em apostilas emprestadas, passou nas três universidades estaduais de São Paulo: Unicamp, USP
e Unesp”. Além disso, segundo a matéria: “Joana conta sobre essa fase de sua vida sem nenhum
traço de amargura”. Dessa colocação, podemos destacar a resiliência e a flexibilidade, a
capacidade de “contornar obstáculos”, características que também fazem parte do discurso
neoliberal, conforme indica Saraiva (2014): Os corpos dóceis e adaptados a uma rotina sobre a qual eles não têm quase nenhuma
ingerência já não servem para a empresa. Os trabalhadores agora devem ser proativos,
autônomos e empreendedores. Os corpos dóceis devem ser substituídos por cérebros flexíveis
(p. 147). Acerca da competitividade inerente à racionalidade neoliberal, Carpenedo (2011)
afirma que, ao mesmo tempo em que as políticas públicas para equidade de gênero são
indispensáveis para mudanças de cenário, a concepção de igualdade refere-se, muitas vezes,
ao fato de todos e todas estarem inseridos em um contexto de competição, mobilizando o
discurso da meritocracia: Jogar o jogo da igualdade de oportunidades implica na necessidade de as mulheres
responderem às demandas capitalistas exigidas pelo contexto contemporâneo às performances
masculinas (isentas das funções de cuidado e da conhecida dupla jornada) para o sucesso no
mundo do trabalho (CARPENEDO, 2011, p. 117). As mulheres entrevistadas narram-se como vitoriosas, capazes de superar as dificuldades
a partir de valores familiares ou de dedicação aos estudos. Sobre o ato de narrar-se, Larrosa
(2008) afirma: Aqui os sujeitos não são posicionados como objetos silenciosos, mas como sujeitos falantes; não
como objetos examinados, mas como sujeitos confessantes; não em relação a uma verdade
sobre si mesmos que lhes é imposta de fora, mas em relação a uma verdade sobre si mesmos
que eles mesmos devem contribuir ativamente para produzir (p. 54). POLLIANE TREVISAN NUNES E FERNANDA WANDERER Embora uma entrevista não se trate necessariamente de uma confissão, no sentido
proposto por Larrosa (2008), ainda assim, coloca em circulação significados para as trajetórias
que reverberam discursos. Desse modo, tanto a narrativa dos jornais quanto a autonarrativa
das entrevistadas articulam-se em um processo de constituição de subjetividades, nas quais se
evidencia a presença do discurso do empresariamento de si. ç
Nas falas das entrevistadas, tanto as que já têm uma carreira consolidada, quanto as
que estão ainda no Ensino Médio ou nos primeiros anos da graduação/pós-graduação, está
presente a ideia da autonomia, seja para trabalhar ou para estabelecer seus próprios métodos de
estudo, como indicam os excertos abaixo: “Encontrou seu próprio método de estudos e chegou
a acompanhar aulas em uma universidade.”; “Estudar nunca foi uma obrigação. No começo,
não era apaixonada por matemática, mas nunca tive dificuldade.”; “No quinto ano, a professora
percebeu que eu tinha um ótimo desempenho e rapidez para aprender. Ela propôs, então, que
eu pulasse o sexto ano. (...) Foi por causa disso que virei autodidata.”; “Já não preciso apenas
do que o professor fala em sala de aula. Essa habilidade me ajudou muito na preparação para
as olimpíadas de que participei.”; “O método que eu descobri que funciona comigo é fazer
provas simuladas.”; “Eu desenvolvi raciocínio lógico e conseguia simplificar os problemas na
minha cabeça para resolvê-los. Depois da primeira medalha, comecei a descobrir sozinha outras
olimpíadas.”; “A engenheira diz que não gostava muito da escola na infância – ela aprendeu a
estudar com a irmã, que fez matemática na USP, no vestibular.”; “Para eu ficar quietinha, minha
mãe me ensinou a ler o jornal que chegava na casa. Sem estudo, minha mãe foi minha primeira
professora. Ela só tinha até a 4ª série.”; “A independência e autonomia agradam a cientista.”. A valorização da autonomia é uma característica importante da sociedade
contemporânea, mobilizada pela racionalidade neoliberal que, como visto, constitui modos
de ser que ultrapassam o âmbito econômico. Acerca desse tema, Saraiva (2014) afirma que a
racionalidade neoliberal pretende estabelecer cada sujeito como empreendedor de si mesmo,
ou seja, responsável pela sua própria inserção na sociedade de consumo. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Enfatiza-se a ideia de que os esforços árduos têm recompensas,
como expressa Joana: “Cheguei a passar fome, mas decidi vencer pelos estudos. Meu pai dizia:
para atingir seus objetivos, tem que passar pelo sacrifício”. Esta ideia que destaca e premia o esforço individual como forma central de atingir
objetivos, a despeito de obstáculos estruturais, faz parte do que Foucault (2008) define como
racionalidade neoliberal. Acerca dessa noção, Karla Saraiva (2014) afirma que o liberalismo (e, posteriormente, o neoliberalismo também) é compreendido pelo filósofo
como uma racionalidade que estabelece determinadas práticas. Essa racionalidade – que
Foucault chama de governamentalidade –, ainda que possa aparecer de modo condensado
nas formas de governar um Estado, atravessa toda a sociedade e implica em práticas de
condução de condutas que extrapolam as ações estatais e se desdobram de modo muito
mais amplo (p. 142). Nesta perspectiva, a lógica neoliberal não fica restrita apenas às esferas governamentais
ou do mercado, mas constitui práticas e funcionamentos em toda a sociedade. Um desses
funcionamentos seria considerar a educação como um investimento, bem como a inclusão
de todos como consumidores em algum grau, conforme abordado anteriormente por meio
do imperativo da inclusão. Saraiva e Veiga-Neto (2009) afirmam, na esteira de Foucault, que a
racionalidade neoliberal expressa uma diferença em relação ao liberalismo: A governamentalidade neoliberal intervirá para maximizar a competição, para produzir
liberdade para que todos possam estar no jogo econômico. Dessa maneira, o neoliberalismo
constantemente produz e consome liberdade. Isso equivale a dizer que a própria liberdade
transforma-se em mais um objeto de consumo (SARAIVA; VEIGA-NETO, 2009, p. 189). Ainda de acordo com os autores, uma marca potente da racionalidade neoliberal
relaciona-se à noção de que a liberdade de mercado e a competividade não são naturais,
elas precisam ser estimuladas: No neoliberalismo a ênfase se desloca para a concorrência. Uma concorrência que atravessa
a sociedade em todos os seus níveis e em todas as esferas. Concorrência entre empresas, mas
também entre indivíduos. Concorrência no âmbito dos assuntos considerados econômicos,
mas também naqueles que estariam fora do estrito campo da economia (SARAIVA, 2014, p. 145). A permeabilidade da competitividade na constituição dos sujeitos, neste caso, nas
mulheres com carreiras consolidadas, é observável em algumas de suas falas, em que também Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 7 ,
p
,
( )
DOI: 10.1590/1806-9584-2021v29n268120 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS são geridos pelo próprio sujeito” (SARAIVA, 2014, p. 148). Assim, no processo de criação de novas
formas de produzir e se relacionar com o trabalho, outras subjetividades entram em jogo para
adequar-se ao novo funcionamento. Segundo Sylvio de Sousa Gadelha Costa (2009), acompanhando este processo de
mudanças na sociedade, houve uma expansão dos valores e ideias inicialmente presentes
apenas no universo empresarial para os demais contextos sociais. Nas palavras do autor, determinados valores econômicos, à medida que migraram da economia para outros domínios
da vida social, disseminando-se socialmente, ganharam um forte poder normativo, instituindo
processos e políticas de subjetivação que vêm transformando sujeitos de direitos em indivíduos-
microempresas-empreendedores (COSTA, 2009, p. 172). Desse modo, segundo Costa (2009), estes funcionamentos empresariais não apenas se
expandem como também passam a compor, normativamente, a subjetividade dos indivíduos,
constituindo-os enquanto sujeitos empreendedores de si mesmos. Desde a perspectiva do
empresariamento de si, é possível considerar que a educação e os processos de aprendizagem
são centrais nesta nova configuração social. Esse entendimento, segundo Osvaldo Javier López-
Ruiz (2004), é possível a partir da leitura da teoria do capital humano feita por Foucault. Esta noção
de capital humano, consolidada nos Estados Unidos pelos economistas da Escola de Chicago
na década de 1960, postula que a formação educacional dos sujeitos pode ser tomada como
um investimento, que, se bem administrado, pode render frutos no futuro (LÓPEZ-RUIZ, 2004). Esse
autor afirma, ainda, que a grande inflexão proposta pela teoria do capital humano é tornar o
investimento em si próprio, enquanto capital, um processo desejado e compreendido por todos: A ciência econômica, nesse caso, não cria só uma teoria sobre a economia; cria um repertório
de interpretação que nos permite pensar e pensar-nos de maneira tal que não nos resulte
repulsiva a imagem do humano como riqueza – como o havia sido em tempos de J. S. Mill. A
partir de seus postulados “cientificamente verificáveis”, o humano passa a ser entendido como
uma forma de capital e, portanto, o “capital humano” e tudo o que se faça para incrementá-lo
é investido de um valor positivo: cada pessoa deve – porque é economicamente conveniente,
mas também porque é “moralmente bom” – aumentar suas habilidades, competências e
destrezas a partir de “investimentos” constantes (LÓPEZ-RUIZ, 2004, p. 38). MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Considerando a valorização do capital humano pela racionalidade neoliberal, Saraiva
e Veiga-Neto (2009) afirmam que “o aprender a aprender significaria tornar-se empresário de si,
colocando-se num processo de gestão daquilo que, segundo Foucault, é chamado de capital
humano pelo neoliberalismo” (SARAIVA; VEIGA-NETO, 2009, p. 199). Dessa forma, as escolas, além
de ensinarem saberes, ficam encarregadas de ensinar os sujeitos a investirem em si mesmos por
meio da educação, já que “gerir seu capital humano é buscar estratégias de multiplicá-lo. À
escola caberia ensinar essas técnicas de gestão” (SARAIVA; VEIGA-NETO, 2009, p. 199). Reverberações desse discurso podem ser observadas nas falas das mulheres entrevistadas. Mariana, por exemplo, afirma que desenvolveu seu próprio método de estudos para as olimpíadas
de que participa: “Já não preciso apenas do que o professor fala em sala de aula. Essa habilidade
me ajudou muito na preparação para as olimpíadas de que participei”. Sua autonomia
para estudar é tanta que, segundo ela, precisa cada vez menos da escola para manter seu
desempenho. O caso é emblemático ao mostrar o quanto a capacidade de aprender por conta
própria e administrar a própria rotina de estudos é uma disposição que acompanha esses sujeitos
ao longo da vida. Segundo Iolanda Montano dos Santos e Viviane Klaus (2013, p. 64): A forma contemporânea de viver requer um sujeito aprendente por toda a vida que pode
recriar continuamente o seu eu ao se tornar um agente de resolução de problemas; um sujeito
que seja responsável pelo progresso social e pela realização pessoal de sua própria vida, ou
seja, um sujeito empresário de si, um sujeito ‘gestor’. Mariana afirma, ainda, o quanto a autonomia constituiu sua forma de construir
conhecimento: “Eu desenvolvi o raciocínio lógico e conseguia simplificar os problemas na
minha cabeça para resolvê-los. Depois da primeira medalha, comecei a descobrir sozinha
outras olimpíadas”. Assim como desenvolver o raciocínio lógico é uma habilidade valorizada
na contemporaneidade, a capacidade de resolver problemas é uma habilidade-chave para
o discurso do empreendedorismo de si, pois, contemporaneamente, são priorizados “enfoques
que, em geral, promovem a iniciativa dos alunos, incentivando-os a fazer uma gestão de sua
aprendizagem” (SARAIVA, 2014, p. 150). Neste sentido, os aprendizados não devem ser estanques,
mas estarem a serviço de uma flexibilização das habilidades para a resolução de problemas. POLLIANE TREVISAN NUNES E FERNANDA WANDERER Ao apontar as
mudanças nas relações de trabalho, que tendem para o que Saraiva (2014) chama de trabalho
imaterial, essa autora observa as modificações ocorridas nos próprios sujeitos, no sentido de
se constituírem como autônomos e flexíveis: “os corpos e os cérebros que o trabalho imaterial
requer estão de acordo com esse princípio: já não priorizam a obediência a regulamentos, mas 8 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 Considerações fi nais Finalizando o artigo, temos o propósito de apresentar algumas implicações do estudo
para a área da Educação. Como esperamos ter apontado ao longo do texto, as discussões
de gênero e ciência, na atualidade, não escapam à lógica neoliberal presente em nossa
sociedade, com ênfase aos processos de inclusão, valorização do capital humano individual e do
empresariamento de si. Como visto anteriormente, boa parte das estratégias de enfrentamento
pelas mulheres dos problemas no campo científico se dá desde uma perspectiva individual e
não coletiva. Cada mulher cientista encontra seu próprio caminho de superação, apesar de a
discussão de gênero ser bastante publicizada nas redes sociais. Nota-se, desse modo, o fortalecimento, ou o “empoderamento”, de indivíduos e não
de coletivos. Não cabe, aqui, um julgamento acerca das estratégias individuais ou coletivas
como mais ou menos eficazes para combater as discriminações contra as mulheres. Entretanto,
é importante salientar que o individualismo faz parte da racionalidade neoliberal que constitui
a sociedade contemporânea, na qual a liberdade individual está conjugada com outros
interesses (do mercado, por exemplo) e pode, portanto, ser problematizada. Como afirmam
Santos e Klaus (2013, p. 71), no mundo contemporâneo a “autonomia” dos sujeitos e das instituições (...), o aumento da
“liberdade de escolha”, a constituição de comunidades autogovernáveis – fragmentação
social do todo social –, a proliferação dos discursos sobre o respeito e a tolerância para com
a diversidade, as discussões sobre a equidade social e o avanço da democracia nos fazem
acreditar que vivemos em uma sociedade mais “libertadora”. Porém, somos cada vez mais
regulados. A realização desta pesquisa permitiu observar dois movimentos sendo produzidos pelas
redes sociais a respeito da posição das mulheres no campo científico. O primeiro aponta que
a emergência do debate sobre gênero e ciências está para além dos espaços acadêmicos
ou militantes, relacionando-se com o compartilhamento de informações que as redes sociais
possibilitam. Essa rede alimenta ainda mais o debate, ao mesmo tempo em que é alimentada
pelo que ocorre “off-line”. Esta circulação de enunciados é um dos movimentos que possibilita
o rompimento de uma barreira invisível de silêncio, fazendo com que as informações sobre as
condições de inserção das mulheres no meio científico e tecnológico cheguem a um número
maior de pessoas. Com isso, esse movimento possibilita também que as mulheres encontrem
outras pessoas em situações semelhantes, o que pode produzir encontros, tensionamentos e
mudanças de conjuntura. MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS ç
ç
p
ç
p
As reportagens que compõem esta seção lançam o olhar para as diversas faces do que
constituiria o espaço da mulher na área de exatas. As narrativas contam o caminho percorrido
até o momento pelas entrevistadas, as dificuldades enfrentadas no contexto acadêmico e no
mercado de trabalho, as mudanças ocorridas ao longo do tempo, as conquistas profissionais
que ocorrem a despeito dos problemas, bem como as perspectivas de futuro. Pode-se afirmar Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 9 POLLIANE TREVISAN NUNES E FERNANDA WANDERER que a mídia produz a relação entre mulheres e ciência, enfatizando as trajetórias de sucesso,
com foco no indivíduo. Situa as mulheres não apenas como vítimas de processos discriminatórios,
mas como resilientes e otimistas em relação ao futuro que depende, apenas, de si. que a mídia produz a relação entre mulheres e ciência, enfatizando as trajetórias de sucesso,
com foco no indivíduo. Situa as mulheres não apenas como vítimas de processos discriminatórios,
mas como resilientes e otimistas em relação ao futuro que depende, apenas, de si. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 Considerações fi nais ç
O segundo movimento diz respeito à presença do discurso do empresariamento de si
nas narrativas e trajetórias das mulheres que se destacam na área das ciências exatas. Divulgar
os casos de sucesso era uma ação recorrente nas páginas acompanhadas ao longo da
realização desta pesquisa. O principal argumento para o compartilhamento de suas histórias é
que elas podem servir como exemplo para outras mulheres, especialmente as mais jovens, que
poderiam ver que existe espaço para elas nesse campo de trabalho. Porém, o que foi possível
observar é que essas trajetórias são marcadas pelo enfrentamento individual – e, por vezes,
solitário – das dificuldades para consolidar uma carreira e lidar com os preconceitos de gênero. Esse enfrentamento aparece no material analisado como uma superação positiva, que pode
ser associada à racionalidade neoliberal em que o indivíduo é o único responsável por sua
educação e qualificação profissional. Desse modo, nas palavras de Zygmunt Bauman e David Lyon (2014, p. 132), as pessoas
são estimuladas a buscar, sem muitas possibilidades de falhas, “soluções individuais para
problemas socialmente gerados”. Os autores ressaltam que a sociedade contemporânea institui
os indivíduos de direito, mas que nem sempre isso se efetiva na prática, porque os problemas
com que esses indivíduos se deparam apenas podem ter uma solução coletiva. Constitui-se,
então, um sujeito em dívida com a solução desses problemas coletivos. Portanto, propagar “casos de sucesso” em que a ênfase da narrativa é apenas na
superação individual reforça este discurso meritocrático e enfraquece a coletividade que, a
princípio, é necessária para realizar modificações culturais e estruturais na sociedade. A análise
que realizamos não pretende minimizar os esforços individuais que constituem as trajetórias das
mulheres apresentadas pelas reportagens, uma vez que a relação entre indivíduo e sociedade é
bastante complexa. Não se trata, também, de afirmar que o empreendedorismo é algo apenas
negativo, pois, embora seus possíveis efeitos não sejam objeto desta pesquisa, entendemos que
a divulgação dessas histórias pode atuar, sim, como elemento catalisador de mudanças. 10 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Outro aspecto que se pode depreender da análise realizada neste trabalho e a partir
dos apontamentos de Lyon (2014) é que a política não necessariamente acompanhou as
modificações na sociedade contemporânea, veloz e global: O poder agora existe num espaço global e extraterritorial, mas a política, que antes ligava
interesses individuais e públicos, continua local, incapaz de agir em nível planetário. Sem
controle político, o poder torna-se fonte de grande incerteza, enquanto a política parece
irrelevante para os problemas e temores da vida das pessoas (LYON, 2014, p. 13). Por isso, talvez, a ênfase atual seja nos movimentos virtuais, que dependeriam menos
da política, já que podem ser iniciados por um único indivíduo e provocar uma adesão e
uma publicização extremamente rápidas, sem necessariamente ter uma pauta discutida
coletivamente, o que, nas formas políticas tradicionais, levaria tempo. Não é objetivo deste
trabalho produzir qualquer escrita prescritiva, porém, é importante que as iniciativas que
buscarão enfrentar quaisquer discriminações, neste caso, discriminações de gênero no contexto
científico e tecnológico, levem em consideração que talvez seja importante uma retomada do
político e do coletivo enquanto formas de atuação na sociedade. Ainda a partir de Bauman e
Lyon (2014, p. 133), temos que as memórias ‘quentes’ que poderiam moldar e dirigir o desenvolvimento cultural de formas
apropriadamente éticas são substituídas pela frieza de dedicar atenção ao e-mail recebido, à
atualização do status e ao prognóstico revisado, enquanto eles voam pela nossa consciência. O fluxo incessante de informações, segundo esses autores, tem a capacidade de diluir a
memória do que é culturalmente produzido. Talvez a velocidade que caracteriza a internet e a
sociedade contemporânea não dê tempo para que as memórias coletivas se tornem fortes. Por
isso, não é evidente a continuidade dos movimentos virtuais de gênero, em que a viralização
de um tópico suplanta o anterior no dia seguinte, sem que tenhamos condições de pensar em
ações e análises mais demoradas sobre os diversos temas polêmicos que são compartilhados. Apesar disso, não se pode negar a efetividade desses mecanismos na disputa por espaço e voz. Como afirma Manuel Castells (2003, p. 11): “Nem utopia nem distopia, a internet é a expressão
de nós mesmos através de um código de comunicação específico, que devemos compreender
se quisermos mudar nossa realidade”. Este artigo objetivou compreender alguns aspectos
presentes nas redes sociais para, quem sabe, mudarmos nossa realidade. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 Referências AMANTE, Lúcia. “Facebook e novas sociabilidades: contributos da investigação”. In: PORTO,
Cristiane; SANTOS, Edmea (Orgs.). Facebook e educação: publicar, curtir, compartilhar. Campina
Grande: Editora da Universidade Estadual da Paraíba, 2014. p. 26-46. BAUMAN, Zygmunt; LYON, David. Vigilância líquida: diálogos com David Lyon. Rio de Janeiro:
Zahar, 2014. CARPENEDO, Manoela. Quando a resistência se torna política pública: analisando a produção
de subjetividade(s) nas políticas de equidade de gênero no campo do trabalho. 2011. Mestrado
(Programa de Pós-Graduação em Psicologia Social) – Faculdade de Psicologia da Universidade
Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil. CASEMIRO, Poliana. “Primeira professora negra no ITA, Sônia Guimarães cobra igualdade para
mulheres: conservadorismo já não é mais capaz de nos parar”. G1 Notícias [online], São Paulo,
08/03/2018. Disponível em https://g1.globo.com/sp/vale-do-paraiba-regiao/noticia/primeira-
professora-negra-no-ita-sonia-guimaraes-cobra-igualdade-para-mulheres-conservadorismo-ja-
nao-e-mais-capaz-de-nos- parar.ghtml. Acesso em 21/10/2019. CASTELLS, Manuel. A galáxia da Internet: reflexões sobre a Internet, os negócios e a sociedade. Rio de Janeiro: Zahar, 2003. CORRÊA, Raimunda de Nazaré Fernandes. Gênero, saber e poder: mulheres nas engenharias da
Universidade Federal do Pará. 2011. Mestrado (Programa de Pós-Graduação em Desenvolvimento
Sustentável do Trópico Úmido) – Núcleo de Altos Estudos Amazônicos da Universidade Federal do
Pará, Belém, PA, Brasil. COSTA, Sylvio de Sousa Gadelha. “Governamentalidade neoliberal, teoria do capital humano e
empreendedorismo”. Educação & Realidade, Porto Alegre, v. 34, n. 2, p. 171-186, maio/ago. 2009. ESTARQUE, Marina. “Mulher assume direção da Poli-USP pela primeira vez em 124 anos. Liedi
Bernucci, 59, chefiará uma das maiores escolas de engenharia do país”. Folha de São Paulo Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 11 POLLIANE TREVISAN NUNES E FERNANDA WANDERER [online], 08/03/2018. Disponível em https://www1.folha.uol.com.br/cotidiano/2018/03/mulher-
assume-direcao-da-poli-usp-pela-primeira-vez-em- 124-anos.shtml. Acesso em 21/10/2019. FISCHER, Rosa Maria Bueno. “Foucault e a análise do discurso em educação”. Cadernos de
Pesquisa, São Paulo, n. 114, p. 197-223, nov. 2001. FORECHI, Marcilene. Identidades femininas em comentários no Facebook: uma análise a
partir dos Estudos Culturais em Educação. 2018. Doutorado (Programa de Pós-Graduação em
Educação) – Faculdade de Educação da Universidade Federal do Rio Grande do Sul, Porto
Alegre, RS, Brasil. FOUCAULT, Michel. Nascimento da Biopolítica. São Paulo: Martins Fontes, 2008. FOUCAULT, Michel. A arqueologia do saber. Rio de Janeiro: Forense Universitária, 2004. JUNQUEIRA, Flávia. “PhD em Química por Harvard, brasileira faz pesquisa de ponta com alunos
no ensino médio”. Jornal Extra [online], São Paulo, 25/09/2017. Disponível em https://extra.globo. com/noticias/rio/phd-em-quimica-por-harvard-brasileira-faz-pesquisa-de-ponta-com-alunos-no-
ensino-medio-21863584.html. Acesso em 21/10/2019. LARROSA, Jorge. “Tecnologias do eu e educação”. Referências In: SILVA, Tomaz Tadeu da. O sujeito da
educação: estudos foucaultianos. Petrópolis: Vozes, 2008. p. 35-86. LIBERATO, Tatiane Furukawa; ANDRADE, Thales Haddas Novaes de. “Relações de gênero e
inovação: atuação de mulheres nos NITs paulistas”. Revista Estudos Feministas, Florianópolis,
v. 26, n. 2, ago. 2018. Disponível em https://periodicos.ufsc.br/index.php/ref/article/view/41763. Acesso em 11/09/2019. LOMBARDI, Maria Rosa. Perseverança e resistência: a Engenharia como profissão feminina. 2004. Doutorado (Programa de Pós-Graduação em Educação) – Faculdade de Educação da
Universidade Estadual de Campinas, Campinas, SP, Brasil. LOPES, Maura Corcini. “Políticas de inclusão e governamentalidade”. Educação & Realidade,
Porto Alegre, v. 34, n. 2, p. 153-169, maio/ago. 2009. LÓPEZ-RUIZ, Osvaldo Javier. O ethos dos executivos das transnacionais e o espírito do capitalismo. 2004. Doutorado (Departamento de Sociologia) – Instituto de Filosofia e Ciências Humanas da
Universidade Estadual de Campinas, Campinas, SP, Brasil. LOURO, Guacira Lopes. Gênero, sexualidade e educação: uma perspectiva pós-estruturalista. Petrópolis: Vozes, 2014. LUZZARDI, Luciana. Força e luz: natureza, cultura e pedagogias de gênero na CEEE transmissão. 2017. Mestrado (Programa de Pós-Graduação em Educação) – Faculdade de Educação da
Universidade Luterana do Brasil, Canoas, RS, Brasil. LYON, David. “Introdução”. In: BAUMAN, Zygmunt; LYON, David. Vigilância líquida: diálogos com
David Lyon. Rio de Janeiro: Zahar, 2014. p. 9-24. MELO, Maria Celia Macedo Araújo. Gênero e universidade: a presença da mulher aluna nos
Cursos do Centro de Ciências Exatas e Tecnologia da Universidade Federal do Maranhão. 2013. Mestrado (Programa de Pós-Graduação em Educação) – Faculdade de Educação da
Universidade Federal do Maranhão, São Luís, MA, Brasil. MELO, Maria Celia Macedo Araújo. Gênero e universidade: a presença da mulher aluna nos
Cursos do Centro de Ciências Exatas e Tecnologia da Universidade Federal do Maranhão. 2013. Mestrado (Programa de Pós-Graduação em Educação) – Faculdade de Educação da
Universidade Federal do Maranhão, São Luís, MA, Brasil. MEYER, Dagmar Estermann. “Abordagens pós-estruturalistas de pesquisa na interface educação,
saúde e gênero: perspectiva metodológica”. In: MEYER, Dagmar Estermann; PARAÍSO, Marlucy
Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. Belo Horizonte: Mazza
Edições, 2012. p. 49-64. MEYER, Dagmar Estermann. “Abordagens pós-estruturalistas de pesquisa na interface educação,
saúde e gênero: perspectiva metodológica”. In: MEYER, Dagmar Estermann; PARAÍSO, Marlucy
Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. Belo Horizonte: Mazza
Edições, 2012. p. 49-64. MEYER, Dagmar Estermann; PARAÍSO, Marlucy Alves. “Metodologias de pesquisa pós-críticas ou
sobre como fazemos nossas investigações”. In: MEYER, Dagmar Estermann; PARAÍSO, Marlucy
Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS MULHERES NA CIÊNCIA. “A sociedade espera que uma mulher tenha sonhos pequenos, mas eu
decidi ser astronauta”. Mulheres na Ciência [online], 18/12/2017. Disponível em https://www. paramulheresnaciencia.com.br/noticias/sociedade-espera-que-uma-mulher-tenha-sonhos-
pequenos-mas-eu-decidi-ser-astronauta-diz-jovem-brasileira-de-18-anos-que-visitou-nasa. Acesso em 21/10/2019. PARAÍSO, Marlucy Alves. “Metodologias de pesquisas pós-críticas em educação e currículo:
trajetórias, pressupostos, procedimentos e estratégias analíticas”. In: MEYER, Dagmar Estermann;
PARAÍSO, Marlucy Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. Belo
Horizonte: Mazza Edições, 2012. p. 25-48. PARAÍSO, Marlucy Alves. “Metodologias de pesquisas pós-críticas em educação e currículo:
trajetórias, pressupostos, procedimentos e estratégias analíticas”. In: MEYER, Dagmar Estermann;
PARAÍSO, Marlucy Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. Belo
Horizonte: Mazza Edições, 2012. p. 25-48. SANTOS, Iolanda Montano dos; KLAUS, Viviane. “A inclusão e o sujeito empresário de si”. In: FABRIS,
Elí Henn; KLEIN, Rejane Ramos. Inclusão e biopolítica. Belo Horizonte: Editora Autêntica, 2013. p. 61-78. SANTOS, Iolanda Montano dos; KLAUS, Viviane. “A inclusão e o sujeito empresário de si”. In: FABRIS,
Elí Henn; KLEIN, Rejane Ramos. Inclusão e biopolítica. Belo Horizonte: Editora Autêntica, 2013. p. 61-78. SARAIVA, Karla. “A aliança biopolítica educação-trabalho”. Revista Pro-Posições, Campinas, v. 25, n. 2, p. 139-156, maio/ago. 2014. SARAIVA, Karla; VEIGA-NETO, Alfredo. “Modernidade Líquida, Capitalismo Cognitivo e Educação
Contemporânea”. Educação & Realidade, Porto Alegre, v. 34, n. 2, p. 187-202, maio/ago. 2009. SCHIEBINGER, Londa. “Mais mulheres na ciência: questões de conhecimento”. Revista História,
Ciências, Saúde, Rio de Janeiro, v. 15, p. 269-282, jun./set. 2008. SCHIEBINGER, Londa. O feminismo mudou a ciência? Bauru: Editora da USC, 2001. SILVA, Fabiane Ferreira da. Mulheres na ciência: Vozes, tempos, lugares e trajetórias. 2012. Doutorado (Programa de Pós-Graduação em Educação em Ciências) – Faculdade de Química
da Universidade Federal do Rio Grande, Rio Grande, RS, Brasil. SPERB, Paula. “Atleta de cálculos, gaúcha de 14 anos se prepara para mundial”. Folha de
São Paulo [online], São Paulo, 21/02/2016. Disponível em https://www1.folha.uol.com.br/
educacao/2016/02/1741518-medalhista-de-matematica-gaucha-de-14-anos-estuda-para-
mundial.shtml. Acesso em 21/10/2019. SPERB, Paula. “Atleta de cálculos, gaúcha de 14 anos se prepara para mundial”. Folha de
São Paulo [online], São Paulo, 21/02/2016. Disponível em https://www1.folha.uol.com.br/
educacao/2016/02/1741518-medalhista-de-matematica-gaucha-de-14-anos-estuda-para-
mundial.shtml. Acesso em 21/10/2019. VEIGA-NETO, Alfredo; LOPES, Maura Corcini. “Inclusão e governamentalidade”. Educação &
Sociedade, Campinas, v. 28, n. 100, p. 947-964, out./dez. 2007. VEIGA-NETO, Alfredo; LOPES, Maura Corcini. “Inclusão e governamentalidade”. Educação &
Sociedade, Campinas, v. 28, n. 100, p. 947-964, out./dez. 2007. WALKERDINE, Valerie. “Ciência, razão e a mente feminina”. Educação & Realidade, Porto Alegre,
v. 32, n. 1, p. 7-24, jan-jun. 2007. Referências Belo Horizonte: Mazza
Edições, 2012. p. 17-24. MEYER, Dagmar Estermann; PARAÍSO, Marlucy Alves. “Metodologias de pesquisa pós-críticas ou
sobre como fazemos nossas investigações”. In: MEYER, Dagmar Estermann; PARAÍSO, Marlucy
Alves (Orgs.). Metodologias de pesquisas pós-críticas em educação. Belo Horizonte: Mazza
Edições, 2012. p. 17-24. MORAES, Adriana Zomer de. Relações de gênero e a formação de engenheiras e engenheiros. 2016. Mestrado (Programa de Pós-Graduação em Educação) – Faculdade de Educação da
Universidade do Sul de Santa Catarina, Tubarão, SC, Brasil. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 12 Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 MULHERES DE SUCESSO NO CAMPO CIENTÍFICO: UMA ANÁLISE DE REDES SOCIAIS Polliane Trevisan Nunes (pollianenunes@yahoo.com.br) é licenciada em Ciências
Sociais e mestre em Educação pela Universidade Federal do Rio Grande do Sul. Atualmente,
exerce funções pedagógicas e administrativas na Pró-Reitoria de Extensão da Universidade
Federal do Rio Grande do Sul. Fernanda Wanderer (fernandawanderer@gmail.com) é licenciada em Matemática pela
Universidade Federal do Rio Grande do Sul, mestre e doutora em Educação pela Universidade
do Vale do Rio dos Sinos. Professora permanente do Programa de Pós-Graduação em Educação
da Universidade Federal do Rio Grande do Sul, vinculada à Linha de Pesquisa: Estudos Culturais
em Educação. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 13 POLLIANE TREVISAN NUNES E FERNANDA WANDERER COMO CITAR ESTE ARTIGO DE ACORDO COM AS NORMAS DA REVISTA NUNES, Polliane Trevisan; WANDERER, Fernanda. “Mulheres de sucesso no campo científico: uma análise de
redes sociais”. Revista Estudos Feministas, Florianópolis, v. 29, n. 2, e68120, 2021. Revista Estudos Feministas, Florianópolis, 29(2): e68120
DOI: 10.1590/1806-9584-2021v29n268120 CONTRIBUIÇÃO DE AUTORIA Polliane Trevisan Nunes: concepção, coleta de dados e análise de dados, elaboração do manuscrito,
redação, discussão de resultados. Fernanda Wanderer: concepção, elaboração do manuscrito, redação, discussão de resultados. FINANCIAMENTO
Não se aplica. CONSENTIMENTO DE USO DE IMAGEM
Não se aplica. APROVAÇÃO DE COMITÊ DE ÉTICA EM PESQUISA
Não se aplica. CONFLITO DE INTERESSES
Não se aplica. LICENÇA DE USO
Este artigo está licenciado sob a Licença Creative Commons CC-BY 4.0 International. Com essa licença você
pode compartilhar, adaptar, criar para qualquer fim, desde que atribua a autoria da obra. HISTÓRICO
Recebida em 21/10/2019
Aceita em 30/10/2020 14
|
https://openalex.org/W3036812544
|
https://hal.inrae.fr/hal-03456641/document
|
English
| null |
Chemical description and organoleptic evaluation of Pinot noir wines from different parts of Italy: a three year investigation
|
OENO One
| 2,020
|
cc-by
| 11,956
|
Chemical description and organoleptic evaluation of
Pinot noir wines from different parts of Italy: a three
year investigation f
p
y
year investigation
Enrico Serni, Ulrich Pedri, Josep Valls, Christof Sanoll, Nikola Dordevic, Eva
Überegger, Peter Robatscher To cite this version: Enrico Serni, Ulrich Pedri, Josep Valls, Christof Sanoll, Nikola Dordevic, et al.. Chemical description
and organoleptic evaluation of Pinot noir wines from different parts of Italy: a three year investigation. OENO One, 2020, 54 (2), pp.393-410. 10.20870/oeno-one.2020.54.2.3098. hal-03456641 Distributed under a Creative Commons Attribution 4.0 International License A B S T R A C T Aim: this work gives a chemical description and sensory evaluation of several Pinot noir wines from different parts
of Italy. For three subsequent years (2016-2018) the wine samples were submitted for in an Italian annual national
Pinot noir competition aiming to define the best Pinot noir red wine from Italy. All of the wine samples were 3-years
old (from vinification) at the moment they were analysed and evaluated; they were also registered for the
competition the same year they were put on the market. Methods and results: all the wines were evaluated by a tasting panel composed of oenologists and wine experts,
using the overall sensory quality as the descriptor. For the chemical screening, standard oenological chemical
parameters (total acidity, colour, alcohol degree, total phenolic content, tannin indexes, etc.) and the content of the
most abundant phenolic molecules by means of LC-MS analysis (triple quadrupole with internal standards) were
determined. Pinot noir red wines produced from different parts of Italy showed a high variability for most standard
wine chemical parameters considered, while the content of most single phenolic constituents was more retained and
consistent with data from literature; except for t-resveratrol, which was significantly higher in our analyses, and
delphinidin-3-glucoside, which was lower. Moreover, changes regarding the corresponding wines from the three
vintages were noted. A correlation between the chemical parameters and the tasting panel results was also
attempted. The results from a statistical analysis confirmed that alcoholic content, malvidin-3-glucoside and total
anthocyanins had the highest positive impact on quality scores, while gallic acid, color tonality and total phenolic
content had the highest negative. Conclusions: our results indicate that most wine producers have a conservative attitude with very slight differences
found in the corresponding wines over the three years of investigation. The strong effects of agronomical,
winemaking and ageing processes on chemical and sensorial features of Pinot noir red wines from Italy were also
clearly shown. Compared to other monovarietal Pinot noir red wines from the same temperate area, single
polyphenol content tended to be more retained than most standard chemical parameters. p yp
p
Significance and impact of the study: an overall quality assessment of a monovarietal wine, with its typicity as the
main goal of a sensorial investigation, appears to be different from an objective quality assessment carried out by
trained professional personnel using single standardised descriptors. Chemical description and organoleptic evaluation of Pinot noir wines
from different parts of Italy: a three year investigation Enrico Serni1, Ulrich Pedri1, Josep Valls1,2, Christof Sanoll1, Nikola Dordevic1,
Eva Überegger1 and Peter Robatscher1,* 1Laimburg Research Centre, Laimburg 6, Pfatten (Vadena), 39040 Auer (Ora) (BZ), Italy
2 current adress: Unité de recherche Œnologie, EA 4577, USC 1366 Inrae-Axe, ISVV,
33882 Villenave d’Omon, France 1Laimburg Research Centre, Laimburg 6, Pfatten (Vadena), 39040 Auer (Ora) (BZ), Italy
2 current adress: Unité de recherche Œnologie, EA 4577, USC 1366 Inrae-Axe, ISVV,
33882 Villenave d’Omon, France *Corresponding author: peter.robatscher@laimburg.it A B S T R A C T Positive and negative correlations exist between
sensorial judgement and chemical parameters, and the multiple linear regression model revealed relationships
between the wine score and the set of the most important wine score description parameters. red wine, sensory evaluation, polyphenols, tannins, linear modeling Ha: hectare
TPC: Total Polyphenol Content
UHPLC-MS: Ultra High-Performance Liquid Chromatography-Mass Spectrometry
IAC: ionized anthocyanins content
Abs: absorbance
WS: wine score
A B B R E V I A T I O N S HAL Id: hal-03456641
https://hal.inrae.fr/hal-03456641v1
Submitted on 30 Nov 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés. HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Received: 3 February 2020 yAccepted: 10 May 2020 y Published: 19 June 2020
DOI:10.20870/oeno-one.2020.54.2.3098 Received: 3 February 2020 yAccepted: 10 May 2020 y Published: 19 June 2020
DOI:10.20870/oeno-one.2020.54.2.3098 Chemical description and organoleptic evaluation of Pinot noir wines
from different parts of Italy: a three year investigation Received: 3 February 2020 yAccepted: 10 May 2020 y Published: 19 June 2020
DOI:10.20870/oeno-one.2020.54.2.3098 INTRODUCTION much higher percentage of monomers, especially
from seeds. Moreover, a particular composition
of anthocyanin pigments with relatively low total
content and a lack of acylated products has long
been known (Wenzel et al., 1987). When
compared to the average of 64 other red grape
cultivars, Mattivi et al. (2006) reported a much
lower amount of total anthocyanins and a
slightly lower amount of total flavonols. In terms
of environmental and terroir influences, climate
parameters (such as temperature and sun
irradiation) are known to have an affect on the
content of anthocyanins flavan-3-ols and
flavonols in red wines, including Pinot noir. For
example, sun-exposed Pinot noir grape clusters
contain up to 10 times more quercetin glycosides
than those that are shade-exposed (Bertamini et
al., 1998; Price et al., 1995). Tannin and
anthocyanin content and composition vary with
different vine vigour (Cortell et al., 2008); as a
consequence, both temperature and sun exposure
can also have a sensorial-organoleptic impact. Pinot noir is an important international grape
variety which accounts for approximately
112,000 ha of world vine area, thus being one of
the most widespread and important red grape
variety for worldwide wine production (VV.AA. Focus OIV, 2017). In Europe, Italy is the fourth
biggest producer of Pinot noir grape (preceded
by France, Germany and Switzerland), with
around 5046 ha of vine area, of which
approximately 464 are in Alto Adige (South
Tyrol) and 353 are in the Trentino provinces
(Anderson and Aryal, 2013; VV.AA., 2017). Pinot noir vine is known to preferentially grow
in relatively cool environments with a significant
day-to-night temperature excursion, which hilly
and mountain territories can provide (Robinson
et al., 2013). However, it is also considered to be
very sensitive to the exogenous factors to which
it is subjected, so that the wines obtained from
different areas and/or following different
agronomical and oenological procedures (in
other words, from different terroirs) can be very
different (Rigaux, 2010; Vaudour, 2005). The chemical composition of grapes is obviously
reflected in the corresponding wines. Pedri et al. (2019) have recently reported the chemical
composition of several Pinot noir grapes, musts
and wines produced on different sites of
Trentino-Alto Adige from 1996 to 2001. All the
wines described in their paper were 1-year old
monovarietal products, with vines originating
from the same clone and rootstock, and with
similar harvesting time. A B B R E V I A T I O N S Ha: hectare
TPC: Total Polyphenol Content
UHPLC-MS: Ultra High-Performance Liquid Chromatography-Mass Spectrometry
IAC: ionized anthocyanins content
Abs: absorbance
WS: wine score
A B B R E V I A T I O N S Ha: hectare
TPC: Total Polyphenol Content
UHPLC-MS: Ultra High-Performance Liquid Chromatography-Mass Spectrometry
IAC: ionized anthocyanins content
Abs: absorbance
WS: wine score
A B B R E V I A T I O N S Supplementary data can be downloaded through: https://oeno-one.eu/article/view/3098 393
© 2020 International Viticulture and Enology Society - IVES
393
© 2020 International Viticulture and Enology Society - IVES OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. © 2020 International Viticulture and Enology Society - IVES
394
© 2020 International Viticulture and Enology Society - IVES
394 INTRODUCTION Moreover, they did not
undergo fining processes on wood supports
(barrels and/or chips) during winemaking. The
study was conducted to define the features of this
wine variety, especially when produced in a
particular area and, as far as possible, separately
from any other agronomical and oenological
variable. Some particular definite features of the
wines emerge in this work: for example, total
phenolic content (TPC) ranged from 1442 to
2126 mg/L, alcoholic grade from 12.1 to 13.2 %
and titrable acidity from 4.3 to 5.0 g/L. Mawdsley et al. (2019) recently reported similar
values for 1-year old Pinot noir wines from one
site in California over three vintages (2016-
2018). In contrast, Samoticha et al. (2017)
reported lower alcoholic grade and higher
titrable acidity for 1-year old Pinot noir wines
from Poland produced during the 2014 vintage
with a comparable maceration period. Such
reports confirm that while genotype is crucial,
geography, climate and vintage also affect wine
chemistry and sensorial quality. Neverthless,
values for key chemical parameters in Pinot noir While the Pinot noir grape can be blended with
other varieties for vinification, it is highly
appreciated as a monovarietal grape for the
production of both sparkling white (including
Champagne) and still red wines. Pinot noir is
considered to be an “elegant” red wine due to its
well-balanced structure with fine organoleptic
qualities (light colour intensity, medium to light
body, medium to low phenolic tenor, typical
olfactory profile; Robinson et al., 2013), and it is
also suitable for mid to long aging (Jaffré et al.,
2009). Depending on the growing area, harvest
and clone, the wines of Pinot noir can have
relatively soft tannins, and it has a characteristic
pleasant fruity aroma. In young wines the smell
of cherries and raspberries is dominant with a
wide range of other fruity aromas. As an aged
wine Pinot noir can have aromas of mulch,
truffles or other fungi (Robinson et al., 2013). The profound effects of both genotype and
environment on grape composition is well
known. In terms of genotype, some specific
chemical properties of the Pinot noir grape
cultivar are currently well-established in
scientific literature. Kennedy et al. INTRODUCTION (2002)
reported a higher total proanthocyanidin content
in Pinot noir berries (intended as seed + skin
tannin, including monomers) compared to
Cabernet-Sauvignon from the same area, with a OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 © 2020 International Viticulture and Enology Society - IVES
394
© 2020 International Viticulture and Enology Society - IVES
394 wine can be very different from other important
Italian and international red wines. Van Leeuw et
al. (2014) described the phenolic composition of
several commercial monovarietal red wines
produced in different countries (including Italy)
and how Pinot noir wines were an exception. This was because the range of phenolic
compounds (flavonols, anthocyanidins, flavan-3-
ols, phenolic acids, resveratrol, etc.) was
significantly different from all the others, which
showed limited differences among each other in
most cases; for example, compared to Syrah,
Merlot and Sangiovese wines, anthocyanidin and
flavonol content was lower, while flavan-3-ol
and phenolic acid content was higher. Again,
ranges were wide when the different origins were
considered. Landrault et al. (2001) studied the
composition of several red wines from France,
and their results show relatively higher content
of catechins and acids and lower content of
anthocyanins. When compared to Tannat wines,
lower anthocyanin content and a lack of
acetylated and p-coumarylated derivatives in
Pinot noir wines from Uruguay has recently been
confirmed (Piccardo et al., 2019). All these data
suggest that cultivar genotype is the key factor
for the development of most of the chemical and
organoleptic features of red wines, especially
aroma and phenolic composition, and that
mesoclimatic variability can also have a strong
impact on the characterisation of terroir and the
sensorial characteristics of a wine. Both elements
must be considered when investigating the
concept of typicity of wines obtained from a
single cultivar. evaluation. However, some of these parameters
(like limpidity, persistence and effervescence)
have a more objective and clear definition
compared to others (like quality, general
impression and typicity) which retain a more
indefinite and subjective character (OIV, 2009). Moreover, the different organoleptic and
sensorial attributes that are usually considered
during the evaluation of red wines (aroma,
alcoholic grade, acidity, tannin quantity and
character, colour) may not necessarily lead to the
assessment of “genuine” (i.e., absolute) quality,
especially if the determination of typicity of a
monovarietal wine is the main goal of a sensorial
investigation. INTRODUCTION The aim of this work was to assess the best
Italian Pinot noir red wines, which feature the
highest qualitative organoleptic profile within the
typical characteristics of the variety, among
distinct batches deriving from a national
competition which occurred during three
consecutive vintages (2016-2018), and including
wines with different geographical origins
(regions and/or terroirs). Sensory evaluations and
chemical analyses (standard wine parameters,
tannin amount and characterisation, and single
phenolics content) were performed, and the
resulting data were evaluated in order to
investigate the putative correlation between the
two. 395
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
395 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 1. Chemicals and reagents Delphinidin-3-glucoside chloride (≥ 95 %),
malvidin-3-glucoside chloride (≥ 95 %),
isorhamnetin (≥ 99 %), myricetin (≥ 99 %),
quercetin (≥ 99 %) and t-resveratrol (≥ 99 %)
were purchased from Extrasynthese (Genay, FR). (-)-epicatechin (≥ 99 %), (-)-gallocatechin
(≥90 %), astilbin (≥ 90 %), caftaric acid
(≥90 %), coutaric acid (≥ 65 %), petunidin-3-
glucoside chloride (≥ 95 %), procyanidin B1
(≥90 %), procyanidin B2 (≥ 95 %), procyanidin
C1 (≥ 90 %), quercetin-3-glucuronide (≥ 90 %)
and taxifolin (≥ 99 %) were from Phytolab
(Vestenbergsgreuth, DE). (+)-catechin (≥ 99 %)
and caffeic acid (≥ 98 %) were from Sigma-
Aldrich (St. Louis, US). Gallic acid (≥ 99 %)
was from Roth (Karlsruhe, DE), vanillin (99 %)
and FeCl3 (anhydrous, flushed with N2) were
from Acros (Morris Plains, US). Grape seed
extract solutions (95 % proanthocyanidins
content) were from ArdaNatura (Piacenza, IT). Valentin et al. (2016) investigated colour as a
driver for quality judgement of Pinot noir wines
from New Zealand. From a literature review on
wine evaluation and quality assessment, they
discussed how perceived quality is usually
related to wine attributes other than colour,
including abstract wine concepts such as typicity
and complexity, and to hedonic aspects such as
likability. In particular, the authors underline the
lack of published data and address how wine
professionals, rather than trained panelists using
descriptive ratings, judge wine quality either
within a winery or the wider wine industry (e.g.,
as part of a jury at wine competitions), which is
how they assess overall wine quality precisely. Results from sensory evaluations of a wine and
its overall quality assessment can therefore be
highly subjective and aleatory unless precise
guidelines are provided. Official guidelines
produce standardised descriptors for wine 395
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
395 OENO One 2020, 2, 393-410
OENO One 2020 54 2 393-4 Enrico Serni et al. Sulphuric acid (95-97 %) was from J.T.Baker
(Phillipsburg, US). Hydrochloric acid (≈36 % -
12N) was from Fisher (Loughborough, GB). n-
butanol (HPLC grade) was from Chemlab
(Zedelgem, BE). methanol (gradient grade) was
from VWR (Fontenay-sous-Bois, FR). Acetonitrile (LC-MS grade) was from Panreac
(Barcelona, ES) and formic acid (LC-MS grade)
was from Merck (Darmstadt, DE). Ultrapure
deionized water was from Millipore MilliQ
apparatus (Burlington, US). detailed in evaluation after submission to 12-
14 different commissions (depending on total
number of samples). 2. Sampling Wine samples were provided from wineries for
an Italian annual national Pinot noir competition
which took place in three consecutive years
(2016-2018) in the dedicated sensory room at the
Laimburg Research Centre (see “Sensory
Evaluation” section for details). The
geographical origins of the samples are shown in
Figure 1. Further details (product specifications
and values for all chemical analyses and sensory
evaluation of all wines in each vintage) are listed
in Supplementary Table 1 (ST-1). All wines were
3-years old as from winemaking at the time they
were evaluated and analysed. Most wines were
registered for the competition the same year they
were released on the market. Shortly before
tasting, samples of the same wine (3 to 5 regular
labeled market bottles) were opened and pooled
in a wine jar to ensure homogeneity, and three
aliquots (0.2 L each) were immediately
transferred to dark glass bottles, saturated with
N2 to avoid excessive oxidation, closed with
screw cap and stored at +4 °C until chemical
analyses were performed (standard wine
parameters, tannin-proanthocyanidin analysis,
LC-MS analysis). All the rest was for the
sensory evaluation. - < 59: bad, defective, unacceptable wine; FIGURE 1. Origin of Pinot noir wine samples
from the Italian territory in the three years
of examination. 1. Chemicals and reagents No particular descriptors
were selected or suggested for the analytical
evaluation of each wine: the panelists were asked
to examine the organoleptic profile of wine
samples, using their knowledge and experience
to evaluate the positive and negative sensations
received by their nose, mouth and eyes in
relation to the typical paradigmatic features of
Pinot noir wines. The aim of the evaluation was
not to provide a detailed scientific description of
the wines, but to assess their overall quality. For
harmonisation among the members of the panel,
three blind tastings of randomly chosen
registered wines were performed before the
beginning, followed by open discussion. On
consensus, wines were given an overall quality
score by each commission, with remarks based
on a one hundred-point scale as follows: © 2020 International Viticulture and Enology Society - IVES
396
© 2020 International Viticulture and Enology Society - IVES
396 - < 59: bad, defective, unacceptable wine; © 2020 International Viticulture and Enology Society - IVES
396
© 2020 International Viticulture and Enology Society - IVES
396 3. Sensory evaluation The sensory evaluation took place on 07/04/2016
(2013 vintage), 06/04/2017 (2014 vintage) and
12/04/2018 (2015 vintage) using a tasting panel
consisting of 20 commissions, each composed of
two judges. All the judges were familiar with red
wine evaluation, being professional oenologists
or journalists for food press and possessing
medium to good knowledge of wine tasting and
wine evaluation. Wines were submitted to each
commission in a randomised way and sequence. To avoid saturation of mouthfeel and tasting
capability, each commission evaluated 50 wines,
so that each wine was considered sufficiently FIGURE 1. Origin of Pinot noir wine samples
from the Italian territory in the three years
of examination. OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 © 2020 International Viticulture and Enology Society - IVES
396
© 2020 International Viticulture and Enology Society - IVES
396 meter with thermostatic cuvette holder
(Steroglass, S. Martino in Campo, Italy). Total
polyphenol content (TPC) and ionised
anthocyanins content (IAC) were determined
using specific reaction kits (Steroglass) and
absorbance (Abs) readings at 620 nm and
520 nm respectively. The two assays were based
on the Folin-Ciocalteau (F-C) reaction and
ionisation of anthocyanins in acidic conditions
respectively. For TPC determination, the samples
were diluted 1:4 and the results multiplied by the
dilution factor and expressed as gallic acid
equivalents. For IAC, the results were expressed
as anthocyanins using a proprietary calculation
based on averages from HPLC analyses. Wine
colour was also objectively evaluated by
measuring the absorbances at three different
wavelengths (420 nm, 520 nm and 620 nm). Colour features of wines are expressed as
intensity (the sum of Abs 420, Abs 520 and Abs
620) and tonality (ratio of Abs 420 and Abs
520). Instrument control and data collection were
performed with Steroglass Hi software (version
0.44.5, S. Martino in Campo, Italy). - 60 - 69: wine having no particular defects, but
with some lacks (excessive tannins, acidity,
etc.); - 70 - 79: average quality wine, linear, with no
remarkable attributes; - 80 - 89: fine, neat wine, above the average,
with positive expressions in smell and taste; - 90 - 100: very good to excellent wine, with
outstanding complexity and varietal character. The ability of each commission to recognise the
same wine and discriminate different wines was
evaluated by blind tasting five samples in two
replicates, and F values were calculated (Kobler,
2008). 3. Sensory evaluation Only F values belonging to 95 %
confidence interval were considered for
acceptance of scores from a given commission,
otherwise all scores from that commission were
not considered. The final score for each wine
(wine score, WS) was calculated as the median
of all scores from the different commissions that
evaluated a given wine. 4.2. Fourier-Transform Infra-red (FT-IR)
analysis 5.2. Total flavan-3-ols content (monomeric to
oligomeric proanthocyanidins, including low-
weight condensed tannins) Analyses of the standard wine parameters were
conducted on WineScan™ SO2 Auto instrument
(Foss, Hillerod, Denmark). Wavelengths in the
mid-infrared spectrum (2400 nm–10000 nm)
were used for our method, and the following
parameters were determined: alcohol content (%
v/v), reducing sugars (g/L), pH, total acidity
(g/L), volatile acidity (g/L), total dry extract
(g/L), glycerol (g/L), methanol (% v/v as
anhydrous ethanol), malic acid (g/L) and lactic
acid (g/L). Both internal and external control of
the results were performed daily and monthly
respectively, using standard reference samples
from the laboratory and a proficiency test from
106 other laboratories. Instrument control and
data collection were performed with FOSS
Integrator software (version 1.7.8, Hillerod,
Denmark). The vanillin assay was applied for the
determination of catechins and proanthocyani-
dins-condensed tannins (Sun et al., 1998). In a
2.0 ml plastic tube with screw cap, 20 µL of
wine was mixed with 180 µl MeOH, 500 µL of
sulphuric acid (25 % v/v in methanol) and
500 µL of vanillin 1 % (w/v) in methanol
(sample) or with 500 µL of methanol (blank). Both sample and blank tubes were incubated for
30 minutes at 30 °C. Absorbance was read in a
2 ml Suprasil quartz cuvette (1,0 cm optic path)
immediately after incubation; the difference in
absorbance at 500 nm between the sample and
the blank was compared to a calibration curve
obtained from (+)-catechin solutions (0-
250 mg/L in MeOH, 200 µL each level solution
instead of 20 + 180 µL as for samples), and the
results were expressed as mg catechin
equivalents per L of wine. The analyses were 5. Determination of flavan-3-ols
and tannins content Wine samples were filtered using a syringe filter
(5.0 μm pore size) prior to analyses. No further
treatment (including dilution) were performed
except when specifically indicated. 5.1. Sample preparation Wine samples were centrifuged (20800 x
g, 10 min at +4 °C) prior to analyses. 4.3. Colorimetric analysis Colorimetric analyses were performed using an
automatic Hyperlab Plus UV/VIS spectrophoto- 397
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
397 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. were separated on a Hypersil Gold C18 column
(50 x 2.1 mm, 1.9 µm particle size, Thermo
Fisher Scientific, USA) with pre-column 0.2 µm
filter, using a linear gradient elution with solvent
A (formic acid 2.5 % in deionised ultrapure
water) and solvent B (formic acid 2.5 % in
acetonitrile) as follows: 0-1 min 2.5 % (B),
5-6.5 min 16.5 % (B), 7.5 min 23.5 % (B),
10.5 min 55.0 % (B), 11-12.5 min 95.0 % (B),
13-16 min: 2.5 % (B). Flow rate was 0.5 mL/min
and column thermostated at 40 °C. Injection
volume was 2 μL. All compounds were detected
and quantified in selected reaction monitoring
(SRM) mode using at least one quantifier and
one qualifier transition, which were previously
determined by direct infusion of standard
solutions and by comparison with the literature. The source conditions were as follows: voltage
(both positive and negative) 1500 V, vaporiser
temperature 450 °C, capillary temperature
150 °C, sheath gas 60 (arbitrary unit, Arb),
auxiliary gas 20 (Arb). Retention time and
MS/MS detection parameters for the polyphenols
analysed in this study are summarised in
Supplementary Table 2 (ST-2). All operations
were controlled by Thermo Xcalibur Version 2.2
software (Thermo Scientific, Waltham, USA). conducted in duplicate and the means reported as
a final value. 5.3. Total condensed tannins content (oligomeric
to polimeric proanthocyanidins, all-range
condensed tannin) 5.3. Total condensed tannins content (oligomeric
to polimeric proanthocyanidins, all-range
condensed tannin) Since the vanillin assay can underestimate the
presence of highly-polymerised proanthocyani-
dins (condensed tannins), total condensed tannin
concentration was also measured using the
method of Porter et al. (1986), as described with
modification by Hagermann (2002). In a 15-ml
glass vial with screw cap, 50 µL of sample was
mixed with 950 µL MeOH and 6.0 ml of 5 %
(v/v) HCl (12 N) in n-BuOH, then added with
200 µL of Fe(III)Cl3 solution (1 % in HCl 2 N). An aliquot of 1,5 mL was immediately
transferred in a quartz cuvette and read at
550 nm, for blank evaluation. 5.3. Quantitation and method validation For quantitative analysis of Pinot noir wine
samples, an external calibration curve was built
for each analyte using standard solutions. Wine
samples were first diluted 1:10 in deionised
water, then all samples and calibration solutions
were added with internal standards before
analysis. Each sample was prepared in triplicate
(technical replicates). 100 μL of sample or
standard solution were diluted with 90 μl of
deionized ultrapure water and added with 10 μL
of an IS mix containing cyanidin-3-galactoside
(4 mg/L), (-)-epigallocatechin gallate (20 mg/L),
and quercetin-4-glucoside (20 mg/L). The
internal standards used for each compound are
included in ST-2. All samples and standards
were injected as a single replicate, and a quality
control (QC), prepared by pooling all the
samples, was injected every 12th analysis to
control the absence of chromatographic drift. Quantitative determination was obtained by
comparing the analite/internal standard ratio in
samples and the external calibration curves,
which were obtained by plotting the
analyte/internal standard response (area ratio,
average of triplicate) against the concentration
that was injected (µg/mL). Coutaric acid values 5.1. Sample preparation Wine samples were centrifuged (20800 x
g, 10 min at +4 °C) prior to analyses. 4.3. Colorimetric analysis Next, tightly
closed vials were incubated at 110 °C for 50 min
on a heat block plate; after cooling at room
temperature, the samples were read at 550 nm. Absorbance was read in a 2 ml Suprasil quartz
cuvette (1.0 cm optic path). The difference in
absorbance between sample and blank was
compared to a calibration curve obtained from
grape seed extract solutions (95 % proantho-
cyanidins content; standard solutions 0-
250 mg/L in MeOH, 1.0 ml each level solution
instead of 50 + 950 µL MeOH as for samples),
and results were expressed as mg grape seed
extract equivalents per L of wine. The analyses
were conducted in duplicate and the means
reported as a final value. 6.1. Exploratory analyses The competition was organised by the Research
area Enology of the Laimburg Research Centre,
and both tasting evaluations and chemical
analyses took place on the Laimburg site
(Vadena, BZ). This event aimed to identify and
promote the best and most typical Italian red
wine from Pinot noir grape. It was thus initially
intended as an event with commercial and/or
marketing implications other than a mere
scientific description. It was decided that a jury
which did not include specifically trained
personnel would be more appropriate for this
purpose, in order to give an objective evaluation
corresponding to consumer feelings. All parameters were visualized in box-plots, and
means, standard deviations and ranges were
considered for each vintage separately. To
evaluate statistical differences for all the
parameters among the three vintages, Kruskal-
Wallis and Wilcoxon tests were performed (the
latter being specific for values measured in only
two vintages). Pearson´s correlations coefficients
were calculated between all indicators (standard
chemical analyses, total proanthocyanidins and
total tannins analyses, LC-MS analyses and
score values). 1. General aspects on samples
and sensory evaluation As previously stated, the primary aim of this
work was to assess a ranking based on results
from sensory evaluations of the overall quality of
the organoleptic profiles of Pinot noir wines
produced in Italy, taking into account the typical
features of this red wine. Furthermore, a
chemical description of the wines registered for
the competition is reported, highlighting their
most characteristic features and tentatively
correlating them with the sensory evaluation and
scores. 5.2. UHPLC-MS/MS analysis Identification and quantification of single
polyphenols was performed on a UHPLC-DAD-
MS/MS system (Thermo Scientific, Waltham,
USA) consisting of an Accela 1250 quaternary
pump, an autosampler, a column oven and an
Accela PDA detector coupled with a TSQ
Quantum Access Max triple quadrupole system
equipped with a heated electrospray ionisation
(HESI) ion source. The analysis was based on an
LC–MS/MS method described for the analysis
of Sangiovese wines, with slight modification
(Arapitsas et al., 2012). Briefly, polyphenols © 2020 International Viticulture and Enology Society - IVES
398
© 2020 International Viticulture and Enology Society - IVES
398 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 were corrected because of the purity of the
standard available (≥65 %). Regression factor
(r2) was calculated by means of least-square
analysis for linearity evaluation. Calibration data
are included in ST-1. The lowest limit for the
linear range (instrumental limit of quantification,
ILOQ) was established as the lowest standard
providing a quantifiable signal (i.e., 10 times
signal-to-noise ratio). The intraday precision was
expressed as the Coefficient of Variation (CV %)
of 6 analyses of the QC in the same day, while
the interday precision was also expressed as the
CV % of 6 QC samples analysed on three
consecutive days. Official guidelines were
followed when validating the method. (Magnusson, 2014). All statistical analyses were carried out using
Excel® software (Microsoft, US) and the
statistical software R (VV.AA., 2019). 399
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
399 2. Standard wine analysis and scores
2. Standard wine analysis and scores Ciocalteau reagent, which oxidises hydroxyl
groups of polyphenols present in the wine
sample in a highly alkaline medium. Ionisable
anthocyanins can be determined due to their
capacity to get ionised in an acidic medium. With this method, it is possible to determine the
anthocyanins in an ionised form, but not those
polymerised with tannic substances. Colour
features of wines, together with vanillin and
BuOH-HCl
assays
performed
for
proanthocyanidins-tannin evaluations, are also
based on the colorimetric quantitative
determination of reaction products. Ciocalteau reagent, which oxidises hydroxyl
groups of polyphenols present in the wine
sample in a highly alkaline medium. Ionisable
anthocyanins can be determined due to their
capacity to get ionised in an acidic medium. With this method, it is possible to determine the
anthocyanins in an ionised form, but not those
polymerised with tannic substances. Colour
features of wines, together with vanillin and
BuOH-HCl
assays
performed
for
proanthocyanidins-tannin evaluations, are also
based on the colorimetric quantitative
determination of reaction products. Different approaches were used for the chemical
characterisation of Pinot noir wine samples in
this work. Automatised FT-IR instrumentals
using mid-infrared spectrum (such as
WineScan™ SO2 Auto) are widely used in
routine analyses of standard parameters for
wines, musts and juices, since fast and reliable
results can be obtained for most of the key
components required (Vilanova et al., 2017;
Whitener et al., 2017; Jouanneau et al., 2012). Spectrophotometric/colorimetric assays are also
used to obtain information about important wine
composition parameters in a reliable way; for
example, total phenolic and ionisable
anthocyanins content (Aleixandre-Tudo et al.,
2017). TPC is usually determined using a Folin-
Different approaches were used for the chemical
characterisation of Pinot noir wine samples in
this work. Automatised FT-IR instrumentals
using mid-infrared spectrum (such as
WineScan™ SO2 Auto) are widely used in
routine analyses of standard parameters for
wines, musts and juices, since fast and reliable
results can be obtained for most of the key
components required (Vilanova et al., 2017;
Whitener et al., 2017; Jouanneau et al., 2012). Spectrophotometric/colorimetric assays are also
used to obtain information about important wine
composition parameters in a reliable way; for
example, total phenolic and ionisable
anthocyanins content (Aleixandre-Tudo et al.,
2017). TPC is usually determined using a Folin- The values obtained for standard wine
parameters, total proanthocyanidin- tannin
assays, and sensory evaluation over three years
are summarised in Supplemetary Table 1 (ST-1). 6.2. Linear modelling The number of wines registered for the
competition was 72 in 2016, 56 in 2017 and 83
in 2018, and they were produced in the regions
of Trentino-Alto Adige, Veneto, Friuli-Venezia
Giulia, Valle d´Aosta, Piemonte, Lombardia,
Toscana and Sicilia. Twenty-five of the same
wines were submitted in all three consecutive
years of competition, among which one was
from Piemonte and the remaining 24 were all
from Trentino Alto-Adige region. The highest
percentage (around 75 to 85 %) of Pinot noir
wines from all three vintages was notably
produced in Trentino Alto Adige (Figure 1). In
fact, the production of Pinot noir vines and wines
from this region - adhering in most part to
“Controlled Designation of Origin” (CDO)
product specification - is relatively high when
compared to the whole Italian territory, probably
due to a positive combination of grape attitude
(genotype and phenotype), pedoclimatic features
for its development and historical tradition. The final aim was to describe the wine score
using all available chemical parameters. In order
to have a model which explains relationships
between wine score and the important chemical
parameters, multiple linear regression analysis
was used. The equation for the overall regression model
becomes: WS = b0 + b1X1 + b2X2 + … + bn*Xn + e WS = b0 + b1X1 + b2X2 + … + bn*Xn + e WS = b0 + b1X1 + b2X2 + … + bn*Xn + e where WS is wine score and b0 is intercept
(constant term), b1 to bn are the estimated
regression coefficients, X1 to Xn variables (wine
compounds) and n are number of variables. A
step-wise selection approach was used, starting
with the full model and then eliminating
parameters according to Akaike’s information
criterion (Hastie et Pregibon, 1992; Venables and
Ripley, 2002). 399
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
399 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. Enrico Serni et al. Enrico Serni et al. Enrico Serni et al. © 2020 International Viticulture and Enology Society - IVES
400
© 2020 International Viticulture and Enology Society - IVES
400
© 2020 International Viticulture and Enology Society - IVES
400 2. Standard wine analysis and scores
2. Standard wine analysis and scores It can first be noted that the
evaluation scores and all the parameters that
were taken into account showed significant
differences between the three vintages (p<0.05),
with only the exceptions of vanillin assay
(vintage 2016 was lacking), dry extract residue
and colour tonality. This means that a significant
vintage effect exists in the period of
investigation, which could be mainly due to
different climate conditions affecting grape
development and, to a lesser extent, to
number/origin of samples. Glycerol, lactic acid,
reducing sugars and TPC showed the highest
differentiation and most chemical parameters
showed high variability, as demonstrated by the
wide range of values obtained in comparison
with data reported for monovarietal Pinot noir
wines. Overall quality, indicated by the score and
expressed as an average ± standard deviation,
was highest in 2016 (79.69 ± 3.97) and lowest in
2017 (75.82 ± 5.15), with 2018 being
intermediate (77.07 ± 6.71). However, wines
from Trentino-Alto Adige earned higher scores
in all three vintages compared to the rest of Italy
(80.42 ± 3.77 vs. 76.35 ± 3.11 in 2016; 77.32 ±
3.78 vs. 71.32 ± 6.17 in 2017; 79.20 ± 5.19 vs. 71.18 ± 7.01 in 2018), even if lower
representativity of the other Italian regions has
implications at a statistical level. The spread
existing between Trentino-Alto Adige and the
rest of Italy, especially for vintages 2017 and
2018, can be considered as a valid indicator of a
better expression of Pinot noir wine and its
typical characteristics from this terroir. A similar
pattern to score along the three vintages is proper
of chemical parameters such as IAC and
glycerol. Similar development is displayed by
EtOH, colour intensity, absorbances (420, 520,
620 nm) and volatile acidity, while an opposite
trend is shown by TPC and total acidity. A
relationship between certain chemical parameters
of wine samples and their organoleptic quality is
thus evident in the first instance. All the wines showed very low values for residue
malic acid (<0 01 g/L in most wines) meaning The variability of some parameters is also valid
when only Pinot noir wines from Trentino-Alto
Adige are considered. As an example, the total
polyphenolic
content
(TPC)
for
the
corresponding 25 wines examined over three
consecutive vintages from that area, with values
ranging from 1500 to 4000 mg/L, are shown in
Figure 3. 2. Standard wine analysis and scores
2. Standard wine analysis and scores The values obtained for standard wine
parameters, total proanthocyanidin- tannin
assays, and sensory evaluation over three years
are summarised in Supplemetary Table 1 (ST-1). FIGURE 2. Year-to-year comparison of measured parameters from standard chemical analyses. Groups (vintages) are considered statistically different when p<0.05. FIGURE 2. Year-to-year comparison of measured parameters from standard chemical analyses. Groups (vintages) are considered statistically different when p<0.05. FIGURE 2. Year-to-year comparison of measured parameters from standard chemical analyses. Groups (vintages) are considered statistically different when p<0.05. FIGURE 2. Year-to-year comparison of measured parameters from standard chemical analyses. Groups (vintages) are considered statistically different when p<0.05. OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410
ENO One 2020 54 2 393 410 Stój et al., 2019; Van Leeuw et al., 2014). In
fact, results reported in this work indicate higher
average values for TPC (2224 ± 497 in 2016,
2663 ± 613 in 2017, 2444 ± 507 in 2018),
ethanol content (13.36 ± 0.50 in 2016, 13.24 ±
0.50 in 2017, 13.64 ± 0.59 in 2018) and total
titrable acidity (5.10 ± 0.46 in 2016, 5.37 ± 0.40
in 2017, 5.31 ± 0.48 in 2018). These higher
average values might also be a consequence of
global warming occurring in the past decade. Moreover, it is common practice to “correct”
some features during vinification, such as colour,
aroma and body by adding hexogenous tannins
and/or performing wood-based aging processes. These procedures can be performed both to exalt
typical features of a wine and to render products
more “stable” and/or “balanced”, thus
responding to general consumer tastes,
sometimes at the expense of a wine’s typicity. With the same goals in mind, it is fair to assume
that single winemakers may choose not to
develop Pinot noir as a single-variety product,
but will add, for example, sweeter more
coloured and higher-bodied blendings; such
treatments can reflect the style of a winemaker
and become his/her trademark. Depending on
the procedures adopted for such corrections
during winemaking and the blending
components used - all complying with the
protocol restrictions for production quality -
some parameters may be deeply affected (e.g.,
colour, TPC and tannin indexes) while others
(e.g., total acidity) may not. Standard chemical parameters and sensory
evaluation scores in the three vintages are
summarised as box-plots in Figure 2. The trend
followed by the indicators over the three years is
also visible. 401
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
401 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 2. Standard wine analysis and scores
2. Standard wine analysis and scores High variability in some chemical
parameters is evident, despite the similarities in
microclimatic and pedological features, evoked
by the same area of origin (Trentino Alto-
Adige), and the strict protocol regarding the
production of wines (COD), traceable for the
whole batch considered (see ST-1). As
previously mentioned, monovarietal Pinot noir
wines from the same region had different values
for such parameters (Pedri et al., 2019). This is
likely due to variations in agronomical
(especially harvesting time), winemaking and
ageing processes, which in turn can cause big
discrepancies in both the chemical and
organoleptic profile of Pinot noir, even when All the wines showed very low values for residue
malic acid (<0.01 g/L in most wines), meaning
that they all underwent malolactic fermentation. It is interesting to note that in all three vintages
certain parameters, like ethanol content and TPC,
had different values to the typical averages
and/or ranges for monovarietal Pinot noir wines
from other European terroirs (Pedri et al., 2019; 401
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
401 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. FIGURE 3. Total phenolic content (TPC) in corresponding Pinot noir wine samples from Trentino-Alto
Adige in the three vintages (samples are listed as A–Z; see ST-1, row W). FIGURE 3. Total phenolic content (TPC) in corresponding Pinot noir wine samples from Trentino-Alto
Adige in the three vintages (samples are listed as A–Z; see ST-1, row W). FIGURE 3. Total phenolic content (TPC) in corresponding Pinot noir wine samples from Trentino-Alto
Adige in the three vintages (samples are listed as A–Z; see ST-1, row W). from the same mesoclimatic area. To a lesser
extent, it could reflect the highly sensitive and
mutable character of Pinot noir vine as
previously mentioned. tannins quantitation respectively in Pinot noir
wine samples from 2014 and 2015 vintages
(analysed in 2017 and 2018 respectively). Value
ranges were 355 to 2087 and 668 to 5341 mg/L,
with averages of 1024 and 1879 mg/L
respectively (tab. ST-1). Rigo et al. (2000)
previously reported 416 to 1741 mg/L for
proanthocyanidins, and 669 to 2180 mg/L total
tannin content in monovarietal 4 to 5 year-old
Pinot noir wines from Trentino-Alto Adige (aged
in bottle) using the same methods (ethanol was
used instead of n-butanol for acid-catalyzed total
tannins assay). 2. Standard wine analysis and scores
2. Standard wine analysis and scores The authors suggest that such
wide ranges could be explained by blending with
varieties having high proanthocyanidin content
and/or oenological processes involving addition
of proanthocyanidin-based tannins. They
propose an index of condensation as a
vanillin/BuOH-HCl assays ratio, with values
ranging from 0.62 to 1.04; our values ranged
from 0.44 to 0.84 in 2017 and from 0.34 to 0.69
in 2018. As can be inferred from Figure 3, TPC are highly
retained over the three-year period, with a small
“vintage” effect for most of them. This is
probably the consequence of the winemakers’
conservative attitude and pursuit of the preferred
“style” of their own products, trying to maintain
it over the years for better characterisation and
recognisability; in other words, to promote their
terroir. Nevertheless, these factors do not result in
harmonious results and scores. For example,
“M” sample obtained 77 points in 2017 and 64
points in 2018, while “Y”, having more than
double the content of TPC, obtained 77 points in
2017 and 79.75 points in 2018. The multiplicity
of factors involved in red wine production
processes, the vintage effect and the complexity
of red wine (in terms of organoleptic attributes to
be evaluated) are thus all confirmed and likely to
have affected this variability. The mechanism of reactions leading to the
coloured products to be measured is different in
the two assays. The vanillin reaction is based on
adduct formation with flanan-3-ols C6 and/or C8
positions, depending on the presence of
interflavanic linkages or other substituent
groups, and the reaction yield is maximum for
monomers and oligomers, becoming lower as
proanthocyanidins become highly polymerised © 2020 International Viticulture and Enology Society - IVES
402
© 2020 International Viticulture and Enology Society - IVES
402 3. Flavan-3-ols and tannins content in Pinot
noir wines and scores Vanillin and BuOH-HCl assays are based on
spectrophotometric determinations and were
used in this work for proanthocyanidins and total © 2020 International Viticulture and Enology Society - IVES
402
© 2020 International Viticulture and Enology Society - IVES
402 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 (Sun et al., 1998). The reaction with BuOH-HCl
leads to coloured products after the cleavage of
unit-unit linkage, so that the reaction yield is null
for monomers, increasing with the increasing
polymerisation (Hagerman, 2002). (Sun et al., 1998). The reaction with BuOH-HCl
leads to coloured products after the cleavage of
unit-unit linkage, so that the reaction yield is null
for monomers, increasing with the increasing
polymerisation (Hagerman, 2002). of polymerisation to most wine samples. These
results are coherent with relatively young wines
not yet subjected to aging, which would yield a
higher average degree of polymerisation
together with lower content of monomers and
small oligomers. Both data sets show a high correlation with the
TPC value in 2018 (Figure 4), so that the
hypothesis of trivial results and/or artifacts can
be discarded (the correlation coefficient values
for the 2017 vintage were also >0.85; data not
shown). Only 7 samples out of 83 show higher
values for the BuOH-HCl assay than for the TPC
assay in 2018, and 0 out of 56 in 2017. This is
probably due to a particular composition in total
phenolics and/or degree of polymerisation of
condensed tannins, including the large extent of
adduct formation with anthocyanins or other
molecules, which prevent the Folin-Ciocalteau
reagent from actingto its full potential. 403
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
403 4. LC-MS analysis The values of single polyphenol content from
the LC-MS analyses are discussed in this section
(indicated as mean ± standard deviation, both
mg/L). The complete listing is available in
Supplementary Table 1 (ST-1). All these
compounds are widely known to be main
constituents of red wines and have been selected
for having been previously reported as the most
abundant polyphenols in Pinot noir wines
(Samoticha et al., 2017; Van Leeuw et al., 2014;
Kemp et al., 2011). Data for these parameters
are graphically summarised in Figure 5,
allowing a comparison of the data spread for the
three vintages. The comparison of parameters
can be achieved by considering the scale of
expression of the values. Again, the Kruskal-
Wallis tests were performed to check the
differences between parameters measured over
three different years. For discussion, compounds
have been grouped into their main classes
(catechins and oligomeric procyanidins,
anthocyanins, phenolic acids, flavonols,
resveratrol) and are graphically represented for
the three years in Supplementary Figures 1 to 5
(Figures S1 - 5). It should be highlighted that the
parameters considered for the LC-MS analyses
also showed significant differences in the three The Vanillin assay had a higher correlation with
the TPC assay than with the BuOH-HCl assay,
indicating that flavan-3-ols comprise mostly
monomers and small oligomers. The correlation
between these two assays was high. An average
degree of polymerisation around low-medium
values (2 to 7), to which both assays give high
reaction yield, can be invoked as the main reason
for these results and for the indexes of
condensation obtained. Oenological condensed
tannins used during winemaking would also
show a degree of polymerisation consistent with
such values. Moreover, the grape seed extract
used for the calibration of the BuOH-HCl assay
probably shows a similar distribution in degree FIGURE 4. Correlation between values from vanillin, BuOH-HCl and TPC assays in 2018. Values are expressed as mg/L on both axes. FIGURE 4. Correlation between values from vanillin, BuOH-HCl and TPC assays in 2018. Values are expressed as mg/L on both axes. 403
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
403 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. FIGURE 5. Year-to-year comparison of measured parameters from LC-MS analyses for Pinot noir wine samp
Groups (vintages) are considered statistically different when p<0.05. Values are expressed as mg/L. 4. LC-MS analysis FIGURE 5. Year-to-year of measured parameters from LC-MS analyses for Pinot noir wine samples. Groups (vintages) are considered statistically different when p < 0.05 as expressed as mg/L FIGURE 5. Year-to-year comparison of measured parameters from LC-MS analyses for Pinot noir win
Groups (vintages) are considered statistically different when p<0.05. Values are expressed as mg/L. FIGURE 5. Year-to-year of measured parameters from LC-MS analyses for Pinot noir wine samples. Groups (vintages) are considered statistically different when p < 0.05 as expressed as mg/L 23.4 in 2017, 83.2 ± 31.3 in 2018), and the other
main monomer (-)-epicatechin was slightly
lower (mg/L: 79.5 ± 38.9 in 2016, 51.2 ± 21.7 in
2017, 57.9 ± 25.7 in 2018). In order of
abundance, (+)-catechin is followed by
procyanidin B1, (-)-epicatechin, procyanidin B2,
procyanidin C1 and (-)-gallocatechin, a pattern
which remains the same during the whole period. The content ratio of single monomers is
consistent with previously reported data (Kemp
et al., 2011), while that of dimers and trimers
could not be found in the literature. vintages (p<0.05), with the only exceptions
being
caffeic
acid
and
total
catechin/procyanidins. As previously noted for
scores and results from standard chemical
analyses, a vintage effect exists in the
investigated period. 4.5. Stilbenes Trans-resveratrol was considered as a
representative of the stilbene subclass of
polyphenols. Its range and average content in the
three batches was lowest in 2016 and highest in
2018, with 2017 being intermediate (mg/L: 5.3 ±
2.4 in 2016, 8.8 ± 3.7 in 2017, 13.2 ± 7.1 in
2018). Stervbo et al. (2007) reviewed levels of t-
resveratrol in monovarietal red wines and
discussed how it was found to vary greatly
between varieties and regions of origin. Among
all the samples examined, the highest average
level of t-resveratrol was found in wines made
from Pinot noir grown in France (5.4 mg/L),
followed by wines made from Spanish and
Italian Pinot noir (5.1 and 4.8 mg/L
respectively). This shows that wines of the Pinot
noir variety do indeed contain the highest
average levels of t-resveratrol. Similar values
were also reported for Italian Pinot noir by Van
Leeuw et al. (2014). Our results only partially
confirm these data, with a comparable average
value obtained in the 2016 vintage and much
higher values obtained in 2017 and 2018. Cool
and humid conditions for vine growth are related
to higher levels of t-resveratrol (Kolouchová-
Hanzlı́ková et al., 2004), but vinification
techniques (such as double maceration) are also
concomitant with high levels of t-resveratrol
(Alonso et al., 2002). The high values obtained
in our investigation could be related to both
these factors, including relation with vintages. Since t-resveratrol can undoubtedly be
considered as one of the most promising
polyphenolic compounds in terms of its
antioxidant activity, this particular aspect of 4.1. Catechins-procyanidins The monomer (+)-catechin is the most abundant
of the flavan-3-ols in the three vintages, except
for vintage 2016 (mg/L: 94.4 ± 28.9 in 2016,
144.7 ± 51.7 in 2017, 150.0 ± 85.0 in 2018),
when the values for procyanidin B1 were almost
comparable (mg/L: 92.8 ± 30.3 in 2016, 71.3 ± The Vanillin assay showed stronger correlation
with the sum of flavan-3-ol values from the LC- © 2020 International Viticulture and Enology Society - IVES
404
© 2020 International Viticulture and Enology Society - IVES
404 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 MS (monomers + dimers + trimer) than from the
BuOH-HCl assay (correlation coefficients are
0.6774 and 0.3259, respectively in 2017; 0.5873
and 0.3562 respectively in 2018). Since a
relatively high content of flavan-3-ols monomers
and oligomers has already been reported in Pinot
noir wines (Van Leeuw et al., 2014), which can
also be seen in our data (the different reactivity
of vanillin and BuOH-HCl assays with
monomers and oligomers has already been
discussed), an explanation for these correlation
values can be found, further validating our data
sets. flavonol subfamilies to be quantified in the Pinot
noir samples. Astilbin was found to be by far the
most abundant (mg/L: 24.9 ± 6.5 in 2016, 22.1 ±
6.0 in 2017, 31.7 ± 11.4 in 2018), followed by
quercetin-3-glucuronide, quercetin, taxifolin,
myricetin and isorhamnetin (in that order) in
both 2016 and 2017. In 2018, the pattern was
similar, but quercetin and taxifolin were lower in
content than myricetin. The ranges of values
were very similar over the three years for all the
six compounds investigated. Similar values for
myricetin, quercetin and isorhamnetin in 2018
have previously been reported in Pinot noir wine
from Trentino-Alto Adige (Baroň and Kumšta,
2013). 4.2. Acids As previously reported by other authors, gallic
and caftaric acid are usually the most abundant
acids in red wines, even if their ratios can be
variable (Van Leeuw et al., 2014). Our results
confirmed this evidence: caftaric acid was the
most abundant in all three vintages (mg/L: 102.3
± 33.6 in 2016, 62.9 ± 16.3 in 2017, 93.2 ± 36.1
in 2018), with gallic acid having similar values
in 2017 (mg/L: 54.8 ± 24.9), slightly lower ones
in 2018 (mg/L: 73.1 ± 36.5) and less than half in
2016 (mg/L: 42.9 ± 17.1). Coutaric acid and
caffeic acid came next in terms of abundance. 4.3. Anthocyanins Malvidin-3-glucoside was by far the most
abundant anthocyanin in 2016, 2017 and 2018
(mg/L: 31.6 ± 12.8 in 2016, 19.8 ± 12.6 in 2017,
32.9 ± 16.1 in 2018), followed in order by
petunidin-3-glucoside and delphinidin-3-
glucoside. Ranges were similar in 2016 and
2018, with slightly lower values in 2017 for all
three compounds. While being slightly lower in
2017, relative ratios were also similar in 2016
and 2018, with malvidin-3-glucoside having a
ratio around 8- and 10-fold that of P-3-glu and
D-3-glu respectively. The absolute values and
their ratios are consistent with previously
released data on free anthocyanidin content
(determined after hydrolysis of glycosides)
except for delphinidin-3-glucoside content,
which is 70 to 80 % lower in our investigation
(Van Leeuw et al., 2014). 4.4. Flavonols and dihydroflavonols 405
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
405 4.4. Flavonols and dihydroflavonols Two glycosides, taxifolin-3-rhamnoside
(astilbin) and quercetin-3-glucoside, together
with the four aglycones, taxifolin, quercetin,
isorhamnetin and myricetin were chosen as
representatives of the flavonol and dihydro- 405
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
405 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. Italian Pinot noir wines could be highly
interesting from a nutraceutical point of view. and colour intensity. Obviously, some of these
characteristics (e.g., Abs 420 and colour tonality)
are highly correlated with one another. Moreover, it is a fair assumption that some of
these parameters are more objectively
recognisable since they can directly and strongly
affect tactile, gustative and visual sensations
during sensory evaluation (e.g., glycerol, alcohol
and anthocyanin-colour respectively); therefore,
it is not surprising or accidental that they show
higher correlation with overall quality value,
especially if typical characteristics of a wine are
reflected. © 2020 International Viticulture and Enology Society - IVES
406
© 2020 International Viticulture and Enology Society - IVES
406 6. Linear modeling To describe relationships between wine score
and wine chemical parameters, the multiple
linear regression is used. To avoid
multicolinearity some highly correlated variables
are excluded from the model and are represented
by a single one from that the same group. In fact,
because highly correlated parameters give the
same information in models, it is possible to use
one to represent the others in the same group. Thus, for example, “Abs 420”, “Abs 520”, “Abs
620” and “Intensity” are highly correlated, so
Abs 420”, “Abs 520” and “Abs 620” are
discarded and “Intensity” is used as the
representative
one. “Quercetin”
and
“Isorhamnetin” are highly correlated, so
“Quercetin” is used. “Catechins/procyanidins
total”, “(+)-Catechin”, “Procyanidin B1”,
“Procyanidin B2”, “Procyanidin C1”, and “(-)-
Epicatechin” are highly correlated, so
“catechins/procyanidins total” is used. “Malvidin-3-glu” and “Petunidin-3-glu” are
highly correlated, so “Malvidin-3-glu” is used. In
the final model, 13 representative parameters
have been selected to describe a wine score from
the full set of parameters (Table 1). 5. Correlation between chemical parameters
and sensory evaluation Pearson´s correlation coefficients between all
indicators (standard chemical analyses, LC-MS
analyses and score values) were computed and
are graphically represented as a heat map
(Figure 6). Results from the tannin assays were
not included since vintage 2016 was missing. The list of all correlation coefficients between
score and all other parameters is available in
Supplementary Table 3 (ST-3). Their values
ranged from +0.37 and -0.32. Higher positive
correlation appears between score and alcohol
content (EtOH %), malvidin-3-glucoside, IAC,
petunidin-3-glucoside, caftaric acid, delphinidin-
3-glucoside, astilbin and glycerol. Negative
correlation is highest between score and gallic
acid, colour tonality, TPC, Abs 420, methanol In particular, regarding single vintages,
correlation coefficients between score and
ethanol content were 0.470 in 2016, 0.339 in
2017 and 0.407 in 2018 (data not shown), being
lowest in vintage 2017, which exhibited lowest
ethanol contents. Such correlation of overall
quality with alcohol content has already been
noticed for Pinot noir wines by other authors
(Jaffré et al., 2009). As previously mentioned, FIGURE 6. Pearson’s correlation matrix - between all the chemical parameters and sensory evaluation
score. FIGURE 6. Pearson’s correlation matrix - between all the chemical parameters and sensory evaluation
score. OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 the score represents the summary of different
sensorial features as one number only; for this
reason, weak correlations between such a general
parameter and single characteristics of the wine
are to be expected. Fitting linear models to the wine score led to a
significant model (p value<2.2e-16, r2=0.5),
showing that wine score is influenced by
multiple chemical features. Parameters with a positive estimated value have
a positive effect on wine score, whereas
parameters with negative estimates decrease the
wine score. As expected, parameters with the
highest positive correlation (EtOH and IAC) and
negative correlation (methanol, TPC and
tonality) with the score are selected in the linear
model. Moreover, it can be confirmed that wine
quality, which is represented by wine score only,
is not derived from just one component, but it is
affected by several, as we can see from the
multiple regression model. 407
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
407 OENO One 2020, 2, 393-410
© 2020 International Viticulture and Enology Socie
© 2020 International Viticulture and Enology Society
OENO One 2020, 54, 2, 393-410 CONCLUSIONS Finally, a multiple linear
regression model was applied to select the most
informative set of parameters for the description
of the wine score, where total anthocyanins,
ethanol, reducing sugars, total acidity,
catechins/procyanidins total, caftaric acid and
quercetin-3-glucuronide have a positive
contribution; while total phenolic content,
methanol, color intensity, color tonality,
delphinidin-3-glucoside and gallic acid have a
negative contribution to overall quality wine
score. a general reference scheme provided at the
beginning. Chemical analyses consisted of the
determination of standard wine parameters,
colorimetric analyses (including total
polyphenols and tannin quantification) and
single polyphenols using LC-MS apparatus. Finally, a putative correlation between scores
and results from chemical analyses was
investigated. Most of the Pinot noir wines were from
Trentino-Alto Adige, followed by Piemonte,
Lombardia, Veneto and Friuli Venezia-Giulia in
that order. A total of 25 wines (of which 24 from
Trentino-Alto Adige) were examined in all three
consecutive vintages and showed only very
slight differences over the years, revealing that
most producers had a conservative attitude to
winemaking. Nevertheless,
the
most
representative standard chemical parameters
(like total phenolic content, total anthocyanins,
total proanthocyanidins and tannins, alcohol,
residue sugars, etc.) showed a high variability
among these wines, which was even higher in
the whole batches. It was thus demonstrated that
both agronomical and winemaking processes
(including eventual blending, wood treatments
and aging) have strong effects on the chemical
composition of single Pinot noir red wines
originating from both the same terroir and
different parts of Italy. In terms of the whole
Italian territory, the average overall quality
scores resulting from the sensory evaluation
showed limited variations in the three vintages
considered, but wines from Trentino-Alto Adige
always obtained higher scores than those from
the rest of Italy. This therefore suggests a higher
affinity for the pedoclimatic features of this
region and a consequent expression of typical
organoleptic characteristics. Compared to
monovarietal Pinot noir wines described in
literature, average values obtained in this
investigation were higher for standard wine
parameters like TPC, total titrable acidity and
alcohol content. Meanwhile, single phenolic
constituents showed contents as being consistent
with previously published data on Pinot noir red
wines from the same temperate climatic area,
except for t-resveratrol, for which we obtained
higher values in our analyses, and delphinidin-3-
glucoside, for which we obtained lower values. With respect to vintages, a significant variability
of most parameters, including scores, was also
observed. CONCLUSIONS A chemical description and sensory evaluation
of Pinot noir red wines from different parts of
Italy were performed in three consecutive years
(2016-2018). All wines were 3-years old from
production and were registered for the annual
Italian Pinot nir competition taking place in the
corresponding year. The purpose of the
competition was to assess the best Pinot noir red
wine from Italian territories in terms of overall
quality. This was achieved using a tasting panel
composed of experts in wine evaluation instead
of specifically trained personnel. The panel
applied their knowledge and experience to judge
quality, taking into account the typical
characteristics of Pinot noir wines and following inear regression model output with the selected chemical parameters for wine score TABLE 1. Multiple linear regression model output with the selected chemical parameters for wine score
d
i ti TABLE 1. Multiple linear regression model output with the selected chemical parameters for wine score
description. n. Term
Estimate
Std. error
t-statistic
p.value
(Intercept)
33.2383
10.0025
3.3230
0.0010617
IAC
0.0781
0.0169
4.6261
0.0000067
TPC
-0.0023
0.0009
-2.4930
0.0134892
EtOH (%)
3.3696
0.5908
5.7031
0.0000000
Red. Sugars
0.6229
0.2837
2.1957
0.0292801
Total H+
2.1168
0.7963
2.6582
0.0085016
Methanol
-25.4953
18.3567
-1.3889
0.1664358
Intensity
-2.1510
0.4496
-4.7846
0.0000034
Tonality
-10.2616
4.6519
-2.2059
0.0285467
Catechins/Procyanidins total
0.0149
0.0035
4.3060
0.0000262
Delphinidin-3-glu
-1.1976
0.4493
-2.6657
0.0083214
Caftaric acid
0.0253
0.0091
2.7770
0.0060157
Gallic acid
-0.0340
0.0118
-2.8803
0.0044125
Querc-3-Glucur
0.1629
0.1134
1.4365
0.1524520 OENO One 2020, 2, 393-410
OENO One 2020 54 2 393-4 Enrico Serni et al. Enrico Serni et al. a general reference scheme provided at the
beginning. Chemical analyses consisted of the
determination of standard wine parameters,
colorimetric analyses (including total
polyphenols and tannin quantification) and
single polyphenols using LC-MS apparatus. Finally, a putative correlation between scores
and results from chemical analyses was
investigated. investigated: alcohol content, total anthocyanins
from the colorimetric assay, single anthocyanins
from the LC-MS analysis, caftaric acid and
glycerol content had the strongest positive
correlation, while gallic acid content, colour
tonality and total phenolic content had the
strongest negative correlation with sensory
evaluation scores. CONCLUSIONS Moreover, the correlation between the
overall quality evaluation scores and all the
determined single chemical parameters was Acknowledgements: Laimburg Research Centre
is funded by the Autonomous Province of
Bozen-Bolzano. The Autonomous Province of
Bozen-Bolzano, Department of Innovation,
Research and University is gratefully
acknowledged for its financial support within the
NOI Capacity Building I Funding Frame
(Decision 1472, 07.10.2013), Capacity Building
II Funding Frame (Decision 864, 04.09.2018)
and the Incoming Researcher Project (decree
334, 16.01.2019). © 2020 International Viticulture and Enology Society - IVES
408
© 2020 International Viticulture and Enology Society - IVES
408 REFERENCES Ed. by JM Chambers and TJ Hastie, Wadsworth and
Brooks/Cole. Pedri U., Pertoll G., Thalheimer M. and
Ueberegger E., 2019. The effects of location on the
quality of grapes and wine of the variety Pinot noir. Laimburg Journal, 1. Jaffré J., Valentin D., Dacremont C. and Peyron D.,
2009. Burgundy red wines: Representation of
potential for aging. Food quality and preference
20(7), 505-513. doi: 10.1016/j.foodqual.2009.05.001 Piccardo D., Favre G., Pascual O., Canals J. M.,
Zamora F. and González-Neves G., 2019. Influence
of the use of unripe grapes to reduce ethanol content
and pH on the color polyphenol and polysaccharide
composition of conventional and hot macerated Pinot
noir and Tannat wines. European Food Research and
Technology 245(6), 1321-1335. doi: 10.1007/s00217-
019-03258-4 Jouanneau S., Weaver R.J., Nicolau L., Herbst-
Johnstone M., Benkwitz F. and Kilmartin P.A., 2012. Jouanneau S., Weaver R.J., Nicolau L., Herbst-
Johnstone M., Benkwitz F. and Kilmartin P.A., 2012. Subregional survey of aroma compounds in
Marlborough Sauvignon blanc wines. Australian
Journal of Grape and Wine Research 18(3), 329-343. doi: 10.1111/j.1755-0238.2012.00202.x Subregional survey of aroma compounds in
Marlborough Sauvignon blanc wines. Australian
Journal of Grape and Wine Research 18(3), 329-343. doi: 10.1111/j.1755-0238.2012.00202.x Porter L.J., L.N. Hrstich and B.C. Chan., 1986. The
conversion of procyanidins and prodelphinidins to
cyanidin and delphinidin. Phytochemistry 25, 223-
230. doi:10.1016/S0031-9422(00)94533-3 Kemp B.S., Harrison R. and Creasy G.L., 2011. Effect of mechanical leaf removal and its timing on
flavan-3-ol composition and concentrations in Vitis
vinifera L. cv. Pinot noir wine. Australian journal of
grape and wine research 17(2), 270-279. doi:
10.1111/j.1755-0238.2011.00150.x Price S.F., Breen P. J., Valladao M. and Watson B.T.,
1995. Cluster Sun Exposure and Quercetin in Pinot
noir Grapes and Wine. American Journal of Enology
and Viticulture 46(2), 187–194. Kennedy J.A., Godard M. and Watson B., 2002. Development of anthocyanins and tannins in Pinot
noir grapes and their relative importance in wine. https://ir.library.oregonstate.edu/concern/technical_re
ports/v979v423w Rigaux J., 2010. Le réveil des terroirs: défense et
illustration des climats de Bourgogne. Éd. de
Bourgogne.66-67. Rigo A., Vianello F., Clementi G., Rossetto M.,
Scarpa M., Vrhovšek U. and Mattivi F., 2000. Contribution of proanthocyanidins to the peroxy
radical scavenging capacity of some Italian red
wines. Journal of agricultural and food chemistry
48(6), 1996-2002. doi:10.1021/jf991203d Kobler A., 2008. Limiti e possibili evoluzioni
dell’analisi sensoriale quantitativa dei vini. L’enologo
11, 1-8. Kolouchová-Hanzlı́ková I., Melzoch K., Filip V. and
Šmidrkal J., 2004. REFERENCES Aleixandre-Tudo J.L., Buica A., Nieuwoudt H.,
Aleixandre J.L. and du Toit W., 2017. Spectrophotometric analysis of phenolic compounds
in grapes and wines. Journal of agricultural and food
chemistry 65(20), 4009-4026. doi:10.1021/acs.jafc. 7b01724 Alonso Á.M., Domínguez C., Guillén D.A. and
Barroso C.G., 2002. Determination of antioxidant
power of red and white wines by a new
electrochemical method and its correlation with
polyphenolic content. Journal of Agricultural and
Food Chemistry
50(11), 3112-3115. doi:
10.1021/jf0116101 Anderson K. and Aryal N.R., Database of Regional
National and Global Winegrape Bearing Areas by
Variety 2000 and 2010, Wine Economics Research
Centre University of Adelaide December 2013, last
rev. July 2014) Arapitsas P., Perenzoni D., Nicolini G. and Mattivi F.,
2012. Study of sangiovese wines pigment profile by
UHPLC-MS/MS. Journal of Agricultural and Food
Chemistry 60(42), 10461–10471. doi: 10.1021/
jf302617e © 2020 International Viticulture and Enology Society - IVES
408
© 2020 International Viticulture and Enology Society - IVES
408 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 Food Chemistry 49(7), 3341-3348. doi: 10.1021
/jf010128f Baroň M. and Kumšta M., 2013. Comparison of
North Italian and South Moravian wines on the base
of their antioxidant activity phenolic composition and
sensory quality. Acta Universitatis Agriculturae et
Silviculturae Mendelianae Brunensis 60(8), 9-18. doi:10.11118/actaun201260080009 Magnusson B., 2014. The fitness for purpose of
analytical methods: a laboratory guide to method
validation and related topics. Mattivi F., Guzzon R., Vrhovsek U., Stefanini M. and
Velasco R., 2006. Metabolite profiling of grape:
flavonols and anthocyanins. Journal of agricultural
and food chemistry 54(20), 7692-7702. doi:
10.1021/jf061538c Bertamini M., Mattivi F. and Nicolini G., 1998. L’influenza del clima e delle tecniche di gestione del
vigneto sui polifenoli del vino. L’Enotecnico 34, (10),
S. 31–42. Cortell J.M., Sivertsen H.K., Kennedy J.A. and
Heymann H., 2008. Influence of Vine Vigor on Pinot
noir Fruit Composition Wine Chemical Analysis and
Wine Sensory Attributes. American Journal of
Enology and Viticulture 59(1), 1–10. Mawdsley P.F., Peterson J.C.D. and Casassa L.F.,
2019. Multi-year study of the effects of cluster
thinning on vine performance fruit and wine
composition of Pinot noir (clone 115) in California’s
Edna Valley AVA, USA. Scientia Horticulturae 256,
108631. doi: 10.1016/j.scienta.2019.108631 Hagerman A.E., 2011. The Tannin Handbook
[Online] Miami University. Oxford, OH. http://www.users.miamioh.edu/hagermae/ OIV, 2009. Standard for international wine and
spirituous beverages of vitivinicultural origin
competitions. Resolution OIV/Concours 332A. Zagreb. Hastie T.J. and Pregibon D., 1992. Chapter 6. Statistical models in S. Generalized linear models. REFERENCES Rapid method for resveratrol
determination by HPLC with electrochemical and UV
detections in wines. Food Chemistry 87(1), 151-158. doi: 10.1016/j.foodchem.2004.01.028 Robinson J., Harding J. and Vouillamoz J., 2013. Wine grapes: a complete guide to 1,368 vine
varieties, including their origins and flavours. Penguin UK. 808-815. Landrault N., Poucheret P., Ravel P., Gasc F., Cros G. and Teissedre P.-L., 2001. Antioxidant capacities and
phenolics levels of French wines from different
varieties and vintages. Journal of Agricultural and Samoticha J., Wojdyło A., Chmielewska J. and
Oszmiański J., 2017. The effects of flash release
conditions on the phenolic compounds and 409
© 2020 International Viticulture and Enology Society - IVES
© 2020 International Viticulture and Enology Society - IVES
409 OENO One 2020, 2, 393-410
OENO One 2020, 54, 2, 393-4 Enrico Serni et al. antioxidant activity of Pinot noir red wine. European
Food Research and Technology 243(6), 999-1007. doi: 10.1007/s00217-016-2817-7 VV.AA., 2017. R Core Team, 2019. R: A language
and environment for statistical computing. R Foundation for Statistical Computing Vienna
Austria. URL https://www.R-project.org/. Stervbo U., Vang O. and Bonnesen C., 2007. A
review of the content of the putative chemopreventive
phytoalexin resveratrol in red wine. Food Chemistry
101(2), 449-457. doi: 10.1016/j.foodchem.2006. 01.047 VV.AA., 2017. Relazione agraria e forestale 2017,
Bolzano Italy: Provincia Autonoma di Bolzano – Alto
Adige Bolzano VV.AA. Focus OIV, 2017. Distribution of the world’s
grapevine varieties. International organization of vine
and wine, Paris, France 2017. ISBN: 979-10-91799-
89-8 Stój A., Szwajgier D., Baranowska-Wójcik E. and
Domagala D., 2019. Gentisic acid salicylic acid total
phenolic content and cholinesterase inhibitory
activities of red wines made from various grape
varieties. South African Journal of Enology and
Viticulture 40(1), 1-12. doi: 10.21548/40-1-2885 Vaudour E., 2005. I Terroir. Definizione
caratterizzazione e protezione. (1st ed.) Edagricole,
Bologna, 57-59. Venables W.N. and Ripley B.D., 2002. Modern
Applied Statistics with S. New York, Springer, 4th ed. doi:10.1007/978-0-387-21706-2 Sun B., Ricardo-da-Silva J.M. and Spranger I., 1998. Critical Factors of Vanillin Assay for Catechins and
Proanthocyanidins Journal of Agricultural and Food
Chemistry 46(10), 4267–4274. doi:10.1021/jf980366j Vilanova M., Genisheva Z., Tubio M., Álvarez K.,
Lissarrague J. and Oliveira J., 2017. Effect of vertical
shoot-positioned scott-henry geneva double-curtain
arch-cane and parral training systems on the volatile
composition of Albariño wines. Molecules 22(9),
1500. doi: 10.3390/molecules22091500 Valentin D., Parr W.V., Peyron D., Grose C. and
Ballester J., 2016. © 2020 International Viticulture and Enology Society - IVES
410
© 2020 International Viticulture and Enology Society - IVES
410 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410 REFERENCES Colour as a driver of Pinot noir
wine quality judgments: An investigation involving
French and New Zealand wine professionals. Food
Quality and Preference
48, 251-261. doi:
10.1016/j.foodqual.2015.10.003 Wenzel K., Dittrich H.H. and Heimfarth M., 1987. Zusammensetzung der Anthocyane in den Beeren
verschiedener Rebsorten. Vitis. van Leeuw R., Kevers C., Pincemail J.,
Defraigne J.O. and Dommes J., 2014. Antioxidant
capacity and phenolic composition of red wines from
various grape varieties: Specificity of Pinot noir. Journal of Food Composition and Analysis 36(1-2),
40-50. doi: 10.1016/j.jfca.2014.07.001 Whitener M.B., Stanstrup J., Carlin S., Divol B., Du
Toit M. and Vrhovsek U., 2017. Effect of non-
Saccharomyces yeasts on the volatile chemical profile
of Shiraz wine. Australian journal of grape and wine
research 23(2), 179-192. doi: 10.1111/ajgw.12269 OENO One 2020, 2, 393-410
ENO One 2020, 54, 2, 393-410
|
https://openalex.org/W2987677750
|
https://bmccomplementmedtherapies.biomedcentral.com/track/pdf/10.1186/s12906-019-2704-4
|
English
| null |
Antiaging effect of a Jianpi-yangwei formula in Caenorhabditis elegans
|
BMC complementary and alternative medicine
| 2,019
|
cc-by
| 7,936
|
(2019) 19:313 (2019) 19:313 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313
https://doi.org/10.1186/s12906-019-2704-4 Zeng et al. BMC Complementary and Alternative Medicine
https://doi.org/10.1186/s12906-019-2704-4 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Jianpi-yangwei (JPYW), a traditional Chinese medicine (TCM), helps to nourish the stomach and
spleen and is primarily used to treat functional declines related to aging. This study aimed to explore the antiaging
effects and mechanism of JPYW by employing a Caenorhabditis elegans model. Methods: Wild-type C. elegans N2 worms were cultured in growth medium with or without JPYW, and lifespan
analysis, oxidative and heat stress resistance assays, and other aging-related assays were performed. The effects of
JPYW on the levels of superoxide dismutase (SOD) and the expression of specific genes were examined to explore
the underlying mechanism of JPYW. Results: Compared to control worms, JPYW-treated wild-type worms showed increased survival times under both
normal and stress conditions (P < 0.05). JPYW-treated worms also exhibited enhanced reproduction, movement and
growth and decreased intestinal lipofuscin accumulation compared to controls (P < 0.05). Furthermore, increased
activity of SOD, downregulated expression levels of the proaging gene clk-2 and upregulated expression levels of
the antiaging genes daf-16, skn-1, and sir-2.1 were observed in the JPYW group compared to the control group. Conclusion: Our findings suggest that JPYW extends the lifespan of C. elegans and exerts antiaging effects by
increasing the activity of an antioxidant enzyme (SOD) and by regulating the expression of aging-related genes. This study not only indicates that this Chinese compound exerts antiaging effects by activating and repressing
target genes but also provides a proven methodology for studying the biological mechanisms of TCMs. Keywords: Jianpi-yangwei formula, Traditional Chinese medicine, Caenorhabditis elegans, Aging Antiaging effect of a Jianpi-yangwei
formula in Caenorhabditis elegans Liling Zeng1, Zhimin Yang2*, Tianchan Yun1, Shaoyi Fan1, Zhong Pei3, Ziwen Chen1, Chen Sun2 and Fuping Xu2* Background effective antiaging treatment has not yet been found since
the mechanisms of aging are complicated. Aging is believed to be an inevitable physiological process
that occurs in all living organisms [1] and has been a con-
cern since ancient times. Some researchers have suggested
that the aging process is affected by environmental [2, 3],
nutritional [4], and genetic factors [5] and have attempted
to explore the mechanisms of aging. In addition, in mod-
ern times, an increasing number of aging-related diseases,
such as cancer, cardiovascular disease, chronic degenera-
tive diseases and other aging-related dysfunctions, have
threatened human health [6, 7]. Even though increasing
evidence has demonstrated that pharmacological interven-
tion may delay the senescence process [8, 9], a definitely In contrast to mainstream modern medicine, traditional
Chinese medicine (TCM) aims to interfere with the aging
process as early as possible, thus preventing and delaying
the occurrence and development of aging-related diseases,
and has begun to draw increasing research interest [10–
12]. TCM has been used as a complementary medicine for
5000 years and has garnered much attention as a result of
its high medical efficacy and its preventative functions [10,
11, 13]. In recent years, many studies have suggested that
lots of TCMs exhibit an array of antiaging effects [12, 14,
15]. According to TCM theory, Jianpi-yangwei (JPYW)
therapy is one of the main treatment modalities for
aging and has been clinically demonstrated to be effect-
ive [16–20]; however, further research on the nature of
JPYW is necessary due to the complexity of its com-
position. JPYW is a TCM formula that is mainly * Correspondence: yangyo@vip.tom.com; xufuping163@163.com
2The Second Affiliated Hospital of Guangzhou University of Chinese
Medicine, 111 Da De Rd., Yuexiu District, Guangzhou, Guangdong Province,
People’s Republic of China510120
Full list of author information is available at the end of the article Page 2 of 10 Page 2 of 10 Page 2 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 composed of 8 ingredients: Panax ginseng C. A. Mey,
Radix Paeoniae Alba, Codonopsis Radix, Poria cocos,
Rhizoma Atractylodis Macrocephalae, Crataegus pin-
natifida, Pericarpium Citri Reticulatae, and Cinnamo-
mum cassia Presl. In TCM theory, JPYW is based on
the Sijunzi decoction, which is a classic Chinese medi-
cine that has been demonstrated to be beneficial for
the spleen and stomach as a result of its antiaging ef-
fects [19, 21]. In a previous study, a JPYW capsule was
proven to have therapeutic effects on gastric precancer-
ous lesions and cancer-related fatigue [22]. In the
present study, we found that JPYW exhibited a spleen-
fortifying and stomach-nourishing effect that helped to
replenish energy and recover functions that were de-
clining as a result of aging. Moreover, we drew our
conclusions from ten years of clinical experience show-
ing that JPYW has strong antiaging effects. Notably,
previous studies suggested that Caenorhabditis elegans
was a comparatively ideal model for aging research [23,
24]. Assessment of stress resistance Assessment of stress resistance
Age-synchronized N2 worms were bred on NGM plates
with or without 150 μg/ml JPYW. For a heat tolerance
assay, day 4 adult worms (on the 4th day after the
worms reached adulthood, n = 50) were transferred to
fresh plates containing 150 μg/ml JPYW or a vehicle
control and then incubated at 37 °C. Survival was re-
corded every hour until all worms had died. The toler-
ance to oxidative stress was measured as reported
previously [25]. Briefly, day 4 adult worms (n = 50) were
placed on plates with various concentrations of hydro-
gen peroxide (from 0 mM to 1 mM, intervals of 0.2 mM)
as well as 150 μg/ml JPYW or a vehicle control, and then
the survival was recorded after 15 h. Each test was re-
peated at least twice. This study aimed to explore the antiaging effects and
the mechanism of JPYW in wild-type C. elegans N2
worms (Bristol). Lifespan assays, stress resistance assays
and other aging-related factors and properties were
assessed to evaluate antiaging effects. The activity of
superoxide dismutase (SOD) and the expression levels of
aging-related genes were assessed to illustrate the poten-
tial mechanisms. Preparation of JPYW p
JPYW mainly consists of 8 crude herbs: P. ginseng C. A. Mey, Radix Paeoniae Alba, Codonopsis Radix, P. cocos,
Rhizoma Atractylodis Macrocephalae, C. pinnatifida, Peri-
carpium Citri Reticulatae, and C. cassia Presl. For this
study, we used a mixture of water extracts of the crude
herbs. The water extracts were provided by Kangmei
Pharmaceutical Co. (Guangzhou, China), were produced
according to the rigid specifications of the Pharmacopeia
of the People’s Republic of China and were approved by
the China Food and Drug Administration (CFDA). In ac-
cordance with TCM research conventions, all concentra-
tions reported in this study refer to the concentrations of
the crude herbs. The JPYW used in the study was dis-
solved in 1% dimethylsulfoxide (DMSO). Measurement of SOD activity To measure SOD activity, wild-type worms (n = 50) were
collected from plates with M9 buffer on the 5th day of
adulthood (day 5 after the worms reached adulthood)
and washed 3 times. Then, the collected worms were re-
suspended in homogenization buffer (10 mM tris(hy-
droxymethyl)aminomethane
hydrochloride(Tris-HCl),
150 mM NaCl, and 0.1 mM ethylenedinitrilotetraacetic
acid (EDTA), pH 7.5) and homogenized through ultraso-
nication on ice. A total of 0.5 mg protein from every
group was used to measure SOD activity. The SOD ac-
tivity was spectrophotometrically analyzed on the basis
of the decolorization of formazan. A Total Superoxide
Dismutase (T-SOD) Assay Kit (hydroxylamine method)
and a Total Protein Assay Kit (standard: bicinchoninic
acid (BCA) method) were purchased from Nanjing Jian-
cheng Bioengineering Institute (Nanjing, China) and were
used to determine the SOD activity and protein concen-
tration, respectively. The procedures were performed in
strict accordance with the manufacturers’ protocols. Lifespan analysis A bleaching technique was used to synchronize the
worm population in this study. The age-synchronized
N2 nematodes were transferred to NGM plates contain-
ing 150 μg/ml JPYW or a vehicle control (1% DMSO). E. coli OP50 was added to the medium. Two NGM plates
containing 25 worms each were used, and the worms
were transferred to new NGM plates every day for the
first 7 days so that the new eggs did not have a disrup-
tive effect. Then, the survival rate was assessed every
other day until the worms died. The survival fraction
was calculated by recording the number of surviving
worms. We considered the nematodes to be dead when
there was no respond after touching them with a plat-
inum loop (failed to exhibit touch-provoked movement). At least three independent trials of the lifespan assay
were performed. Effects of JPYW on lifespan extension and stress
resistance The fluorescence intensity of lipofuscin and autofluo-
rescence were assessed in the worms on the 10th day of
adulthood, and were quantified using ImageJ to deter-
mine the average pixel intensity. All tests were repeated
more than 2 times. To
determine
the
lifespan-extending
properties
of
JPYW, lifespan assays were performed using wild-type
worms with or without 150 μg/ml JPYW treatment. We found significantly more worms in the old-age
phase among the JPYW-treated worms than among
the controls (Fig. 1a). Therefore, we hypothesized that
JPYW may affect the lifespan of worms without af-
fecting worm development. We subsequently used
aged wild-type worms (7-day-old adult worms) as the
experimental models for the lifespan assay. Interest-
ingly, after 7 days of treatment, there was a significant
difference between the JPYW group and the control
group for every day; in addition, compared to control
worms, JPYW-treated worms displayed significant in-
creases in lifespan (11.86 ± 4.24 vs. 14.49 ± 4.78 days, P
< 0.05) (Fig. 1b). To evaluate stress resistance, we
performed heat stress assays and oxidative stress as-
says using wild-type worms with or without 150 μg/
ml JPYW treatment. As shown in Fig. 2a, compared
to control worms, 150 μg/ml JPYW-treated worms
had a significantly increased mean lifespan during
heat stress (5.82 ± 0.62 vs. 6.49 ± 0.81 h, P < 0.01). Thermotolerance was also elevated in aged worms. As
shown in Fig. 2b, compared to the control treatment,
JPYW treatment significantly increased the survival
rate in aged worms (4.50 ± 1.20 vs. 5.29 ± 0.97 h, P <
0.01). Then,
we
determined
whether
JPYW
also
exerted
protective
effects
on
wild-type
and
aged
worms under oxidative stress conditions. Interestingly,
compared to the control treatment, JPYW treatment
improved survival under mild to moderate oxidative
stress but did not improve survival under severe oxi-
dative stress. The results showed that JPYW-treated Quantitative analysis of aging-related genes in C. elegans
Age-synchronized N2 worms were treated with 150 μg/
ml JPYW or vehicle at 20 °C until the 4th day after the
worms reached adulthood. Total RNA was extracted
from approximately 600 worms per group with TRIzol
(TaKaRa, Beijing, China). For RNA extraction and quan-
titative real-time polymerase chain reaction (qRT-PCR),
more detailed steps have been described in the previous
study [26]. Measurement of aging-related factors (reverse); daf-16, 5′- CCAGACGGAAGGCTTAAACT-
3′ (forward) and 5′-ATTCGCATGAAACGAGAATG-3′
(reverse). The cDNA was produced using random 6-
mers and oligo (dT) primers. qRT-PCR was performed
using SYBR green as the detection method. The com-
parative 2−ΔΔCT method was used to assess the expres-
sion levels of each mRNA relative to those of act-1. The
test was performed in triplicate. (reverse); daf-16, 5′- CCAGACGGAAGGCTTAAACT-
3′ (forward) and 5′-ATTCGCATGAAACGAGAATG-3′
(reverse). The cDNA was produced using random 6-
mers and oligo (dT) primers. qRT-PCR was performed
using SYBR green as the detection method. The com-
parative 2−ΔΔCT method was used to assess the expres-
sion levels of each mRNA relative to those of act-1. The
test was performed in triplicate. For a pharyngeal pumping assay, age-synchronized N2
worms (n = 10) were treated with 150 μg/ml JPYW or ve-
hicle until the 4th day after the worms reached adulthood,
and then their pharynx contractions were counted under
an inverted microscope for 10 s in the fresh plates. For reproduction assay, worms (n = 5) were cultured
from eggs. Worms were individually moved to a fresh
plate every day once they became adults. The progeny
were counted at the L2 or L3 stage. (2019) 19:313 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 Statistical analyses For a growth alteration assay, on the 4th day of
adulthood, worms were photographed and their body
length was analyzed by using Nikon software (Nikon,
Japan). All the datas in the study were analyzed by using Graph-
Pad Prism 6.0. Kaplan-Meier survival analysis and log-
rank test were conducted for the lifespan assay. Student’s
t-test was used for comparing two datasets. For all the
datas, the mean and standard error of the mean (SEM)
were analyzed. P values < 0.05 were considered to indi-
cate significance. For a body movement assay, age-synchronized N2
worms (n = 10) were bred on NGM plates with or with-
out 150 μg/ml JPYW. On the 7th day of adulthood, their
body movements expressed as the travel distance were
recorded under an inverted microscope for 20 s in fresh
plates, and were analyzed by using Nikon software. Effects of JPYW on lifespan extension and stress
resistance Briefly, the collected worms were moved to
1.5-ml RNase-free microfuge tubes to extract RNA and
the RNA concentration was quantified using a Nano-
Drop spectrophotometer. Complementary DNA (cDNA)
was synthesized by reverse transcription using a Prime-
Script RT Reagent Kit with gDNA Eraser (Perfect Real
Time; TaKaRa, Beijing, China) according to the manu-
facturer’s protocol. Quantitative real-time polymerase
chain reaction (qRT-PCR) was performed using TB
Green Premix Ex Taq II (Tli RNase H Plus; TaKaRa,
Beijing, China) with SuperReal PreMix Plus (SYBR
Green; TaKaRa, Beijing, China). The primers were as
follows: act-1, 5-TCCCTCTCCACCTTCCAACA-3 (for-
ward) and 5-GCACTTGCGGTGAACGATG-3 (reverse);
skn-1,
5-CCAGTGACAACGAGCTTCCA-3
(forward)
and 5-GTGACGATCCGTGCGTCTTT (reverse); clk-2,
5-ACTCCGATCTACTCGCCTCA-3 (forward) and 5-
GATGCAGGCAGTCCGTAGTT-3 (reverse); sod-3, 5′-
CCAACCAGCGCTGAAATTCAATGG-3′
(forward)
and
5′-
GGAACCGAAGTCGCGCTTAATAGT-3′ C. elegans: strains and maintenance The wild-type C. elegans N2 worms (Bristol) and E. coli
OP50 were provided by the Caenorhabditis Genetics
Center (CGC) (Minneapolis, MN, USA). The C. elegans
strains were cultured at 20 °C on solid nematode growth
medium (NGM) plates seeded with E. coli OP50. The
wild-type C. elegans N2 worms (Bristol) were aged and
were considered adults at 7 days. Page 3 of 10 Quantitative analysis of aging-related genes in C. elegans Medicine (2019) 19:313
Page 4 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313
Page 4 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 1 (2019) 19:313 Page 4 of 10 Fig. 1 Effect of JPYW on the lifespan of C. elegans N2 worms under normal conditions. a The worms were treated with JPYW beginning at the
larval stage. The curves show the percentages of surviving worms on different days after treatment with a vehicle control (1% DMSO) or 150 μg/
ml JPYW. JPYW did not significantly prolong the lifespan of wild-type worms, but it caused a positive trend in the number of surviving aged
worms (n = 50). b The aged worms were exposed to JPYW beginning on the 7th day of adulthood. The curves show the percentages of
surviving worms on different days after treatment with a vehicle control (1% DMSO) or 150 μg/ml JPYW. JPYW significantly prolonged the
lifespan of aged wild-type worms; n = 50–51, P < 0.05 Fig. 1 Effect of JPYW on the lifespan of C. elegans N2 worms under normal conditions. a The worms were treated with JPYW beginning at the
larval stage. The curves show the percentages of surviving worms on different days after treatment with a vehicle control (1% DMSO) or 150 μg/
ml JPYW. JPYW did not significantly prolong the lifespan of wild-type worms, but it caused a positive trend in the number of surviving aged
worms (n = 50). b The aged worms were exposed to JPYW beginning on the 7th day of adulthood. The curves show the percentages of
surviving worms on different days after treatment with a vehicle control (1% DMSO) or 150 μg/ml JPYW. JPYW significantly prolonged the
lifespan of aged wild-type worms; n = 50–51, P < 0.05 Effects of JPYW on antioxidant enzyme activity
To verify the possible mechanism by which JPYW mediated
longevity extension and elevated stress tolerance, the activ-
ity of individual stress resistance proteins was investigated
in wild-type worms and aged worms. In this study, we
assessed the activity of antioxidant enzymes such as SOD. Effects of JPYW on antioxidant enzyme activity wild-type worms lived longer than control vehicle-
treated
worms
under
0.6
to
0.8 mM
hydrogen
peroxide-induced oxidative stress (Fig. 2c). Significant
differences were also observed between aged wild-type
worms and aged control worms under 0.4 to 1.0 mM
hydrogen peroxide-induced oxidative stress (Fig. Quantitative analysis of aging-related genes in C. elegans Aged worms that were incubated at a constant temperature (37 °C)
were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO). Survival was assessed every hour after heat stress treatment. JPYW
treatment significantly prolonged the lifespan of aged wild-type worms under heat stress compared to the control treatment (n = 50–55, P <
0.05). c Oxidative stress resistance in C. elegans N2 worms. Wild-type worms were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO)
and were exposed to various concentrations of hydrogen peroxide (0, 0.2, 0.4, 0.6, 0.8, and 1 mM). Survival was assessed after 15 h of each
treatment. d Oxidative stress resistance in aged C. elegans N2 worms. Aged worms were pretreated with 150 μg/ml JPYW or vehicle control (1%
DMSO) and were exposed to various concentrations of hydrogen peroxide (0, 0.2, 0.4, 0.6, 0.8, and 1 mM). Survival was assessed after 15 h of
each treatment As shown in Fig. 3a and b, SOD was significantly upregu-
lated in the presence of 150 μg/ml JPYW in both wild-type
and aged worms compared to controls (P < 0.05). As shown in Fig. 3a and b, SOD was significantly upregu-
lated in the presence of 150 μg/ml JPYW in both wild-type
and aged worms compared to controls (P < 0.05). this aging-associated decline was attenuated by JPYW treat-
ment compared to the control treatment (Fig. 4c). Then,
we measured the body movements to estimate the health-
span of aged worms (worms that had been adults for more
than 7 days) by recording the distances the worms traveled
over 20 s. As shown in Fig. 4d, worm body movement was
significantly higher in the JPYW group than in the un-
treated control group (0.92 ± 0.08 vs. 2.13 ± 0.18 mm, n =
10, P < 0.01), suggesting that the functional aging of worms
is strongly delayed by JPYW. As shown in Fig. 4e, the fluor-
escence intensity of intestinal lipofuscin was significantly at-
tenuated in the JPYW group compared to the control
group (37.29 ± 0.54 vs. 26.32 ± 0.35, n = 20, P < 0.01). Quantitative analysis of aging-related genes in C. elegans 2d). To verify the possible mechanism by which JPYW mediated
longevity extension and elevated stress tolerance, the activ-
ity of individual stress resistance proteins was investigated
in wild-type worms and aged worms. In this study, we
assessed the activity of antioxidant enzymes such as SOD. Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313
Page 5 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 (2019) 19:313 Page 5 of 10 Fig. 2 The effect of JPYW on stress resistance in C. elegans N2 worms. a Heat stress resistance in wild-type larvae. Wild-type worms that were
incubated at a constant temperature (37 °C) were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO). Survival was assessed every
hour after heat stress treatment. JPYW significantly prolonged the lifespan of wild-type worms under heat stress compared to the vehicle control
(n = 50–55, P < 0.05). b Heat stress resistance in aged C. elegans N2 worms. Aged worms that were incubated at a constant temperature (37 °C)
were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO). Survival was assessed every hour after heat stress treatment. JPYW
treatment significantly prolonged the lifespan of aged wild-type worms under heat stress compared to the control treatment (n = 50–55, P <
0.05). c Oxidative stress resistance in C. elegans N2 worms. Wild-type worms were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO)
and were exposed to various concentrations of hydrogen peroxide (0, 0.2, 0.4, 0.6, 0.8, and 1 mM). Survival was assessed after 15 h of each
treatment. d Oxidative stress resistance in aged C. elegans N2 worms. Aged worms were pretreated with 150 μg/ml JPYW or vehicle control (1%
DMSO) and were exposed to various concentrations of hydrogen peroxide (0, 0.2, 0.4, 0.6, 0.8, and 1 mM). Survival was assessed after 15 h of
each treatment Fig. 2 The effect of JPYW on stress resistance in C. elegans N2 worms. a Heat stress resistance in wild-type larvae. Wild-type worms that were
incubated at a constant temperature (37 °C) were pretreated with 150 μg/ml JPYW or vehicle control (1% DMSO). Survival was assessed every
hour after heat stress treatment. JPYW significantly prolonged the lifespan of wild-type worms under heat stress compared to the vehicle control
(n = 50–55, P < 0.05). b Heat stress resistance in aged C. elegans N2 worms. Effects of JPYW on aging-related factors Previous study has indicated that lifespan was associated
with reproduction, pharyngeal pumping, body size and mo-
tility in many species, such as C. elegans [27]. In this study,
we found that JPYW treatment significantly increased the
total number of progeny compared to the control treatment
(297.4 ± 15.3 vs. 223.8 ± 6.3 progeny, n = 5, P < 0.01, Fig. 4a). In addition, a significant change in worm body length was
detected after JPYW exposure (0.953 ± 0.035 vs. 1.108 ±
0.024 mm, n = 10, P < 0.05, Fig. 4b), suggesting that JPYW
activity affects growth as well as fertility (Fig. 4a). Then, we
assessed the muscle activity and the movement ability of
the worms by recording the rate of pharyngeal pumping. The graph in Fig. 4c showed that the rate of pharyngeal
contractions declined gradually with increasing age, and Discussion In the present study, one control group (1% DMSO) and
one experimental group (150 μg/ml) were used to explore
the antiaging effects of JPYW and their underlying mecha-
nisms in a C. elegans model. Since the experiments were
not designed as noninferiority tests or superiority tests, a
positive control group was not used. Each test in the study
was performed at least two times to control for random
effects and to ensure the repeatability and accuracy of the
results. We found that JPYW treatment significantly pro-
longed the lifespan of wild-type worms under stress condi-
tions. In addition, the lifespan of aged worms increased
more significantly than that of wild-type worms under
both normal and stress conditions. This result indicates
that JPYW may have a strong antiaging effect and that
JPYW therapy may be a useful antiaging treatment. As
previously reported, most of the plants in JPYW have anti-
aging effects. For instance, P. ginseng C. A. Mey, one of the
main herbs in this formula, has been proven to be very ef-
fective in delaying senility [31], and ginsenosides, the ac-
tive ingredients in P. ginseng, have been proven to
promote development and growth and to prolong lifespan
of C. elegans [32]. In addition, ginsenoside Rg1, the main
active pharmaceutical ingredient in P. ginseng, has been
found
to
improve
the
antiaging
ability
of
the
hematopoietic microenvironment by enhancing the anti-
oxidant and anti-inflammatory capacities of bone marrow
stromal cells in a D-galactose-induced aged rat model and
also to act on hematopoietic cells to protect them from
aging [33, 34]. Pachymic acid, a main compound in P. cocos, can induce autophagy via the IGF-1 signaling path-
way in aged cells to delay the aging process [35]. Addition-
ally, nobiletin, an active ingredient in Pericarpium Citri
Reticulatae, may ameliorate isoflurane-induced cognitive
impairment and delay the aging process through antioxi-
dant, anti-inflammatory and antiapoptotic effects via
modulation of Akt, Bax, pCREB and BDNF in aging rats
[36]. Finally, C. cassia Presl can increase C. elegans life-
span via insulin signaling and stress-response pathways
[37], and the major chemical components of C. cassia,
cinnamates, may promote adiponectin production during
adipogenesis in human adipose tissue-derived mesenchy-
mal stem cells and prevent skin aging [38]. JPYW may Fig. 3 Effect of JPYW on SOD activity in C. elegans N2 worms. a SOD
activity in C. elegans N2 worms. Effects of JPYW on aging-related gene expression Effects of JPYW on aging-related gene expression
Pathways for the induction of stress-response genes that
affect lifespan have been identified in C. elegans. JPYW
treatment might improve survival by activating these
genes. Treatment with JPYW can increase C. elegans (2019) 19:313 Page 6 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 Page 6 of 10 Fig. 3 Effect of JPYW on SOD activity in C. elegans N2 worms. a SOD
activity in C. elegans N2 worms. Quantitative comparisons showed
that SOD levels were significantly higher in JPYW-pretreated worms
than in control worms (25.44 ± 0.22 vs. 30.96 ± 1.53 U/mg of protein,
P < 0.05). b SOD activity in aged C. elegans N2 worms. Quantitative
comparisons showed that SOD levels were significantly higher in
JPYW-pretreated aged worms than in control worms (15.54 ± 1.09 vs. 21.35 ± 0.52 U/mg of protein, P < 0.05) control treatment, suggesting that JPYW may act in a
manner that is dependent on these genes (Fig. 5a). JPYW
treatment also significantly downregulated the expres-
sion level of clk-2 compared to the control treatment,
which may have slowed the shortening of telomere
length in the JPYW-treated worms, resulting in in-
creased lifespan. Surprisingly, compared to the vehicle
control, JPYW significantly increased SOD activity, but
it did not increase the expression of the sod-3 gene. Discussion Quantitative comparisons showed
that SOD levels were significantly higher in JPYW-pretreated worms
than in control worms (25.44 ± 0.22 vs. 30.96 ± 1.53 U/mg of protein,
P < 0.05). b SOD activity in aged C. elegans N2 worms. Quantitative
comparisons showed that SOD levels were significantly higher in
JPYW-pretreated aged worms than in control worms (15.54 ± 1.09 vs. 21.35 ± 0.52 U/mg of protein, P < 0.05) lifespan through sir-2.1, which regulates this effect
through kat-1-mediated fatty acid oxidation [28]. As
shown in Fig. 5a, the expression level of the sir-2.1 gene
was significantly upregulated in JPYW-treated worms
compared to control-treated worms. In C. elegans, two
transcription factors, daf-16 and skn-1, promote the ex-
pression of antioxidant or detoxification enzymes, en-
hance stress resistance and increase lifespan [29, 30]. JPYW treatment significantly increased the expression
levels of the daf-16 and skn-1 genes compared to the lifespan through sir-2.1, which regulates this effect
through kat-1-mediated fatty acid oxidation [28]. As
shown in Fig. 5a, the expression level of the sir-2.1 gene
was significantly upregulated in JPYW-treated worms
compared to control-treated worms. In C. elegans, two
transcription factors, daf-16 and skn-1, promote the ex-
pression of antioxidant or detoxification enzymes, en-
hance stress resistance and increase lifespan [29, 30]. JPYW treatment significantly increased the expression
levels of the daf-16 and skn-1 genes compared to the Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 Zeng et al. BMC Complementary and Alternative Medicine Page 7 of 10 Fig. 4 Effect of JPYW on aging-related factors. a Daily and total reproductive outputs. The progeny were counted at the L2 or L3 stage. JPYW
treatment significantly increased the total progeny number (297.4 ± 15.3 vs. 223.8 ± 6.3, n = 5, P < 0.01) compared to the control treatment. b For
the growth alteration assay, photographs were taken of the worms, and the body length of each animal was analyzed. A small but significant
change in body length was detected after JPYW treatment compared to the control treatment (0.953 ± 0.035 vs. 1.108 ± 0.024 mm, n = 10,
P < 0.05). c JPYW slowed the decline in pharyngeal pumping during aging. Worms were treated with 150 μg/ml JPYW and the pumping rates
(pumps per 10 s) of 10 animals were scored in two trials (untreated vs. Discussion treated: day 6, P < 0.05; day 8, P < 0.05; day 10, P < 0.05; n = 10). d Body
movement in wild-type N2 nematodes. Worm body movement was evaluated under a dissecting microscope for 20 s. The differences between
the JPYW-treated worms and controls were significant (0.92 ± 0.08 vs. 2.13 ± 0.18 mm, n = 10, P < 0.01). e Fluorescence intensity of lipofuscin and
autofluorescence on the 10th day of adulthood. Compared to that in control worms, the intestinal lipofuscin accumulation in JPYW-treated
worms was reduced (37.29 ± 0.54 vs. 26.32 ± 0.35, n = 20, P < 0.01)
Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313
Page 7 of 10 Fig. 4 Effect of JPYW on aging-related factors. a Daily and total reproductive outputs. The progeny were counted at the L2 or L3 stage. JPYW
treatment significantly increased the total progeny number (297.4 ± 15.3 vs. 223.8 ± 6.3, n = 5, P < 0.01) compared to the control treatment. b For
the growth alteration assay, photographs were taken of the worms, and the body length of each animal was analyzed. A small but significant
change in body length was detected after JPYW treatment compared to the control treatment (0.953 ± 0.035 vs. 1.108 ± 0.024 mm, n = 10,
P < 0.05). c JPYW slowed the decline in pharyngeal pumping during aging. Worms were treated with 150 μg/ml JPYW and the pumping rates
(pumps per 10 s) of 10 animals were scored in two trials (untreated vs. treated: day 6, P < 0.05; day 8, P < 0.05; day 10, P < 0.05; n = 10). d Body
movement in wild-type N2 nematodes. Worm body movement was evaluated under a dissecting microscope for 20 s. The differences between
the JPYW-treated worms and controls were significant (0.92 ± 0.08 vs. 2.13 ± 0.18 mm, n = 10, P < 0.01). e Fluorescence intensity of lipofuscin and
autofluorescence on the 10th day of adulthood. Compared to that in control worms, the intestinal lipofuscin accumulation in JPYW-treated
worms was reduced (37.29 ± 0.54 vs. 26.32 ± 0.35, n = 20, P < 0.01) Fig. 4 Effect of JPYW on aging-related factors. a Daily and total reproductive outputs. The progeny were counted at the L2 or L3 stage. JPYW
treatment significantly increased the total progeny number (297.4 ± 15.3 vs. Discussion overexpression of sir-2.1 can extend the longevity of C. elegans by suppressing the IIS pathway or activating daf-
16. The third, skn-1 [44], involves in regulating oxidative
stress resistance and lifespan by encoding a worm homo-
log of Nrf2. The fourth key gene, clk2 [45], reduces lon-
gevity and telomere length. Recently, antiaging medicine has aimed at not only sim-
ply increasing longevity but also extending healthspan. In
this study, we showed that JPYW treatment effectively de-
layed
aging-related
declines
in
function,
such
as
pharyngeal pumping, body movement, egg laying and de-
velopment, compared with the control treatment, indicat-
ing that JPYW can enhance the healthspan of worms. g
y
g
In the present study, JPYW upregulated the activity
of the antioxidant enzyme SOD but did not signifi-
cantly increase the expression of the relevant gene sod-
3. This finding indicates that protein expression did not
correlate with gene expression, which is an intriguing
and unexplained phenomenon. The precise mecha-
nisms underlying these results are uncertain, but it is
known that some proteins are not encoded by only sin-
gle genes. For example, SOD is encoded not only by the
gene sod-3 but also by the genes sod-2, sod-1, etc. In
addition, the process of gene regulation is complex and
unclear. This issue requires further study, and this dis-
crepancy is one of the limitations of our study. In
addition, JPYW is a Chinese herbal compound that
contains many complex components, such as steroid-
like compounds, but no specific compound extracted
from JPYW was tested in this study. Hence, it is not
clear how many ingredients were related to the ob-
served antiaging effects or how these active ingredients
may have interacted. This uncertainty is another limita-
tion of the present study. Further studies are warranted
to identify the active ingredients in JPYW. To explore the potential mechanisms by which JPYW
exerts antiaging effects, SOD activity and aging-related
gene expression were assessed in C. elegans. As was re-
ported in the previous studies [39, 40], the oxidative
stress caused by oxygen free radicals played an import-
ant role in aging, and eliminating free radical and enhan-
cing oxidative stress resistance could delay senility. Our
research indicated that compared to the control treat-
ment, JPYW treatment elevated the activity of an anti-
oxidant enzyme (SOD), which resulted in elimination of
oxygen free radicals that might contribute to aging. Discussion 223.8 ± 6.3, n = 5, P < 0.01) compared to the control treatment. b For
the growth alteration assay, photographs were taken of the worms, and the body length of each animal was analyzed. A small but significant
change in body length was detected after JPYW treatment compared to the control treatment (0.953 ± 0.035 vs. 1.108 ± 0.024 mm, n = 10,
P < 0.05). c JPYW slowed the decline in pharyngeal pumping during aging. Worms were treated with 150 μg/ml JPYW and the pumping rates
(pumps per 10 s) of 10 animals were scored in two trials (untreated vs. treated: day 6, P < 0.05; day 8, P < 0.05; day 10, P < 0.05; n = 10). d Body
movement in wild-type N2 nematodes. Worm body movement was evaluated under a dissecting microscope for 20 s. The differences between
the JPYW-treated worms and controls were significant (0.92 ± 0.08 vs. 2.13 ± 0.18 mm, n = 10, P < 0.01). e Fluorescence intensity of lipofuscin and
autofluorescence on the 10th day of adulthood. Compared to that in control worms, the intestinal lipofuscin accumulation in JPYW-treated
worms was reduced (37.29 ± 0.54 vs. 26.32 ± 0.35, n = 20, P < 0.01) Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 (2019) 19:313 Page 8 of 10 Fig. 5 Effects of JPYW treatment on the expression of aging-related genes. The expression levels of aging-related genes were determined by
qRT-PCR using the 2−ΔΔCT method in worms with or without 150 μg/ml JPYW treatment at 20 °C. The graph shows the mean and SEM values
from two independent experiments. Compared to the control treatment, JPYW treatment significantly changed the expression levels of the
genes daf-16, clk-2, skn-1 and sir-2.1 (P < 0.05), but not those of the gene sod-3 (P > 0.05) Fig. 5 Effects of JPYW treatment on the expression of aging-related genes. The expression levels of aging-related genes were determined by
qRT-PCR using the 2−ΔΔCT method in worms with or without 150 μg/ml JPYW treatment at 20 °C. The graph shows the mean and SEM values
from two independent experiments. Compared to the control treatment, JPYW treatment significantly changed the expression levels of the
genes daf-16, clk-2, skn-1 and sir-2.1 (P < 0.05), but not those of the gene sod-3 (P > 0.05) thus exert antiaging effects through the combined effects
of all of its components. Discussion Not-
ably, previous studies have revealed that gene expression
can change during aging in C. elegans. Using qRT-PCR,
we confirmed that compared to control-treated worms,
JPYW-treated worms exhibited upregulated expression
of the antiaging genes daf-16, skn-1, and sir-2.1 and
downregulated expression of the proaging gene clk-2,
while they did not exhibit changes in the antiaging gene
sod-3. Overall, four key genes are involved in the ameli-
orative effects of JPYW on the aging pathway. The first,
daf-16 [41], is a part of FOXO-family transcriptional fac-
tor, which can regulate many target genes that can im-
prove stress resistance and increase longevity. The
second, sir-2.1 [42, 43] belongs to NAD+-dependent
histone deacetylases, which involves in regulating li-
fespan
conservatively. As
was
previously
reported, Funding 16. Fan GR, Zong WJ, Wang XL. Effect of yishen jianpi drugs on T lymphocyte
subsets, soluble interleukin-2 receptor and red blood cell immunity of senile
deficiency syndrome patients. Zhongguo Zhong Xi Yi Jie He Za Zhi. 1995;
15(1):18–20. This study was supported by the Guangdong Provincial Hospital of the
Traditional Chinese Medicine Scientific and Technological research of China
(no. YN2015QN08), the National Key Research and Development Plan for the
13th Five-Year Plan (no. 2018YFC1705600), and the Natural Science Founda-
tion of China (no. 81503515). None of the funding agencies participated in
the study. 17. Yang JY, Tao DQ, Liu S, Zhang S, Ma W, Shi ZH. Effects of three Wenyang
Jianpi Tang on cell proliferation and apoptosis of nonalcoholic fatty liver
cells. Zhongguo Zhong Yao Za Zhi. 2017;42(8):1591–6. 18. Peng W, Zhang S, Zhang Z, et al. Jianpi Jiedu decoction, a traditional
Chinese medicine formula, inhibits tumorigenesis, metastasis, and
angiogenesis through the mTOR/HIF-1alpha/VEGF pathway. J
Ethnopharmacol. 2018;224:140–8. Consent for publication
Not applicable. 21. Gong YX, Sun Y, Lin AP. Comparative study on the anti-free-radical damage
by vital energy-reinforcing method and blood-tonifying method. Zhongguo
Zhong Yao Za Zhi. 1993;18(7):438–41 448. Not applicable. Acknowledgments 10. Liu J, Peng L, Huang W, et al. Balancing between aging and Cancer:
molecular genetics meets traditional Chinese medicine. J Cell Biochem. 2017;118(9):2581–6. This study was conducted at the Key Laboratory of the Neurology
Department, The First Affiliated Hospital, Sun Yat-sen University for Model Or-
ganisms and the Lingnan Medical Research Center of Guangzhou University
of Chinese Medicine, Guangzhou, China. The authors are grateful to Professor
Qiuying Xu, Dr. Simei Long and Fengyin Liang for their technical support
and assistance. 11. Chen KJ. Reflections on human longevity and Chinese medicine prevention
and treatment of chronic diseases. Chin J Integr Med. 2015;21(9):643–7. 12. Liang ZH, Yin DZ. Preventive treatment of traditional Chinese medicine as
antistress and antiaging strategy. Rejuvenation Res. 2010;13(2–3):248–52. 13. Zhou DH. Preventive geriatrics: an overview from traditional Chinese
medicine. Am J Chin Med. 1982;10(1–4):32–9. 13. Zhou DH. Preventive geriatrics: an overview from traditional Chinese
medicine. Am J Chin Med. 1982;10(1–4):32–9. Abbreviations 5. Villa F, Carrizzo A, Spinelli CC, et al. Genetic analysis reveals a longevity-
associated protein modulating endothelial function and angiogenesis. Circ
Res. 2015;117(4):333–45. 5. Villa F, Carrizzo A, Spinelli CC, et al. Genetic analysis reveals a longevity-
associated protein modulating endothelial function and angiogenesis. Circ
Res. 2015;117(4):333–45. BCA: Bicinchoninic acid; C. elegans: Caenorhabditis elegans;
cDNA: complementary DNA; CFDA: China food and drug administration;
CGC: Caenorhabditis genetics center; DMSO: Dimethylsulfoxide;
EDTA: Ethylenedinitrilotetraacetic acid; JPYW: Jianpi-yangwei;
NGM: Nematode growth medium; qRT-PCR: quantitative real-time polymer-
ase chain reaction; SEM: Standard error of the mean; SOD: Superoxide
dismutase; TCM: Traditional Chinese medicine; Tris- 6. Krut'ko VN, Dontsov VI, Khalyavkin AV, Markova AM. Natural aging as as a
sequential poly-systemic syndrome. Front Biosci (Landmark Ed). 2018;23:909–20 6. Krut'ko VN, Dontsov VI, Khalyavkin AV, Markova AM. Natural aging as as a
sequential poly-systemic syndrome. Front Biosci (Landmark Ed). 2018;23:909–20 7. Gladyshev TV, Gladyshev VN. A disease or not a disease? Aging as a
pathology. Trends Mol Med. 2016;22(12):995–6. 8. Bellantuono I. Find drugs that delay many diseases of old age. Nature. 2018;
554(7692):293–5. HCl: Tris(hydroxymethyl)aminomethane hydrochloride; T-SOD: Total
superoxide dismutase 9. Savage N. New tricks from old dogs join the fight against ageing. Nature. 2017;552(7684):S57–9. Conclusions In conclusion, this study demonstrated that JPYW, a TCM
formula, increases stress resistance and promotes longevity
in C. elegans by activating and repressing target genes re-
lated to aging, including daf-16, sir-2.1, skn-1 and clk-2. Page 9 of 10 Page 9 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 (2019) 19:313 Authors’ contributions ZLL, XFP and YZM conceived and designed the study. ZLL, YTC, CZW, SC
and FSY performed the experiments. ZLL and XFP wrote the manuscript. ZLL, YZM and PZ analyzed the data. ZLL, FSY and YTC searched and
reviewed the literature. All authors read and approved the final manuscript. 14. Hsu YC, Chiu CJ, Wray LA, Beverly EA, Tseng SP. Impact of traditional
Chinese medicine on age trajectories of health: evidence from the Taiwan
longitudinal study on aging. J Am Geriatr Soc. 2015;63(2):351–7. 14. Hsu YC, Chiu CJ, Wray LA, Beverly EA, Tseng SP. Impact of traditional
Chinese medicine on age trajectories of health: evidence from the Taiwan
longitudinal study on aging. J Am Geriatr Soc. 2015;63(2):351–7. 15. Wan F, Zhi D, Liu D, et al. Lifespan extension in Caenorhabiditis elegans by
several traditional Chinese medicine formulas. Biogerontology. 2014;15(4):377–87. 15. Wan F, Zhi D, Liu D, et al. Lifespan extension in Caenorhabiditis elegans by
several traditional Chinese medicine formulas. Biogerontology. 2014;15(4):377–87. Competing interests 22. Shi XY, Zhao FZ, Dai X, Ma LS, Dong XY, Fang J. Effect of jianpiyiwei capsule on
gastric precancerous lesions in rats. World J Gastroenterol. 2002;8(4):608–12. The authors declare that they have no competing interests. Author details
1 23. Guarente L, Kenyon C. Genetic pathways that regulate ageing in model
organisms. Nature. 2000;408(6809):255–62. 1The Second Clinical College of Guangzhou University of Chinese Medicine,
Guangzhou, China. 2The Second Affiliated Hospital of Guangzhou University
of Chinese Medicine, 111 Da De Rd., Yuexiu District, Guangzhou, Guangdong
Province, People’s Republic of China510120. 3Department of Neurology,
National Key Clinical Department and Key Discipline of Neurology, the First
Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 1The Second Clinical College of Guangzhou University of Chinese Medicine,
Guangzhou, China. 2The Second Affiliated Hospital of Guangzhou University
of Chinese Medicine, 111 Da De Rd., Yuexiu District, Guangzhou, Guangdong
Province, People’s Republic of China510120. 3Department of Neurology,
N ti
l K
Cli i
l D
t
t
d K
Di
i li
f N
l
th Fi t 24. Sluder AE, Baumeister R. From genes to drugs: target validation in
Caenorhabditis elegans. Drug Discov Today Technol. 2004;1(2):171–7. 25. Munoz MJ, Riddle DL. Positive selection of Caenorhabditis elegans mutants
with increased stress resistance and longevity. Genetics. 2003;163(1):171–80. National Key Clinical Department and Key Discipline of Neurology, the First
Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. 26. Zeng L, Sun C, Pei Z, et al. Liangyi Gao extends lifespan and exerts an
antiaging effect in Caenorhabditis elegans by modulating DAF-16/FOXO. Biogerontology. 2019. Received: 10 March 2019 Accepted: 9 October 2019 Received: 10 March 2019 Accepted: 9 October 2019 27. Xian B, Shen J, Chen W, et al. WormFarm: a quantitative control and
measurement device toward automated Caenorhabditis elegans aging
analysis. Aging Cell. 2013;12(3):398–409. Availability of data and materials The datasets generated and analysed during the current study are not
publicly available since a follow-up study is undergoing, but are available
from the corresponding author on reasonable request. 19. Huang C, Zhu Z, Cao X, et al. A Pectic Polysaccharide from Sijunzi
Decoction Promotes the Antioxidant Defenses of SW480 Cells. Molecules. 2017;22(8):1341-12. Ethics approval and consent to participate
Not applicable. 20. Zhao AG, Zhao HL, Jin XJ, Yang JK, Tang LD. Effects of Chinese Jianpi herbs
on cell apoptosis and related gene expression in human gastric cancer
grafted onto nude mice. World J Gastroenterol. 2002;8(5):792–6. 1.
Harman D. Aging: a theory based on free radical and radiation chemistry. J
Gerontol. 1956;11(3):298–300. References 28. Berdichevsky A, Nedelcu S, Boulias K, Bishop NA, Guarente L, Horvitz HR. 3-
Ketoacyl thiolase delays aging of Caenorhabditis elegans and is required for
lifespan extension mediated by sir-2.1. Proc Natl Acad Sci U S A. 2010;
107(44):18927–32. 1. Harman D. Aging: a theory based on free radical and radiation chemistry. J
Gerontol. 1956;11(3):298–300. 2. MSN N, Longo-Mbenza B, Adeniyi OV, et al. Ageing, exposure to pollution,
and interactions between climate change and local seasons as oxidant
conditions predicting incident hematologic malignancy at KINSHASA
University clinics, Democratic Republic of CONGO (DRC). BMC Cancer. 2017;17(1):559. 29. Mukhopadhyay A, Oh SW, Tissenbaum HA. Worming pathways to and from
DAF-16/FOXO. Exp Gerontol. 2006;41(10):928–34. 30. Chavez V, Mohri-Shiomi A, Maadani A, Vega LA, Garsin DA. Oxidative stress
enzymes are required for DAF-16-mediated immunity due to generation of
reactive oxygen species by Caenorhabditis elegans. Genetics. 2007;176(3):1567–77. 3. Bijnens EM, Zeegers MP, Derom C, et al. Telomere tracking from birth to
adulthood and residential traffic exposure. BMC Med. 2017;15(1):205. 4. Calder PC, Bosco N, Bourdet-Sicard R, et al. Health relevance of the
modification of low grade inflammation in ageing (inflammageing) and the
role of nutrition. Ageing Res Rev. 2017;40:95–119. 31. Yang Y, Ren C, Zhang Y, Wu X. Ginseng: an nonnegligible natural remedy
for healthy aging. Aging Dis. 2017;8(6):708–20. Page 10 of 10 Zeng et al. BMC Complementary and Alternative Medicine (2019) 19:313 32. Lee JH, Choi SH, Kwon OS, et al. Effects of ginsenosides, active ingredients
of Panax ginseng, on development, growth, and life span of Caenorhabditis
elegans. Biol Pharm Bull. 2007;30(11):2126–34. 33. Hu W, Jing P, Wang L, Zhang Y, Yong J, Wang Y. The positive effects of
Ginsenoside Rg1 upon the hematopoietic microenvironment in a
D-Galactose-induced aged rat model. BMC Complement Altern Med. 2015;15:119. 34. Zhou Y, Liu J, Cai S, Liu D, Jiang R, Wang Y. Protective effects of ginsenoside
Rg1 on aging Sca-1(+) hematopoietic cells. Mol Med Rep. 2015;12(3):3621–8. 35. Lee SG, Kim MM. Pachymic acid promotes induction of autophagy related
to IGF-1 signaling pathway in WI-38 cells. Phytomedicine. 2017;36:82–7. to IGF-1 signaling pathway in WI-38 cells. Phytomedicine. 2017;36:82– 36. Bi J, Zhang H, Lu J, Lei W. Nobiletin ameliorates isoflurane-induced
cognitive impairment via antioxidant, anti-inflammatory and anti-apoptotic
effects in aging rats. Mol Med Rep. 2016;14(6):5408–14. 37. Yu YB, Dosanjh L, Lao L, Tan M, Shim BS, Luo Y. References Cinnamomum cassia bark
in two herbal formulas increases life span in Caenorhabditis elegans via
insulin signaling and stress response pathways. PLoS One. 2010;5(2):e9339. 38. Rho HS, Hong SH, Park J, et al. Kojyl cinnamate ester derivatives promote
adiponectin production during adipogenesis in human adipose
tissue-derived mesenchymal stem cells. Bioorg Med Chem Lett. 2014;24(9):2141–5. 39. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408(6809):239–47. 39. Finkel T, Holbrook NJ. Oxidants, oxidative stress and the biology of ageing. Nature. 2000;408(6809):239–47. 40. Bokov A, Chaudhuri A, Richardson A. The role of oxidative damage and
stress in aging. Mech Ageing Dev. 2004;125(10–11):811–26. 40. Bokov A, Chaudhuri A, Richardson A. The role of oxidative damage and
stress in aging. Mech Ageing Dev. 2004;125(10–11):811–26. 41. Murphy CT, McCarroll SA, Bargmann CI, et al. Genes that act downstream of
DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature. 2003;424(6946):277–83. 42. Tissenbaum HA, Guarente L. Increased dosage of a sir-2 gene extends
lifespan in Caenorhabditis elegans. Nature. 2001;410(6825):227–30. 43. Berdichevsky A, Viswanathan M, Horvitz HR, Guarente L. C. elegans SIR-2.1
interacts with 14-3-3 proteins to activate DAF-16 and extend life span. Cell. 2006;125(6):1165–77. 44. Park SK, Tedesco PM, Johnson TE. Oxidative stress and longevity in
Caenorhabditis elegans as mediated by SKN-1. Aging Cell. 2009;8(3):258–69. 45. Benard C, McCright B, Zhang Y, Felkai S, Lakowski B, Hekimi S. The C. Elegans maternal-effect gene clk-2 is essential for embryonic development,
encodes a protein homologous to yeast Tel2p and affects telomere length. Development. 2001;128(20):4045–55. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
|
https://openalex.org/W4376634361
|
https://zenodo.org/record/7939543/files/349-355.pdf
|
Russian
| null |
РОЛЬ ЦИТОКИНОВ В МЕХАНИЗМЕ РАЗВИТИЯ ВОСПАЛИТЕЛЬНЫХ ЗАБОЛЕВАНИЙ ПАРОДОНТА У РАБОТНИКОВ НЕФТЕГАЗОПЕРЕРАБАТЫВАЮЩЕГО ЗАВОДА КАШКАДАРЬИНСКОЙ ОБЛАСТИ
|
Zenodo (CERN European Organization for Nuclear Research)
| 2,023
|
cc-by
| 2,185
|
Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023 Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/pass Сейдеметова Эльмира Оралбай кизи
Управление таможенного комитета при Министерстве экономики и
финансов Республики Узбекистан по Республике Каракалпакстан,
старший инспектор Сейдеметова Эльмира Оралбай кизи
Управление таможенного комитета при Министерстве экономики и
финансов Республики Узбекистан по Республике Каракалпакстан,
старший инспектор Аннотация. В данной статье был изучен и проанализирован опыт
Всемирной таможенной организации по использованию цифровой таможни
и цифровых технологий, которые в настоящее время являются актуальными
и широко применяются. В статье рассмотрены содержание и отличия
понятий, таких как цифровая таможня, электронная таможня, а также
приведены основные причины и необходимость использования этих понятий,
высказаны автором основные идеи и предложения с целью улучшения
эффективности деятельности в таможне. Ключевые слова. Цифровая таможня, электронная таможня, Big data,
телематика, облачные технологии, Всемирная таможенная организация. THE EXPERIENCE OF THE WORLD CUSTOMS ORGANIZATION IN
USING DIGITAL TECHNOLOGIES IN CUSTOMS SERVICE’S
ACTIVITIES Seydemetova Elmira Oralbay qizi
Department of the Customs Committee under the Ministry of Economy and
Finance of the Republic of Uzbekistan for the Republic of Karakalpakstan,
senior inspector Annotation. This article examines and analyzes the experience of the World
Customs Organization in using digital customs and digital technologies, which are
currently relevant and widely applied. The article discusses the content and differences
of concepts such as digital customs and electronic customs, and provides the main
reasons and necessity for using these concepts. The author presents key ideas and
suggestions aimed at improving the efficiency of customs activities. Keywords. Digital customs, electronic customs, Big data, telematics, cloud
technologies, World Customs Organization. //t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
349 349 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
349 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 На сегодняшнее время кардинальные изменения в сфере экономики,
экономических связей между странами и внутренней экономической политикой
каждого государства, а также широта, обмен и анализ экономической
информации привели к коренному изменению общества и необходимости
автоматизации этой отрасли. По этой причине в настоящее время наиболее
актуальной
проблемой
становится
цифровизация
экономики,
особенно
таможенного сектора, являющегося одной из ее отраслей. Важнейшим
процессом в данной области является прежде всего изучение мирового опыта. Ведущей организацией мирового уровня в таможенной сфере всех развитых или
развивающихся стран является - Всемирная таможенная организация (далее в
тексте – именуемая ВТО). Поэтому в данной статье будут рассмотрены способы
применения цифровых технологий на таможне на основе опыта ВТО. Поскольку ситуация в сфере технологий в нашем обществе стремительно
меняется, необходимо учитывать современные тенденции в таких важных
областях, как облачные технологии, мобильные технологии, передовые средства
анализа и управления информацией. Ведь каждая из этих технологий может
оказать особое влияние на деятельность таможенных органов. В целом они
создают большие возможности для трансграничного сотрудничества между
таможенными органами, торговыми операторами и другими ведомствами,
повышая эффективность и, как следствие, ускоряя экономический рост [1]. Поэтому
использование
цифровых
технологий
в
регулировании
внешнеторговой и таможенной деятельности в рамках Всемирной таможенной
организации является актуальным вопросом на сегодняшний день. Поэтому
объявление ВТО 2016 года годом «Цифровой таможни» (Digital Customs) также
подтверждает этот факт. В этом направлении работа, проводимая ВТО, является
основной частью цифровой трансформации в таможенной сфере. Цифровизация таможенной сферы призвана повысить эффективность
таможенной деятельности, облегчить международную торговлю, бороться с
контрабандой и другими преступлениями в таможенной сфере, упростить
таможенное
администрирование
с
помощью
информационно-
коммуникационных технологий. Появление предложенной ВТО «Цифровой таможни» также связано с
растущими объемами Интернет-торговли и необходимостью таможенного
контроля за перемещением таких товаров. «Цифровая таможня» подразумевает
использование информационно-коммуникационных технологий, базы данных,
облачных технологий, информации, полученной с помощью информационно- https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
350 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
350 350 Multidisciplinary Scientific Journal коммуникационных технологий (ИКТ), а также сети Интернет, средств массовой
информации и сетей мобильной связи [2]. Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 По мнению Кожанкова А.Ю., «Электронная таможня» представляет собой
комплекс информационных и коммуникационных технологий, используемых
таможенными органами для получения информации о товарах и транспортных
средствах, а также для автоматизированного обмена данными с другими
пограничными органами в целях ускорения таможенного оформления
торговыми операторами для упрощения процедур торговли [3]. При этом цель цифровой таможни — обеспечить глобальную безопасность
цепочки поставок этих товаров. Для осуществления эффективного таможенного
контроля в рамках цифровой таможни предусмотрено взаимодействие с другими
таможенными сетями с использованием ИКТ, анализа баз данных, и
использование телематики, облачных технологий и глобальной сети Интернет
[4]. Однако следует также отметить, что основной целью внедрения всех этих
понятий
в
таможенную
сферу
является,
во-первых,
экономическая
эффективность, во-вторых, улучшение мировой экономической ситуации,
стимулирование процессов глобализации, сокращение бедности, борьба с
террористическими угрозами. Исследователи А. Ю. Кожанков и К. И. Бабенко провели сравнительный
анализ понятий «Электронная таможня» и «Цифровая таможня» и сделали
следующие научно обоснованные выводы: 1. «Электронная таможня» и «цифровая таможня» характеризуются
следующими квалификациями: а) электронная таможня: электронная обработка документов, упрощение
и дематериализация; электронная оплата пошлин и налогов; система
таможенного
декларирования
(для
целей
электронной
коммерции);
предварительная дополнительная информация перед погрузкой товара;
автоматизация работы таможни в режиме «24/7»; электронный платежный
калькулятор; услуги мобильной связи (информация о статусе товара);
электронный возврат товара. б) цифровая таможня: гиперкоммуникабельность; обработка больших
данных (Big Data); использование сети Интернет и средств массовой
информации (включая социальные сети); телематика; транспортная телематика
(спутниковый мониторинг транспорта); автоматизация зданий (организация
производства); телематика услуг (бизнес, коммерция, логистика, правительство); https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
351 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
351 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
351 351 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 облачные технологии; Интернет вещей; мобильные технологий и сотовой сети
(для отслеживания местоположения беспилотных транспортных средств) [4]. облачные технологии; Интернет вещей; мобильные технологий и сотовой сети
(для отслеживания местоположения беспилотных транспортных средств) [4]. Стоит отметить, что основной целью и функцией внедрения этих понятий
в таможенную деятельность, как мы уже упоминали выше, является улучшение
мировой экономической ситуации и облегчение международной торговли,
повышение производительности таможенного администрирования, повышение
эффективности борьбы с нарушениями таможенного и иного законодательства. В частности, ВТО предложил лозунг «Анализ данных для эффективного
управления границами» в развитии темы цифровой таможни в 2017 году [5]. Согласно предложению ВТО, анализ и изучение данных в таможенном
секторе будут иметь важное значение для модернизации сектора и упрощения
таможенного
администрирования. Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Поэтому
необходимо
развивать
у
таможенных сотрудников соответствующие навыки анализа данных и
использования ИТ-инструментов для улучшения пограничного таможенного контроля. ВТО — это таможенная сеть право охранения, которая использует коды
государственной службы для расследования сроков выпуска товаров, сравнения
импорта и экспорта и выявления изменений в количестве, весе или стоимости
товаров. Он будет продолжать продвигать такие инструменты, как модель
данных ВТО, которая поддерживает анализ данных за счет улучшения сбора
данных
и
обеспечения
обмена
данными
между
государственными
учреждениями. В последние годы, наряду с движением товаров в международной
торговле, возросли объем и значимость собираемой о них информации. Наличие
такой информации позволило соблюдать таможенные правила и облегчить
международную торговлю. В целях совершенствования таможенного контроля и
улучшения взаимодействия таможенных органов и субъектов хозяйствования
необходимо провести анализ данных о перемещении товаров через таможенные
границы. По мнению экспертов ВТО, потребность в данных в режиме реального
времени включает в себя возможность преобразования большего количества
данных для выявления таможенных нарушений. Достижения в области информационных и коммуникационных технологий
(ИКТ) упростили сбор данных, но при специальной обработке и анализе таких
больших объемов данных возникает реальная проблема. В таможенном
администрировании информация должны собираться не только из таможенной
грузовой
декларации,
поданной
участником
внешнеэкономической https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
352 352 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 деятельности, или путем обмена информацией с другими таможенными
службами и государственными органами, но и из открытых источников (СМИ,
базы данных, официальные сайты компаний, веб-сайты и т.д.). Такие
информации помогают таможенным сотрудникам расставлять приоритеты,
принимать решения, оценивать эффективность, разрабатывать стратегии по
борьбе с коррупцией и комплаенсу, планировать и прогнозировать бюджет, а
также выполнять повседневные операции [5]. Анализируя элементы инновационной модели управления данными в
рамках цифровой таможни, разработанной ВТО, считаем, что необходимо
начинать исследование с определения характеристик понятия и содержания «Big
Data». В управлении данными «эталонная модель» дает следующее определение
данных: данные — это информация, представленная в формализованной форме,
пригодной для передачи, интерпретации или обработки человеком или
автоматической машиной [6]. Исследовательская и консалтинговая компания Gartner определяет
большие данные следующим образом: «Большие данные — это автоматизация
высокопроизводительных,
высокоскоростных
и/или
высокоуровневых
информационных ресурсов, которые требуют экономичных и инновационных
форм обработки данных, улучшающих понимание, принятие решений.» [7]. В последние годы появились новые инструменты, дающие еще больше
возможностей для получения максимальной отдачи от имеющейся информации. Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Таможенные службы все чаще обращаются к анализу данных и вероятностному
анализу, что означает нахождение смысла в неоднозначных данных с
использованием специализированных компьютерных систем, использующих
передовые алгоритмы для анализа неструктурированных данных, или даже
когнитивных технологий. Данные в сочетании с аналитикой и другими новыми
технологиями дают нам новые возможности для достижения организационных
целей. Ключевым требованием для успешной организации когнитивного анализа
данных является оцифровка данных. Если данные не используются когнитивной
системой, они не могут быть эффективно проанализированы. Таким образом, цифровизация данных и процессов и перенос данных в
облако или другую удобную платформу — одно из ключевых условий успеха в
использовании аналитики данных. Кроме того, пограничные органы должны согласовать общие данные
(используя модель данных ВТО) и развивать навыки для решения любых
вопросов, связанных с информационными технологиями. Кроме того,
таможенные органы должны обеспечить соблюдение законодательства, https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
353 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
353 353 Multidisciplinary Scientific Journal Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 касающегося конфиденциальности персональных данных, коммерческой,
банковской, налоговой и иных видов тайны, в целях сохранения доверия
населения к использованию этой информации [8]. касающегося конфиденциальности персональных данных, коммерческой,
банковской, налоговой и иных видов тайны, в целях сохранения доверия
населения к использованию этой информации [8]. В заключение следует отметить, что в настоящее время в Республике
Узбекистан, в период совершенствования таможенной системы и деятельности,
для выявления скрытой информации, анализ данных должен иметь доступ к
различным базам данных (например, конфискованным базам данных, базам
данных трейдеров, базы данных таможенной оценки и т. д.). Другими словами,
автоматизация поиска и анализа таможенных данных, в том числе внешних,
также необходима. Таможенные органы должны сделать анализ данных своим
стратегическим приоритетом, внедряя передовые технологии, устанавливая
соответствующие политики автоматизации, привлекая экспертов по сбору и
анализу данных и действуя на основе данных. Сотрудники таможни также
должны постепенно развивать соответствующие навыки для анализа данных и
использования возможностей инструментов информационных технологий. Конечно,
своевременное
получение
качественных
данных
важно
для
оптимизации использования аналитики данных. Это одно из основных
направлений взаимодействия с бизнесом по обеспечению своевременного
поступления достоверной информации. Список использованной литературы 1. Официальный сайт: Facilitating e-commerce (Журнал Всемирной таможенной
организации) – 2015.–№78.–С.32–36.facilitating-ecommerce-wco-news-78-october-
2106.pdf (wcoomd.org); 2. Официальный сайт: Альта-Софт / “Электронная таможня”: итоги заседания
рабочей группы в ЕЭК, https://www.alta.ru/ts_news/52792; 2. Официальный сайт: Альта-Софт / “Электронная таможня”: итоги заседания
рабочей группы в ЕЭК, https://www.alta.ru/ts_news/52792; 3. Официальный сайт: Евразийская экономическая комиссия «Выработка
концепции определения понятия ««Электронная таможня» в праве Евразийского
экономического союза с учётом международных стандартов и практики,
http://www eurasiancommission org/ru/act/tam sotr/edinoe okno/Pages/intconfecust 3. Официальный сайт: Евразийская экономическая комиссия «Выработка
концепции определения понятия ««Электронная таможня» в праве Евразийского 3. Официальный сайт: Евразийская экономическая комиссия «Выработка
концепции определения понятия ««Электронная таможня» в праве Евразийского
экономического союза с учётом международных стандартов и практики,
http://www.eurasiancommission.org/ru/act/tam_sotr/edinoe_okno/Pages/intconfecust
oms.aspx; экономического союза с учётом международных стандартов и практики,
http://www.eurasiancommission.org/ru/act/tam_sotr/edinoe_okno/Pages/intconfecust
oms.aspx; 4. Кожанков А.Ю., Бабенко К.И. Новая парадигма применения информационных
коммуникационных технологий всемирной таможенной организацией // Вестник
Российской таможенной академии. –2017; https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
354 https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
354 354 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 Innovative Development in Educational Activities ISSN: 2181-3523 VOLUME 2 | ISSUE 9 | 2023
Scientific Journal Impact Factor (SJIF): 5.938 http://sjifactor.com/passport.php?id=22323 5. Официальный сайт: WCO (World Customs Organization) / Сообщение
Всемирной таможенной организации в Международный день таможенника в
2017
году,
http://www.wcoomd.org/-/media/wco/public/global/pdf/about-
us/international-customs-day/2017/message-from-the-world-customs-organization-
ru.pdf?db=web; 5. Официальный сайт: WCO (World Customs Organization) / Сообщение
Всемирной таможенной организации в Международный день таможенника в
2017
году,
http://www.wcoomd.org/-/media/wco/public/global/pdf/about-
us/international-customs-day/2017/message-from-the-world-customs-organization-
ru.pdf?db=web; 6. Официальный сайт: Гартнер dictionary / “Big data”, https://www.gartner.com/it-
glossary/big-data; 6. Официальный сайт: Гартнер dictionary / “Big data”, https://www.gartner.com/it-
glossary/big-data; 7. Официальный
сайт:
Gartner
glossary
/
“Big
data”
Available
at:
https://www.gartner.com/it-glossary/big-data; 8. Официальный сайт: WCO (World Customs Organization) / Инструменты
содействия
торговли
Всемирной
таможенной
организации,
http://www.wcoomd.org/-/media/wco/public/ru/pdf/topics/key-issues/revenue-
package/catalog-of-revenue-package_rus.pdf. https://t.me/openidea_uz Multidisciplinary Scientific Journal May, 2023
355 355 Multidisciplinary Scientific Journal
|
https://openalex.org/W2985648138
|
https://journal.iainlangsa.ac.id/index.php/enlighten/article/download/1069/827
|
English
| null |
Social Value Orientation Effects On Adolescents Friendship Quality
|
Enlighten
| 2,019
|
cc-by-sa
| 5,036
|
Enlighten: Jurnal Bimbingan dan Konseling Islam
Vol. 2 No. 1 (Jan-Jun 2019), 24-34
https://doi.org/10.32505/enlighten.v2i1.1069 Enlighten: Jurnal Bimbingan dan Konseling Islam
Vol. 2 No. 1 (Jan-Jun 2019), 24-34
https://doi.org/10.32505/enlighten.v2i1.1069 Social Value Orientation Effects on Adolescents Friendship
Quality Syiva Fitria1, Sabine Peters2, Syiva Fitria1, Sabine Peters2,
1Faculty of Usluhuddin, Adab, and Dakwah, IAIN Langsa, 2Faculty of Behavioral Sciences, Leiden
University
1syivafitria@iainlangsa.ac.id, 2 s.peters@fsw.leidenuniv.nl tria@iainlangsa.ac.id, 2 s.peters@fsw.leidenuniv.n Final Accepted:
15 April 2019 Abstract The current study intended to explore the association between Social Value Orientation (SVO) and
friendship quality in adolescence, the development as well as gender and age differences. Participants between ages 12 and 25 (N = 292) completed a series of games to measure their SVO
and Friendship Quality Scale in order to assess their friendship quality. Analysis of covariance
confirmed that SVO did not affect friendship quality. No age and gender differences were found in
SVO. However, the results revealed that there was a significant gender difference in friendship
quality, where girls have higher positive friendship quality. There was also an age effect on
friendship quality, such that, as age increased, negative friendship quality decreased. In conclusion,
there was no evidence that SVO influences someone friendship quality. It is possible that SVO only
influences the number of friends that someone has. Keywords: social value orientation SVO friendship quality adolescents gender age Keywords: social value orientation, SVO, friendship quality, adolescents, gender, age study intended to assess how friendship
quality related to SVO. INTRODUCTION It also
suggested that prosocial behavior increased
with
age
while
individualist
and
competitiveness decreased. Au and Kwong
(2004) agreed, that more adults fall into
prosocial
category
followed
by
individualistic and competitive, although,
some of them have inconsistent SVO. However, is it also suggested that the
differences in social interaction experiences,
from early childhood to young adulthood,
resulted in different patterns of SVO during
that period (Van Lange, et al., 1997). Additionally, results from child studies
showed some inconsistency. One study
found that 4 to 9-year-olds children are
becoming more competitive as they get
older
(Kagan
&
Madsen,
1971). Accompanying this study, Knight, Dubro, &
Chao (1985) also found that 8 to 10-year-
olds children were more competitive. Nonetheless, another study mentioned that
more children from 5 to 8-year-olds were
fall into cooperative type than other SVO
types (McClintock & Moskowitz, 1976). Conversely, a different study measuring
SVO, found that 8 to 11 year-olds were more
individualistic (Knight et al., 1981). Thus,
based on the studies mentioned above, it
could be concluded that there is no fixed
pattern of the development of SVO across
different
stages
of
childhood
and
adolescents. One study reported that SVO might
influence individual affect and cognition as
well as behavior in daily functioning (Van
Lange & Folmer, 2007). Previous studies
have shown that prosocials increases
helping behavior (McClintock & Allison,
1989) and willingness to make sacrifices in
close relationships (Van Lange et al., 1997). Also, SVO could be used to understand
relationships as it is related to attachment
security, which is important in continuing
healthy relationships (Van Lange, Otten, De
Bruin & Joireman. 1997). Therefore, how
individual SVO influences the shape and
form of relationships in everyday life is
becomes one of the foci of this study. Additionally, adolescence is known as
an important stage where the transition to
adulthood happens and is characterized by
changes in many aspects such as cognitive,
physical, and social (Mann, Harmoni &
Power, 1989). One distinctive characteristic
of mature adolescents is the improved
ability in decision-making (Mann et al.,
1989). Many factors could influence how
adolescents
make
decisions
in
social
interactions, including SVO. Nonetheless,
SVO has not been widely measured in
adolescents, as one of the social factors
influencing decision-making. In relation to gender differences, a
study
found
non-significant
gender
differences in social orientation (Kuhlman &
Marshello, 1975). INTRODUCTION It is suggested that once children
become adolescents, their social behavior
and interaction become more complicated
and meaningful due to physical and
environmental factors (Derks, Lee, &
Krabbendam, 2014). One noticeable social
factor
that
constantly
changes
along
development is friendship. In addition,
people have different social motives when
making a decision and these motives are
known as Social Value Orientation (SVO). It
is stated that this SVO principle could be
used
in
order
to
understand
social
interaction and interpersonal behavior (Van
Lange, De Cremer, Van Dijk, & Van Vugt,
2007). However, little is known regarding
how adolescents’ SVO influence their
friendship quality. Therefore, the current Social Value Orientation (SVO) is an
approach defining individual differences in
consideration of outcomes for oneself and
another
individual
when
allocating
resources (McClintock, 1972). McClintock
and Van Avermaet (1982) stated that SVO is
a motivational orientation of outcome
distribution for oneself and others. SVO
significantly influences an individual’s
social behavior in a setting where the
outcome is dependent on others (Librand,
Jansen, Rijken, & Suhre, 1986). Thus, SVO
explains
how
individuals
differ
motivationally and whether they will make
a decision based on their own or mutual
importance. There are three distinctive types of SVO
recognized; prosocial, individualistic, and 24 Social Value Orientation – Fitria and Peters competitive
(Eek
&
Gärling,
2008). Prosocial SVO is defined by maximizing
mutual gains as the goal. Kuhlman, Camac,
and Cunha (1986) stated prosocials value
cooperation and put forward fairness. Individualistic SVO is maximizing one’s
own benefit without concern for the other’s
outcome, whereas, competitive SVO is
maximizing the difference between own
and others’ outcomes. Additionally, De
Dreu & Boles (1998) suggested that SVO
could
affect
cognition
and
influence
behavior related to decision making, such as
negotiating. competitive
(Eek
&
Gärling,
2008). Prosocial SVO is defined by maximizing
mutual gains as the goal. Kuhlman, Camac,
and Cunha (1986) stated prosocials value
cooperation and put forward fairness. Individualistic SVO is maximizing one’s
own benefit without concern for the other’s
outcome, whereas, competitive SVO is
maximizing the difference between own
and others’ outcomes. Additionally, De
Dreu & Boles (1998) suggested that SVO
could
affect
cognition
and
influence
behavior related to decision making, such as
negotiating. Referring to SVO study in adults and
children, Van Lange, et al. (1997) found that
there was a parallel relationship between
prosocial behavior and age where prosocial
behavior increased as age increased. INTRODUCTION Nevertheless, one study
stated that there was a significant difference
in social value orientation distribution
between boys and girls where boys were
more individualistic compared to girls 25 Social Value Orientation – Fitria and Peters features
of
friendship
are
prosocial
behavior, self-esteem support, intimacy,
and loyalty, while the negative features of
friendship
are
conflicts,
dominance
attempts, and rivalry. It is suggested that
high-quality friendships are characterized
by high levels of positive features and low
levels of negative features. Furthermore, a
study by Kuttler, La Greca, and Prinstein
(1999) found significant gender differences
in friendship qualities. Girls reported that
they have a higher quality of friendship
than boys, marked by greater levels of
support, intimacy, and companionship. while girls were categorized as more
prosocial oriented than boys (Iedema &
Poppe, 199). Correspondingly, other studies
have confirmed that compared to boys, girls
are more pro-socially oriented. (Eisenberg,
Cumberland, Guthrie, Murphy, & Shepard,
2005; Derks et al., 2014) In addition, given that many variables
change during adolescence, friendship is
one variable that is important to assess. Rubin, Bukowski, & Parker (2006) defined
friendship as positive affect shared in a
voluntary dyadic relationship that is
intimate and both parties accept each other. Berndt (1982) stated that there is a
significant
change
in
adolescents’
friendship
characteristics
and
its
importance. Moreover, friendship is also
one social factor that develops during
adolescence that consists of peer network
growth,
increased
close
friendship
importance
and
romantic
relationship
appearance (La Greca & Prinstein, 1999). Crockett,
Losoff,
&
Petersen
(1984)
identified that adolescents increasingly
spend
time
with
their
friends. Consequently, in adolescents, close friends
start to become the primary social support
instead of parents, which also influences the
development of their self-concept and well-
being
(Furman
&
Buhrmester,
1992). Moreover, friendship plays an important
part in the social arena and fulfills the need
for affection, togetherness, and closeness
(Furman & Collins, 2009). Therefore,
friendship as a social factor is important
topic to investigate in adolescence. Nevertheless, few available studies
provide an established link between SVO
and friendship quality. Recent research, in
an online user study, reported that social
value orientation influences the number of
friends people make (Chesney, Chuah, &
Hoffmann, 2016). Additionally, one study
reported that prosocial behavior was
significantly related to friendship. McGuire
and Weisz (1982) implied that compared to
adolescents who do not have friends, those
with friends are more likely to show
prosocial behavior. Participants This study was part of a larger
project
on
cognitive
and
affective
development
(i.e. Peters,
Peper,
Van
Duijvenvoorde, Braams, & Crone, 2016). The current study involved 292 participants
(female: 153, male: 139) with an age range
from 12 to 25 (M = 14.06, SD = 3.61) who
were recruited trough local schools and
advertisements. All
participation
was
voluntary. Participants signed informed
consent at the beginning of the study and
were allowed to withdraw from the study at
any time without any penalties. The
procedures in this study were approved by
the
Ethical
Review
Board. After
participation in the study, children received
presents and parents received 30 euros for
travel compensation. Friendship Quality Scale Participants were asked to complete
the Friendship Quality Scale (FQS) that has
been found to be a valid and reliable
measure of friendship quality (Bukowski,
Hoza, & Boivin, 1994). FQS consist of 23-
items that belong to 5 subscales; conflict (4
items), closeness (5 items), companionship
(4 items), receiving help (5 items) and
security (5 items). The item examples are
“my friend would help me if I needed it”
and “my friend and I spend all our free time
together”. Participants were provided with
5-point Likert scale response option ranging INTRODUCTION The current study intended to examine
the
relationship
between
SVO
and
friendship quality with a specific focus on
adolescence. This study also sought to
address the development of both SVO and
friendship quality in adolescents as well as
gender differences in both variables. It was
hypothesized that first, adolescents who are
prosocially oriented have higher friendship
quality
compared
to
those
that
are
individualist or competitive. Second, that
girls are more prosocially oriented than
boys. Third, that girls would have a higher
quality of friendship than boys. Lastly, it
was expected that as age increases,
friendship quality also increases and Friendship is a complex construct that
consists of many components. One of the
components is friendship quality. Berndt
(2002) argued that high-quality friendship
strengthens
individual
development. Accordingly, there are positive and negative
features of a good friendship. Positive 26 Social Value Orientation – Fitria and Peters among options of outcomes for oneself or
another person. An example of decomposed
game options are Option A, 480 points for
self and 80 points for other (competitive; the
completer obtains more point than the other
person but less than in the individualistic
option), Option B, 540 points for self and 280
points
for
other
(individualistic;
the
completer obtains more points than the
other person) and Option C, 480 points for
self and 480 points for other (prosocial; the
completer and the other person get the same
amount of points). Six consistent choices of
one social value would determine whether
participants
classified
as
competitive,
individualistic or prosocial. In this study, it
was decided to also categorize SVO into just
two types; prosocial, and proself. Proself is
the combination of individualist and
competitive. adolescents
become
more
prosocially
oriented, rather than the other types of SVO. from 1= not true to 5 = really true. to make a choice e Social Value Orientation Participants’ SVO was measured by
asking participants to complete a series of
games (Messick & McClintock, 1968). This
measure has been found to be a reliable
measure of SVO (Kuhlman et al., 1986). Participants received nine scenarios with three alternative options for each scenario. Participants were asked to make a choice
from 1= not true to 5 = really true. 27 Social Value Orientation – Fitria and Peters RESULTS
First I examined the relationship
age did not vary significantly with the type
of SVO (prosocial, proself), F(1, 187)= .903,
p= .343. RESULTS
First I examined the relationship
age did not vary significantly with the type
of SVO (prosocial, proself), F(1, 187)= .903,
p= .343. RESULTS It revealed there was no
significant effect of SVO type (prosocial,
individualist, competitive) on FQS Positive
was no significant effect of SVO type
(prosocial, proself) on FQS Positive scale
after controlling for age and gender, F(1,
185) = .12, p = .734 and there was no
significant effect of SVO type (prosocial,
proself) on FQS Negative scale after
controlling for age and gender, F(1, 185) =
.07, p = .785. A two-way ANCOVA was conducted to
determine the effect of different types of
SVO (prosocial, individualist, competitive)
and gender (female, male) on friendship SVO (prosocial, proself) and gender, X2 (1) =
.02, p = .884. was no significant effect of SVO type
(prosocial, proself) on FQS Positive scale SVO (prosocial, proself) and gender, X2 (1) =
.02, p = .884. Finally I tested the hypothesis that
adolescents who are prosocially oriented
have higher friendship quality compared to
was no significant effect of SVO type
(prosocial, proself) on FQS Positive scale
after controlling for age and gender, F(1,
185) = .12, p = .734 and there was no
significant effect of SVO type (prosocial,
proself) on FQS Negative scale after Finally I tested the hypothesis that
adolescents who are prosocially oriented
have higher friendship quality compared to those that are individualist or competitive. A one-way ANCOVA was conducted to
determine the difference between types of
SVO on friendship quality controlling for
age and gender. It revealed there was no
significant effect of SVO type (prosocial,
individualist, competitive) on FQS Positive
scale after controlling for age and gender,
F(2, 184) = .40, p = .673. There was no
significant effect of SVO type (prosocial,
individualist, competitive) on FQS Negative
scale after controlling for age and gender,
F(2, 184) = .42, p = .656. Also, it revealed there those that are individualist or competitive. A one-way ANCOVA was conducted to
determine the difference between types of
SVO on friendship quality controlling for
age and gender. It revealed there was no
significant effect of SVO type (prosocial,
individualist, competitive) on FQS Positive
scale after controlling for age and gender,
F(2, 184) = .40, p = .673. There was no
significant effect of SVO type (prosocial,
individualist, competitive) on FQS Negative
scale after controlling for age and gender,
F(2, 184) = .42, p = .656. RESULTS age did not vary significantly with the type
of SVO (prosocial, proself), F(1, 187)= .903,
p= .343. RESULTS
First I examined the relationship
age did not vary significantly with the type
of SVO (prosocial, proself), F(1, 187)= .903,
p= .343. between the two main variables and age. Next I investigated sex differences in
FQS and SVO. An independent sample t-
test revealed that there was a significant
difference between females (M = 57.92, SD =
5.30) and males (M = 53.87, SD = 6.09) on
FQS positive scale, t(187) = 4.89, p < .001 and
there was no significant difference between between the two main variables and age. The descriptive statistics for age separated
for friendship quality and SVO are
described in Table 1. A Pearson’s r revealed
that there is no significant relationship
between FQS Positive (M= 56.01, SD= 6.02)
and age (M= 15.82, SD= 3.13), r(285)= .06 , p= .312. However, there is a significant
negative
relationship
between
FQS
Negative and age, such that as age (M=
females (M = 11.48, SD = 3.87) and males (M
= 12.43, SD = 393 ) on FQS negative scale,
t(187) = -1.67, p = .097. .312. However, there is a significant
negative
relationship
between
FQS
Negative and age, such that as age (M=
15.82, SD= 3.13) increases, the FQS negative
scale (M= 11.93, SD= 3.92) decreases, r(285)=-
.12, p= .045. females (M = 11.48, SD = 3.87) and males (M
= 12.43, SD = 393 ) on FQS negative scale,
t(187) = -1.67, p = .097. Next, I investigated the relationship
between SVO and gender. A chi-square test
of independence revealed that there was no
significant relationship between type of
SVO (prosocial, individualist, competitive)
and gender, χ2 (2) = 2.37, p = .306. There was
no significant relationship between type of A one-way ANOVA revealed that
age did not vary significantly with type of
SVO (prosocial, individualist, competitive),
F(2, 186)= 1.028, p= .360. It also revealed that 28 Social Value Orientation – Fitria and Peters SVO (prosocial, proself) and gender, X2 (1) =
.02, p = .884. Finally I tested the hypothesis that
adolescents who are prosocially oriented
have higher friendship quality compared to
those that are individualist or competitive. A one-way ANCOVA was conducted to
determine the difference between types of
SVO on friendship quality controlling for
age and gender. RESULTS Also, it revealed there A two-way ANCOVA was conducted to
determine the effect of different types of
SVO (prosocial, individualist, competitive)
and gender (female, male) on friendship
quality controlling for age. For FQS positive
scale, it revealed that there was no
significant main effect of SVO, F(2, 182) =
0.97, p = .380. However, there was a
significant main effect of gender, F(1, 182) =
19.91, p = .00. There was no significant 29 Social Value Orientation – Fitria and Peters interaction between SVO types and Gender
on FQS Positive scale after controlling for
age, F(2, 182) = 1.32, p = .269. For FQS negative scale, there was no significant main
effect of SVO and gender, F(2, 182) = 0.18, p
= .835, F(1, 182) = 0.69, p = .408. There interaction between SVO types and Gender
on FQS Positive scale after controlling for
age, F(2, 182) = 1.32, p = .269. For FQS
was no significant interaction between the
effect of SVO and gender on FQS Negative
scale after controlling for age, F(2, 182) =
1.82, p = .834. features that include factors such as
prosocial behavior, self-esteem support,
intimacy, loyalty, conflicts, dominance
attempts, and rivalry. Therefore, it might be
that other factors are also responsible for
individual friendship quality. was no significant interaction between the
effect of SVO and gender on FQS Negative
scale after controlling for age, F(2, 182) =
1.82, p = .834. was no significant interaction between the
effect of SVO and gender on FQS Negative
scale after controlling for age, F(2, 182) =
1.82, p = .834. For the other SVO type (prosocial, proself),
it revealed that there was no main effect of
SVO on FQS positive, F(1, 184) = 0.10, p =
.755. However, there was a main effect of
gender, F(1, 184) = 24.69, p = .00. There was
no significant interaction between the effect
of SVO type and gender on FQS Positive
scale after controlling for age, F(1, 184) =
1.46, p = .703. For FQS negative, there was no
main effect of SVO and gender, F(1, 184) =
0.07, p = .794, F(1, 184) = 3.44, p = .065. There
was no significant interaction between the
effect of SVO and Gender on FQS Negative
scale after controlling for age, F(1, 184) = .23,
p = .87. The next hypothesis predicted that
girls would be more prosocially oriented
compared to boys. RESULTS However, the results
rejected the hypothesis as it showed that
there
was
no
relationship
between
adolescents’ type of SVO and their gender. This indicated that gender differences did
not influence SVO. This result confirmed a
previous study by Kuhlman and Marshello
(1975) that proposed that there are no
gender differences in SVO. However, the
present
study
results
disagree
with
previous studies, which stated that boys
were more individualistic, while girls were
more prosocially oriented (Eisenberg et al.,
2005; Derks et al., 2014; Iedema & Poppe,
1999). DISCUSSIONS The objective of the current study
was to examine the relationship between
SVO and friendship quality, specifically the
effect
of
adolescents’
SVO
on
their
friendship quality. For the first hypothesis, I
tested
whether
prosocially
oriented
adolescents are more likely to have higher
friendship
quality. In
contrast,
the
hypothesis was rejected as the results
disclosed that SVO did not affect their
positive and negative friendship quality. It
could be that SVO only affects the quantity
of friends that individuals make, but not
friendship quality, as previous studies
specified that adolescents with more friends
apparently show more prosocial behavior
(Chesney et al, 2016; McGuire & Weisz,
1982). Furthermore,
Berndt
(2002)
suggested that high quality friendship are
characterized by positive and negative The third hypothesis was that girls
are more likely to have higher friendship
quality than boys. Confirmed by the results
of present study, girls demonstrated higher
positive friendship quality compared to
boys. However, no differences were found
in negative friendship quality. This might
be explained by looking at a previous study
by Kuttler et al. (1999), which stated that
girls had greater positive features of
friendship than boys. This possibly leads to
girls reporting higher friendship quality. Lastly, I examined the relationship
between SVO and friendship quality and
age. The hypothesis was, as they grow
older, the friendship quality increases and
they become prosocially oriented. However, 30 Social Value Orientation – Fitria and Peters the results rejected the hypotheses. This
study found, that there was a significantly
weak negative relationship between age
and negative friendship quality, such that,
as age increase, negative friendship quality
decreases. Nonetheless, there was no
relationship between positive friendship
quality and age. This might explain
friendship as a factor that changes during
adolescence, as teenagers are more likely to
spend an increased amount of time with
their friends (Crockett et al., 1984; La Greca
& Prinstein, 1999). This might be the reason
for a decrease in negative friendship
quality. attachment as one of the variables to assess. It might be essential to consider including
attachment in the future study, since
attachment security is a part of SVO and is
essential in a lasting healthy relationship
(Van Lange et al., 1997). In summary, the present study
showed that there was no relationship
between SVO and friendship quality in
adolescents. DISCUSSIONS Also, there was no gender
effect in SVO but there was a gender effect
in friendship quality, such that girls have
higher
positive
friendship
quality
compared to boys. The results also indicated
that
adolescents’
negative
friendship
quality decreased as age increased and there
was no significant difference in SVO with
age. Conclusively, the present study results
provide information that someone’s SVO
does not affect the quality of their
friendship, but it is possible that it
influences the number of friends they have. Furthermore, the current results
shows that there was no relationship
between SVO and age. This confirmed
previous study results conducted by Van
Lange, et al. (1997), which suggested that
different social interaction experiences
influences the development of SVO and
resulted in different type SVO that someone
has during specific period. This findings
might be explained by Kelley and Thibaut’s
(1978) study, who proposed that prosocial,
individualistic and competitive orientations
were established based on different forms of
social interactions experienced from early
childhood to young adulthood, which later
are also shaped by experiences during
adulthood. Thus, it could be concluded that
there is no development of SVO in
adolescence. REFERENCES Au, W. T., Kwong, J. Y. Y. (2004). Measurements and effects of social-
value orientation in social dilemmas:
A review. In R. Suleiman, D. Budescu, I. Fischer & D. Messick
(eds.), Contemporary Psychological
Research on Social Dilemmas (pp. 71–
98). Cambridge:
Cambridge
University Press. Berndt, T. J. (1982). The features and effects
of friendships in early adolescence. Child Development, 53, 1447-1460. There are a number of limitations in this
study. First, the ranges of participants’ age
are large, meaning that this study did not
capture
the
results
that
specifically
represent adolescents. For future study, it
might be useful to just include participants
within adolescents’ age range to assess
exclusively, the exact pattern of SVO and
friendship quality
within
adolescence. Second, this study did not consider Berndt, T. J. (2002). Friendship quality and
social
development. Current
Direction in Psychological Science,
11(1), 7-10. Bukowski, W. M., Hoza, B., & Boivin, M. (1994). Measuring friendship quality
during pre- and early adolescence: 31 Social Value Orientation – Fitria and Peters The development and psychometric
properties
of
the
Friendship
Qualities Scale. Journal of Social and
Personal Relationships, 11, 471-484. Journal of Research on Adolescence,
15(3), 235-260. Furman, W., & Buhrmester, D. (1992). Age
and sex differences in perceptions of
networks of personal relationships. Child Development, 63, 103–11 Chesney, T., Chuah, S., & Hoffmann, R. 2016. How user Personality and
social Value orientation influence
avatar-mediated
friendship. Information Technology & People,
29(4), 688-716. Furman, W., & Collins, W. A. (2009). Adolescent romantic relationships
and experiences. In Poorthuis, A. M. G., Thomaes, S., Denissen, J. J. A.,
van Anken, M. A. G., & de Castro, B. O. (2012). Prosocial
tendencies
predict friendship quality, but not
for popular children. Journal of
Experimental Child Psychology, 112,
378-388. Crockett, L. , Losoff, M., & Petersen, A. C. (1984). Perceptions of the peer group
and friendship in early adolescence. Journal of Early Adolescence, 4, 155-
181. De Dreu, C.K.W., & Boles, T. ( 1998). Share
and share alike or winner take all? Impact of social value orientation on
the choice and recall of decision
heuristics
in
negotiation. Organizational Behavior and Human
Decision Processes, 76, 253-267. Iedema, J. & Poppe. M. (1999). Expectations
of others’ social value orientations in
specific and general populations. Personality and Social Psychology
Bulletin, 25(12), 1443 – 1450. Kagan, S., & Madsen, M. C. (1971). REFERENCES Cooperation and competition of
Mexican, Mexican-American, and
Anglo-American children of two
ages under four instuctional sets. Developmental Psychology, 5, 31–39. Derks, J., Lee, N. C., & Krabbendam, L. (2014). Adolescent
trust
and
trustworthiness, role of gender and
social value orientation. Journal of
Adolescence, 37, 1379-1386. Kelley, H. H., & Thibaut, J. W. (1978). Interpersonal relations: a theory of
interdependence. New York: Wiley Eek, D., & Gärling, T. (2008). A new look at
the
theory
of
social
value
orientations:
Prosocials
neither
maximize
joint
outcome
nor
minimize outcome differences but
prefer equal outcomes. In A. Biel, D. Eek, M. Gustafsson, & T. Gärling
(Eds.), New issues and paradigms in
research on social dilemmas (pp. 10-26). New York: Springer. Knight, G. P., Dubro, A. F., & Chao, C. C. (1985). Information processing and
the development of cooperative,
competitive,
and
individualistic
social
values. Development
Psychology, 21, 37–45. Kuhlman, D. M., Camac, C. R., Cuhna, D. A. (1986). Individual differences in
social orientation. In H. A. M. Wilke,
D. M. Messick & C. G. Rutte (eds.),
Experimental Social Dilemmas (pp. Eisenberg, N., Cumberland, A., Guthrie, I. K., Murphy, B. C., & Shepard, S. A. (2005). Age changes in prosocial
responding and moral reasoning in
adolescence and early adulthood. 32 Social Value Orientation – Fitria and Peters 151–176). Frankfurt: Verlag Peter
Lang. McClintock, C. G., & Moskowitz, J. M. (1976). Children’s preferences for
individualistic, coopera- tive, and
competitive outcomes. Journal of
Personality and Social Psychology, 34,
543–555. Kuhlman, D. M., & Marshello, A. (1975). Individual
differences
in
game
motivation
as
moderators
of
preprogrammed strategic effects in
prisoner’s
dilemma. Journal
of
Personality and Social Psychology, 32,
922-931. McClintock, C. G., & Van Avermaet, E. (1982). Social values and rules of
fairness: A theoretical perspective. In Liebrand, W. B. G., Jansen, R. W. T. L., Rijken, V. M., & Suhre, C. J. M. (1986). Might over morality: Social
values and the perception of other
players in experimental games. Journal
of
Experimental
Social
Psychology, 22, 203-215. Kuttler, A. F., La Greca, A. M., & Prinstein,
M. J. (1999). Friendship qualities and
social-emotional
functioning
of
adolescents with close, cross-sex
friendships. Journal of Research on
Adolescence, 9(3), 339-366. La Greca, A. M., & Prinstein, M. J. (1999). The peer group. In W. K. Silverman
&
T. H. Ollendick
(Eds.),
Developmental issues in the clinical
treatment of children and adolescents
(pp. 171–198.) Needham Heights,
MA: Allyn & Bacon. McGuire, K. REFERENCES D., & Weisz, J. R. (1982). Social
cognition and behavior correlates of
preadolescense
chumship. Child
Development, 53, 1478-1484. Messick, D. M., & McClintock, C. G. (1968). Motivational bases of choice in
experimental
games. Journal
of
Experimental Social Psychology, 4, 1-
25. Liebrand, W. B. G., Jansen, R. W. T. L.,
Rijken, V. M., & Suhre, C. J. M. (1986). Might over morality: Social
values and the perception of other
players in experimental games. Journal
of
Experimental
Social
Psychology, 22, 203-215. Rubin, K., Bukowski, W., & Parker, J. (2006). Peer interactions, relationships, and
groups. In N. Eisenberg, W. Damon
& R.M. Lerner (Eds). Handbook of
child
psychology:
Vol. 3,
Social,
emotional, and personality development
(6th ed.) (pp. 571-645). John Wiley &
Sons Inc, Hoboken, NJ US. Mann, L., Harmoni, R., & Power, C. (1989). Adolescent decision-making: the
development of competence. Journal
of Adolescence, 12, 265-278. Peters, S., Peper, J. S., Van Duijvenvoorde,
A. C. K., Braams, B. R., & Crone, E. A. (2016). Amygdala–orbitofrontal
connectivity predicts alcohol use
two years later: a longitudinal
neuroimaging study on alcohol use
in
adolescence. Developmental
Science, 1-11. McClintock, C. G. (1972). Social motivation-
a set of propositions. Behavioral
Science, 17, 438-454. McClintock, C. G., & Allison, S. (1989). Social value orientation and helping
behavior. Journal of Applied Social
Psychology, 19, 353- 362. 33 Social Value Orientation – Fitria and Peters Van Lange, P. A. M. (1999). The pursuit of
joint outcomes and equality in
outcomes: An integrative model of
social value orientation. Journal of
Personality and Social Psychology,
77,337-349. Van Lange, P. A. M., & Folmer, C. P. R. (2007). Social value orientation. In R. F. Baumeister, & K. D. Vohs.(Ed.)
Encyclopedia of social psychology (pp
924-926). Van Lange, P. A. M., Otten, W., De Bruin, E. N. M., & Joireman, J. A. (1997). Development
of
prosocial,
individualistic,
and
competitive
orientations:
Theory
and
preliminary evidence. Journal of
Personality and Social Psychology, 73,
733-746. Van Lange, P. A. M., De Cremer, D., Van
Dijk, E., & Van Vugt, M. (2007). Self-
interest and beyond: Basic principles
of social interaction. In A. W. Kruglanski & E. T. Higgins (Ed.),
Social psychology: Handbook of basic
principles (pp. 540–561). New York:
Guilford Press. 34 34
|
https://openalex.org/W2592443350
|
https://www.nature.com/articles/srep43534.pdf
|
English
| null |
Effects of different kinds of essentiality on sequence evolution of human testis proteins
|
Scientific reports
| 2,017
|
cc-by
| 14,247
|
Julia Schumacher, Hans Zischler & Holger Herlyn received: 25 August 2016
accepted: 25 January 2017
Published: 08 March 2017 We asked if essentiality for either fertility or viability differentially affects sequence evolution of
human testis proteins. Based on murine knockout data, we classified a set of 965 proteins expressed
in human seminiferous tubules into three categories: proteins essential for prepubertal survival
(“lethality proteins”), associated with male sub- or infertility (“male sub-/infertility proteins”), and
nonessential proteins. In our testis protein dataset, lethality genes evolved significantly slower than
nonessential and male sub-/infertility genes, which is in line with other authors’ findings. Using tissue
specificity, connectivity in the protein-protein interaction (PPI) network, and multifunctionality as
proxies for evolutionary constraints, we found that of the three categories, proteins linked to male sub-
or infertility are least constrained. Lethality proteins, on the other hand, are characterized by broad
expression, many PPI partners, and high multifunctionality, all of which points to strong evolutionary
constraints. We conclude that compared with lethality proteins, those linked to male sub- or infertility
are nonetheless indispensable, but evolve under more relaxed constraints. Finally, adaptive evolution
in response to postmating sexual selection could further accelerate evolutionary rates of male sub- or
infertility proteins expressed in human testis. These findings may become useful for in silico detection of
human sub-/infertility genes. Almost four decades ago, Wilson et al.1 hypothesized that proteins essential for an organism’s viability or fer-
tility should evolve at lower rates than those which are more dispensable. This predicted association has been
studied extensively in mammalian (see, e.g., refs 2–4) and non-mammalian species (see, e.g., refs 5–7), but the
respective investigations yielded inconsistent outcomes. In the mentioned studies on mammals essentiality was
defined as indispensability for either viability or both survival and fertility. In contrast, Torgerson et al.8 differ-
entiated between fertility and viability proteins and reported that murine proteins important to either male or
female reproduction evolve at higher rates than proteins indispensable for survival and also than a representative
genomic sample. This observation is in line with previous studies’ findings whereupon reproduction-related pro-
teins show accelerated rates of evolution9 and are oftentimes subject to positive selection10. In particular, male
reproductive proteins have been described to evolve rapidly (see, e.g., refs 11 and 12). Furthermore, genes with
testis-specific expression evolve at overall higher rates relative to female-specific genes or those unrelated to
reproduction in Drosophila13 and relative to genes with expression maxima in other rodent tissues14. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 Results and Discussion Characteristics of the human testis protein dataset. The initial dataset contained 2,986 proteins
expressed in human seminiferous tubules according to information from Human Protein Atlas version 12
(see Materials and Methods). Although testes consist of various cell and tissue types, we focused on proteins from
seminiferous tubules including Sertoli cells as well as spermatogenesis and spermiogenesis stages. In the course
of mapping IDs from Ensembl version 73 to 82 (see Materials and Methods), four of the 2,986 IDs were mapped
to IDs already existing in the dataset. These redundant IDs were discarded. For further 602 genes, no dN/dS
estimate calculated for human and murine 1-to-1 orthologues was available in Ensembl version 82. Another 344
proteins were not contained in the PPI network used (see Materials and Methods) and lack of murine KO data
(see Materials and Methods) led to exclusion of 964 proteins. Finally, five proteins were excluded due to lacking
gene ontology (GO) biological process data (see Materials and Methods). For 1,067 proteins all of the collected
variables including murine phenotype data for targeted KO mutations were available. Of these, 73 were excluded
because their ablation in mice resulted in both male reproductive anomalies and prepubertal lethality. Another 26
proteins were removed from the dataset because their KO mutants had an unclear fertility status, due to difficult
transferability of the observed phenotype to humans (“decreased litter size”), or because male null mice were
fertile despite reproductive abnormalities (for details, see Materials and Methods). Finally, three more proteins
were excepted from analyses due to further reasons which are outlined in detail in the Materials and Methods
section. After exclusion of these overall 102 proteins, the final dataset contained 965 testis proteins, comprising
57 male sub-/infertility, 502 lethality, and 406 nonessential proteins (for 965 included and 102 excluded proteins,
see Supplementary Table S1). pp
y
None of the included 965 genes coding for human testis proteins had a dN/dS estimate >1 as calculated for
1-to-1 orthologues between human and mouse, indicating that purifying selection prevailed in the evolution of
our dataset. This conclusion is in line with previous studies demonstrating prevalent sequence conservation of
male reproductive proteins in various taxa, e.g., in the Drosophila sperm proteome16, the murine male repro-
ductive tract17, and hominoid seminal proteins29. www.nature.com/scientificreports/ essentiality. Testis proteins were assigned to one of these three categories based on known phenotypes resulting
from targeted knockout (KO) mutations in murine orthologues of human genes. We hypothesized that lethality
proteins evolve under stronger purifying selection due to their functional importance and increased evolutionary
constraints. In contrast, relaxation of constraints as well as sexual selection might accelerate sequence evolution
of more specialized8, but nonetheless important sub-/infertility proteins. p
p
y p
To assess levels of evolutionary constraints, we employed three measures. First, we derived node degree, the
number of links a protein has to other nodes, from a human protein-protein interaction (PPI) network. Second,
tissue specificity was estimated using the index τ, which ranges from 0 to 1, whereby higher values indicate more
tissue-biased expression20. Third, numbers of biological processes in which a protein participates served as a
measurement of its multifunctionality21. Numbers of PPI partners, expression breadth, and multifunctionality
are known to correlate with pleiotropy22,23 and have been previously used to quantify levels of pleiotropy (see,
e.g., refs 24 and 25). Moreover, PPIs also exert structural and functional constraints on proteins (see, e.g., refs
26–28). Hence, the applied properties enable assessment of a broad range of constraints under which proteins
evolve. Magnitude and direction of selection were measured using pairwise dN/dS estimates between human and
mouse orthologues. The dN/dS estimate contrasts nonsynonymous (dN) and synonymous substitution rates (dS). Thereby, dN/dS values >1, <1, and = 1 are associated with positive selection, purifying selection, and neutral
evolution, respectively. Before comparing the three protein categories – lethality, sub-/infertility, and nonessential
proteins – regarding their evolutionary rates and constraints, we evaluated the interrelations among dN/dS, node
degree, multifunctionality, and tissue specificity within our human testis dataset employing rank correlations. In
doing so, we were able to examine the interdependencies among essentiality, evolutionary constraints, and rates
of sequence evolution in a set of human testis proteins. Julia Schumacher, Hans Zischler & Holger Herlyn Thereby, rate
acceleration of male reproductive proteins is assumed to be driven by different forms of postmating sexual selec-
tion, such as sperm competition and sexual conflict (see, e.g., refs 14 and 15). Although sexual selection could
indeed explain enhanced evolutionary rates of some male reproductive proteins, sequences of the majority of
proteins expressed in sperm or the male reproductive tract are evolutionarily conserved (see, e.g., refs 16 and 17). One possible explanation for this prevailing conservation may be proteins’ involvement in basic cellular func-
tions such as metabolism16,18. But while the influence of sperm proteins’ functions on their evolutionary rate has
already been explored (see, e.g., refs 18 and 19), the effects of different forms of essentiality, i.e. for viability or
fertility, on evolutionary rates of male reproductive proteins have not yet been disentangled.hf y
y
p
p
y
g
Therefore, the present study aimed to unveil the impact of different kinds of essentiality on sequence evolution
of testis proteins. Our analyses were conducted on a sample of human testis proteins relying on protein expression
data. We distinguished between proteins associated with prepubertal death (“lethality proteins”), linked to male
sub- or infertility (“male sub-/infertility proteins”), and “nonessential proteins”, associated with neither form of Institute of Anthropology, Johannes Gutenberg University Mainz, Anselm-Franz-von-Bentzel-Weg 7, D-55099,
Germany. Correspondence and requests for materials should be addressed to J.S. (email: julia.schumacher@uni-
mainz.de) or H.H. (email: herlyn@uni-mainz.de) SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 1 www.nature.com/scientificreports/ SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 Results and Discussion aAll p-
values were adjusted with Holm’s procedure (see Materials and Methods). ***Highlight significance at the 0.1%
level; ns, nonsignificant. male infertility category is CREM: Ablation of this gene in mice leads to male sterility43–45 and altered expression
f mRNA or the encoded protein in human spermatids is suspected to underlie some cases of male infertility46,47 Correlates of evolutionary rates in human testis proteins. Spearman’s rank correlations indicated
significant interrelations among a protein’s dN/dS, node degree, level of tissue specificity (τ) and multifunction-
ality in the complete dataset comprising 965 testis proteins (Table 1).i Due to the strong interrelatedness of the incorporated variables, we assumed that the significant correlations
between dN/dS and each of the remaining three properties might at least to some extent reflect effects of the
other considered variables. In order to disentangle the specific role of single variables in sequence evolution, we
employed partial rank correlations between dN/dS and each of the remainder three variables, controlling for the
two other properties. This approach revealed that only node degree had a significant partial correlation with dN/dS
(Table 2). Hence, the seemingly considerable influence of multifunctionality and tissue specificity on dN/dS
values actually reflected variance they shared with node degree. Yet, this does not mean that a gene’s dN/dS is
completely independent of its tissue specificity and the number of biological processes in which the encoded
protein is involved in. Potentially, within our dataset higher multifunctionality and broader expression entail
higher numbers of PPIs, thereby restraining evolutionary rates. This possibility is further addressed in the fol-
lowing sections. Importantly, results of zero-order and partial rank correlations including node degree or τ could
be reproduced using alternative approaches to infer these variables (see Materials and Methods, Supplementary
Tables S2 and S3). Negative correlations of evolutionary rates with number of PPIs as described herein have been reported pre-
viously for other protein samples and species (see, e.g., refs 26, 48 and 49). Apparently, a major factor underlying
this correlation is that highly connected proteins contain a higher proportion of sections with interaction-related
functions, each evolving under constraints26. In addition, essentiality itself might promote sequence conserva-
tion of proteins with high node degree (see, e.g., refs 4, 50 and 51; see also ref. 52). Results and Discussion Results of Spearman’s rank correlations between studied variables. aAll p-values were adjusted with
Holm’s procedure (see Materials and Methods). ***, **, and * highlight significance at the 0.1%, 1%, and 5%
level, respectively. The asterisk in parentheses indicates significance lost after correction against multiple testing. Correlation between | controlling for
Spearman’s partial correlation coefficient; pa
dN/dS, node degree |
multifunctionality, τ
ρ = −0.178***
dN/dS, multifunctionality | node
degree, τ
ρ = −0.049ns
dN/dS, τ | node degree,
multifunctionality
ρ = 0.022ns
Table 2. Results of partial rank correlations between dN/dS estimates and three other variables. aAll p-
values were adjusted with Holm’s procedure (see Materials and Methods). ***Highlight significance at the 0.1%
level; ns, nonsignificant. Correlation between
Spearman’s correlation coefficient; pa
dN/dS, node degree
ρ = −0.229***
dN/dS, multifunctionality
ρ = −0.134***
dN/dS, τ
ρ = 0.088*(*)
node degree, multifunctionality
ρ = 0.398***
node degree, τ
ρ = −0.304***
τ, multifunctionality
ρ = −0.082* node degree, multifunctionality
ρ = 0.398***
node degree, τ
ρ = −0.304***
τ, multifunctionality
ρ = −0.082*
Table 1. Results of Spearman’s rank correlations between studied variables. aAll p-values were adjusted with
Holm’s procedure (see Materials and Methods). ***, **, and * highlight significance at the 0.1%, 1%, and 5%
level, respectively. The asterisk in parentheses indicates significance lost after correction against multiple testing. Correlation between | controlling for
Spearman’s partial correlation coefficient; pa
dN/dS, node degree |
multifunctionality, τ
ρ = −0.178***
dN/dS, multifunctionality | node
degree, τ
ρ = −0.049ns
dN/dS, τ | node degree,
multifunctionality
ρ = 0.022ns
Table 2. Results of partial rank correlations between dN/dS estimates and three other variables. aAll p-
values were adjusted with Holm’s procedure (see Materials and Methods). ***Highlight significance at the 0.1%
level; ns, nonsignificant. Table 1. Results of Spearman’s rank correlations between studied variables. aAll p-values were adjusted with
Holm’s procedure (see Materials and Methods). ***, **, and * highlight significance at the 0.1%, 1%, and 5%
level, respectively. The asterisk in parentheses indicates significance lost after correction against multiple testing. Table 1. Results of Spearman’s rank correlations between studied variables. aAll p-values were adjusted with
Holm’s procedure (see Materials and Methods). ***, **, and * highlight significance at the 0.1%, 1%, and 5%
level, respectively. The asterisk in parentheses indicates significance lost after correction against multiple testing. Table 2. Results of partial rank correlations between dN/dS estimates and three other variables. Results and Discussion But as dN/dS >1 measured over the entire length of a gene is a
very conservative threshold of positive selection30, it is probable that some of the proteins in our sample contain
positions subject to adaptive evolution, although their pairwise dN/dS value between human and mouse was
below one (see also ref. 31). Suitability of the mouse model in present analyses. The approach to assess survival essentiality of
human genes via murine phenotypic KO data has been widely used (see, e.g. refs 32–34). Nonetheless, phenotypic
consequences of gene loss may vary between mouse and human35, which might also apply to an unknown num-
ber of genes in our dataset. However, Kim et al.36 reported that proteins essential in yeast, but not mouse never-
theless engage in significantly more interactions in the murine PPI network than proteins which are nonessential
in both species. The same authors inferred dN/dS estimates across four yeast species: dN/dS estimates of genes
with such differential essentiality status in mouse and yeast closely resembled those of proteins indispensable in
both taxa36. These similarities have been observed in phylogenetically distant models like mouse and yeast and
should hence be even more valid for more closely related taxa such as human and mouse. Therefore, even if some
proteins categorized as lethal herein are essential for murine, but not human viability, their node degrees and
evolutionary rates should approximate those of proteins essential for survival in both species; a similar pattern
should apply to male sub-/infertility proteins. Furthermore, there are several examples for the transferability of gene essentiality between mouse and human
within our dataset. For instance, null mutations in EIF2AK3, DLD, or PDHA1 may result in prepubertal death in
both humans and mice (see dataset S1 of ref. 35; and see, e.g., refs 37–39; for human, see Phenotype MIM num-
bers 246900, 226980, 312170). Deletion of Sycp3, a member of our male sub-/infertility category, causes azoo-
spermia in mice40. In humans, lack of testicular SYCP3 mRNA expression has been implicated in male infertility41
and truncating mutations in this gene have been reported in azoospermic patients42. Another example from our SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 2 www.nature.com/scientificreports/ Correlation between
Spearman’s correlation coefficient; pa
dN/dS, node degree
ρ = −0.229***
dN/dS, multifunctionality
ρ = −0.134***
dN/dS, τ
ρ = 0.088*(*)
node degree, multifunctionality
ρ = 0.398***
node degree, τ
ρ = −0.304***
τ, multifunctionality
ρ = −0.082*
Table 1. SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Variable
H
pa
dN/dS
43.334
***
dN
54.360
***
dS
29.781
***
node degree
45.898
***
τ
60.307
***
multifunctionality
26.273
***
Table 3. Results of Kruskal-Wallis tests among three protein categories for six variables. aFor node degree
and τ, p-values were adjusted with Holm’s procedure (see Materials and Methods). ***highlight significance at
the 0.1% level. Variable
H
pa
dN/dS
43.334
***
dN
54.360
***
dS
29.781
***
node degree
45.898
***
τ
60.307
***
multifunctionality
26.273
***
Table 3. Results of Kruskal-Wallis tests among three protein categories for six variables. aFor node degree
and τ, p-values were adjusted with Holm’s procedure (see Materials and Methods). ***highlight significance at
the 0.1% level. Variable
H
pa
dN/dS
43.334
***
dN
54.360
***
dS
29.781
***
node degree
45.898
***
τ
60.307
***
multifunctionality
26.273
*** Table 3. Results of Kruskal-Wallis tests among three protein categories for six variables. aFor node degree
and τ, p-values were adjusted with Holm’s procedure (see Materials and Methods). ***highlight significance at
the 0.1% level. Table 3. Results of Kruskal-Wallis tests among three protein categories for six variables. aFor node degr
and τ, p-values were adjusted with Holm’s procedure (see Materials and Methods). ***highlight significance
the 0.1% level. testis protein categories. Nonetheless, it should be emphasized that node degree clearly had the strongest inde-
pendent link to dN/dS in our testis dataset, as demonstrated by partial rank correlations (Table 2). Differences in evolutionary rates, network connectivity, multifunctionality, and tissue specificity
among our three protein groups categorized according to their essentiality. We aimed to iden-
tify differences among the three categories – male sub-/infertility, lethality, and nonessential proteins – regarding
evolutionary rates and with respect to several pleiotropy measures potentially associated with their sequence
evolution either directly or indirectly (see above).if Kruskal-Wallis tests revealed highly significant differences among the three categories in their dN/dS, dN,
dS, node degree, tissue specificity (τ), and multifunctionality (Table 3). Post-hoc Mann-Whitney U (MWU) tests
enabled us to determine which categories differed significantly regarding each variable (see below). All presented
results which were based on analyses including τ or node degree were recalculated using the same variables
inferred with alternative approaches (see Materials and Methods). Results remained unchanged, emphasizing
their validity (see Supplementary Table S4 and Fig. S1). Differential selective pressures in three testis protein categories. www.nature.com/scientificreports/ Our results demonstrate that
human testis protein categories associated with various kinds of essentiality evolve under different selective
pressures. Gibbs et al.58 inferred a median KA/KS (also: dN/dS) of 0.10 across 11,084 functionally unspecified
protein-coding genes, based on 1-to-1 orthologues of human and mouse. While median dN/dS of our nonessential
category was only marginally lower than the genome-wide median reported by Gibbs et al.58, median dN/dS values
of lethality and sub-/infertility groups were decreased and increased, respectively (Fig. 1a). Inferring a genome-wide
median calculated from 8,610 human protein-coding genes (see Supplementary Materials and Methods;
dashed line in Fig. 1a; median dN/dS = 0.089) we were able to reproduce these findings with the exception that
nonessential genes showed a median dN/dS value slightly higher than this genome-wide median.i g
g
y
g
g
We observed significantly lower dN/dS in the lethality category compared with both nonessential and male
sub-/infertility genes; the latter two groups did not differ significantly in their dN/dS (Fig. 1a). Inspection of the
underlying numerators and denominators revealed significantly lower dS in genes coding for sub-/infertility than
for nonessential proteins, while their median dN values were of about the same level (Fig. 1b and c). Genes asso-
ciated with male sub- or infertility had significantly higher dN than lethality genes, while the difference regarding
dS was nonsignificant; this result corresponds to the findings reported by Torgerson et al.8. Nonessential genes
had both, significantly higher dN and dS than the lethality category (Fig. 1b and c). These results illustrate that
highest median dN/dS of sub-/infertility genes was not caused by a generally elevated substitution rate, but rather
a decline in dS, although they also displayed significantly higher dN than lethality genes. On the contrary, ele-
vated dN/dS values of genes encoding nonessential proteins compared with lethality genes might partly rely on
the generally accelerated substitution rate of the nonessential category. Still, dN apparently increases more than
dS in nonessential genes. Finally, lowest dN of lethality genes underscores their strong sequence conservation. h l d ff
f d
b
b
d
d
f b
h
d
l While differences of dN among categories may be attributed to disparity of both mutation rate and selection,
variation of dS should theoretically be determined by mutation rates59. However, synonymous mutations may also
be under selection due to their effects on e.g. mRNA stability60, or splicing61 as well as translational efficiency (see,
e.g., ref. SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 Results and Discussion However, Hahn and Kern48
demonstrated on the basis of three eukaryotic PPI networks that the interrelation of evolutionary rates and net-
work centrality, as quantified by betweenness centrality, cannot solely be ascribed to a protein’s essentiality status. Following Promislow53, they instead hypothesized that proteins more central to interaction networks might be
more pleiotropic in terms of functional diversity53 and thus more evolutionarily constrained48. Such negative
relationship between levels of multifunctionality and evolutionary rates, which we also found in zero-order cor-
relations (Table 1), has been attributed to the deleterious effects which substitutions of multifunctional proteins
may have on some of the processes they are involved in, even if they are beneficial to others21,54. As multifunc-
tional proteins are thought to perform their different functions by interaction with varying partners55, the effect
of multifunctionality on sequence evolution could be partly mediated via numbers of PPIs in the present dataset. Likewise, tissue specificity (τ) may be indirectly linked to sequence evolution via its association with network
connectivity. In support of such possibility, broadly expressed proteins are commonly thought to form a core
of interactomes, to which tissue-specific proteins are attached as peripheral components, modulating the basal
processes carried out by ‘hub’ proteins in a compartment-specific manner56,57. Although we cannot definitely rule
out that zero-order correlations of tissue specificity and multifunctionality with dN/dS were largely spurious, we
assume indirect associations of those two variables with sequence evolution (see Table 2). In order to account for
such potential indirect effects we kept multifunctionality and tissue specificity in subsequent comparisons among SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 3 www.nature.com/scientificreports/ (b) Median nonsynonymous substitution rates (dN) of nonessential and male sub-/
infertility genes are largely similar, whereas median dN of lethality genes is significantly lower than that of the
two other categories. (c) While lethality and male sub-/infertility genes are not significantly different regarding
their median synonymous substitution rates (dS), median dS of nonessential genes is significantly higher than
those of both sub-/infertility and lethality genes. Vertical bars define 95% confidence intervals calculated from
100,000 pseudo-replicates. *** and ** highlight significance at the 0.1% and 1% level, respectively. Significances
were corrected against multiple testing using Holm’s procedure (see Materials and Methods). If no asterisk is
given, the result of the MWU test is nonsignificant. Dashed lines indicate genome-wide median values of dN/
dS, dN, and dS (see Supplementary Materials and Methods). Several studies unveiled a functional compartmentalization of the sperm proteome: protein-coding genes with
functions proximate to fertilization evolve more rapidly than those relevant for more basal steps such as spermat-
ogenesis or sperm assembly18,19 (see also ref. 16). Based on nucleotide sequences of different mouse strains and
species, Vicens et al.18 found higher proportions of genes with signals of positive selection in groups linked to
sperm-egg interaction and sperm motility than in four other categories. They concluded that adaptive evolution
of motility-associated proteins could be driven by sperm competition, which might also pertain to a fraction
of our sub-/infertility category, such as the proteins encoded by AKAP4 and ATP2B4, which are both linked to
(hyperactivated) sperm motility68–71. Vicens et al.18 moreover identified candidate sites of positive selection in
murine Clgn whose human orthologue also belongs to our sub-/infertility category. Due to its presumable partic-
ipation in gamete interaction72 (see also ref. 73), coevolution with egg surface proteins could be a factor acceler-
ating its sequence evolution (see, e.g., refs 18 and 74). Hence, coevolutionary processes as well as other forms of
postcopulatory sexual selection such as sperm competition might be some of the forces underlying higher median
dN/dS values of sub-/infertility genes compared with the two other categories in our dataset and especially in
comparison with lethality genes. Sequence evolution of immunity and X-chromosomally encoded testis proteins. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Sequence evolution of three human testis gene groups categorized according to their essentiality. (a) Male sub-/infertility genes display highest dN/dS values, followed by the nonessential and the most
conserved lethality category. (b) Median nonsynonymous substitution rates (dN) of nonessential and male sub-/
infertility genes are largely similar, whereas median dN of lethality genes is significantly lower than that of the
two other categories. (c) While lethality and male sub-/infertility genes are not significantly different regarding
their median synonymous substitution rates (dS), median dS of nonessential genes is significantly higher than
those of both sub-/infertility and lethality genes. Vertical bars define 95% confidence intervals calculated from
100,000 pseudo-replicates. *** and ** highlight significance at the 0.1% and 1% level, respectively. Significances
were corrected against multiple testing using Holm’s procedure (see Materials and Methods). If no asterisk is
given, the result of the MWU test is nonsignificant. Dashed lines indicate genome-wide median values of dN/
dS, dN, and dS (see Supplementary Materials and Methods). igure 1. Sequence evolution of three human testis gene groups categorized according to their essentiality. ) M l
b /i f
ili
di
l
hi h
dN/dS
l
f ll
d b
h
i l
d h Figure 1. Sequence evolution of three human testis gene groups categorized according to their essentiality. (a) Male sub-/infertility genes display highest dN/dS values, followed by the nonessential and the most
conserved lethality category. (b) Median nonsynonymous substitution rates (dN) of nonessential and male sub-/
infertility genes are largely similar, whereas median dN of lethality genes is significantly lower than that of the
two other categories. (c) While lethality and male sub-/infertility genes are not significantly different regarding
their median synonymous substitution rates (dS), median dS of nonessential genes is significantly higher than
those of both sub-/infertility and lethality genes. Vertical bars define 95% confidence intervals calculated from
100,000 pseudo-replicates. *** and ** highlight significance at the 0.1% and 1% level, respectively. Significances
were corrected against multiple testing using Holm’s procedure (see Materials and Methods). If no asterisk is
given, the result of the MWU test is nonsignificant. Dashed lines indicate genome-wide median values of dN/
dS, dN, and dS (see Supplementary Materials and Methods). Figure 1. Sequence evolution of three human testis gene groups categorized according to their essentiality. (a) Male sub-/infertility genes display highest dN/dS values, followed by the nonessential and the most
conserved lethality category. www.nature.com/scientificreports/ 62). These examples contradict the assumed neutrality of synonymous mutations and argue for nearly
neutral evolution of synonymous exchanges (see, e.g. ref. 63). Moreover, dS values may vary in dependence of
chromosomal positions: for instance, Torgerson and Singh64 reported significantly lower synonymous substitu-
tion rates of tissue-specific genes on the X chromosome when compared to those on the autosomes. A similar
pattern might partly account for the relatively low median dS of male sub-/infertility genes (Fig. 1c) (see below). Independent of the selective or neutral forces underlying the differences regarding dS among our three testis gene
categories, the findings of higher dN in both nonessential and male sub-/infertility genes as compared with the
lethality category remain unaffected. Present evidence for strongest sequence conservation of proteins required for prepubertal survival corre-
sponds to the “knockout-rate prediction”2: genes indispensable for viability have been shown to evolve more
slowly than genes nonessential in this regard in various taxa, including, e.g., Escherichia coli65 and mouse4. In
contrast, protein-coding genes associated with male sub- or infertility displayed highest dN/dS values, an increase
that was significant relative to lethality, but not nonessential proteins (Fig. 1a). A trend for accelerated sequence
evolution of some male reproductive proteins is a well-documented phenomenon, probably affected by sexual
selection (see, e.g., refs 66 and 67). SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 4 www.nature.com/scientificreports/ nonessential
sub-/infertility
lethality
Statistical test
immunity
32.0% (130/406)
19.3% (11/57)
27.9% (140/502)
4.687a
ns
X chromosome
6.7% (27/406)
8.8% (5/57)
1.6% (8/502)
***b Table 4. Proportion of proteins in three human testis protein categories with immunity-related functions
or encoded on the X chromosome. Numbers of proteins in each category which are involved in immune
system processes or encoded on the human X chromosome and total numbers of proteins per category are given
in parentheses; aPearson’s Chi2. ***Highlight significance at the 0.1% level; ns, nonsignificant. bp of Fisher’s exact
test. Table 4. Proportion of proteins in three human testis protein categories with immunity-related functions
or encoded on the X chromosome. Numbers of proteins in each category which are involved in immune
system processes or encoded on the human X chromosome and total numbers of proteins per category are given
in parentheses; aPearson’s Chi2. ***Highlight significance at the 0.1% level; ns, nonsignificant. bp of Fisher’s exact
test. Yet, dS values of immunity-related and X-chromosomal protein-coding genes were significantly higher (p < 0.05;
MWU test) and lower (p < 0.001; MWU test) compared with those of all other genes, respectively. The finding
of lower dS values on the X chromosome compared with autosomes corresponds to prior literature (see above
and, e.g., ref. 64). Although a smaller proportion of sub-/infertility than lethality or nonessential proteins were
involved in immune system processes, the differences among the categories were nonsignificant (Table 4). In
contrast, compared with the lethality category, significantly higher proportions of nonessential (post-hoc Chi2,
p < 0.001 after correction against multiple testing) and male sub-/infertility proteins (post-hoc Fisher’s exact,
p < 0.05 after correction against multiple testing) were encoded on the human X chromosome (see also Table 4). The difference between nonessential and sub-/infertility proteins regarding their percentage of X-chromosomal
genes was nonsignificant (post-hoc Fisher’s exact, p > 0.05 after correction against multiple testing). We conclude
that the different composition of the three protein categories regarding X-encoded and, less so, immunity-related
members could have influenced their evolutionary rates, especially their dS values. This might in particular apply
to the sub-/infertility category with its low dS and relatively high and low proportions of X-chromosomal and
immunity-related proteins, respectively. www.nature.com/scientificreports/ However, whether or not the varying fractions of proteins participating
in immune system processes or encoded on the human X chromosome actually explain some differences among
the categories, their influence on the obtained results should only be marginal. In particular, these differences
cannot account for the higher median dN/dS in male sub-/infertility genes relative to the nonessential category. dN/dS values of our three testis protein categories before the background of node degree,
multifunctionality, and tissue specificity. In order to unravel the detailed driving forces behind the
differential evolutionary rates among our three testis protein categories we employed measures of evolutionary
constraints, i.e., node degree, multifunctionality, and tissue specificity. This approach revealed that proteins in
the least divergent lethality category (in terms of dN/dS and dN; see Fig. 1a and b) had significantly higher node
degree and were involved in more biological processes than the remaining two groups, although the latter rela-
tion was significant only in comparison to nonessential proteins (Fig. 2a and b). Additionally, lethality proteins
showed significantly less tissue-biased expression than both, sub-/infertility and nonessential proteins (Fig. 2c). Thus, the lethality category reflected the findings of rank correlations carried out on our entire testis protein sam-
ple, whereupon evolutionary conservation combines especially with increased node degree, but also with high
multifunctionality, and broad expression (see above). Lethality proteins hence evolve under the influence of con-
straints imposed by high network connectivity, as well as engagement in a multitude of biological processes and
expression in a wide range of tissues. In addition, their indispensability for organismal survival probably further
increases the extent of purifying selection operating on their sequences, as outlined above. Therefore, sequence
evolution of proteins in the lethality category is constrained by both factors proposed by Wilson et al.1, namely
functional importance and evolutionary (or functional) constraint (see also ref. 59). p
y
In contrast, sequence conservation due to indispensability should not have played a major role in the evolution
of nonessential proteins. Accordingly, the median dN/dS value of nonessential genes was significantly higher than
that of the lethality category (Fig. 1a), corresponding to findings by other authors (see, e.g. ref. 4; see also ref. 33). Moreover, levels of all measures of evolutionary constraints differed significantly from those in the lethality group
(see Fig. 2). www.nature.com/scientificreports/ Since
immunity-related proteins (see, e.g., refs 75 and 76) and those encoded on the X chromosome (see, e.g., refs 77
and 78) have been described as rapidly evolving or subject to positive selection, we tested whether such proteins
also showed increased rates of sequence evolution in our human testis protein sample. Furthermore, we exam-
ined if immunity-related and X-chromosomally encoded proteins were differentially distributed among our three
categories, which might have influenced their evolutionary rates. Neither genes coding for proteins involved in
immune system processes (GO:0002376; n = 281) nor those encoded on the human X chromosome (n = 40) had
significantly higher dN/dS or dN compared with all other members of the dataset (both p > 0.05; MWU test). SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 5 ificreports/
nonessential
sub-/infertility
lethality
Statistical test
immunity
32.0% (130/406)
19.3% (11/57)
27.9% (140/502)
4.687a
ns
X chromosome
6.7% (27/406)
8.8% (5/57)
1.6% (8/502)
***b
Table 4. Proportion of proteins in three human testis protein categories with immunity-related functions
or encoded on the X chromosome. Numbers of proteins in each category which are involved in immune
system processes or encoded on the human X chromosome and total numbers of proteins per category are given
in parentheses; aPearson’s Chi2. ***Highlight significance at the 0.1% level; ns, nonsignificant. bp of Fisher’s exact
test. www.nature.com/scientificreports/ nonessential
sub-/infertility
lethality
Statistical test
immunity
32.0% (130/406)
19.3% (11/57)
27.9% (140/502)
4.687a
ns
X chromosome
6.7% (27/406)
8.8% (5/57)
1.6% (8/502)
***b
Table 4. Proportion of proteins in three human testis protein categories with immunity-related functions
or encoded on the X chromosome. Numbers of proteins in each category which are involved in immune
system processes or encoded on the human X chromosome and total numbers of proteins per category are given
in parentheses; aPearson’s Chi2. ***Highlight significance at the 0.1% level; ns, nonsignificant. bp of Fisher’s exact
test. nonessential
sub-/infertility
lethality
Statistical test
immunity
32.0% (130/406)
19.3% (11/57)
27.9% (140/502)
4.687a
ns
X chromosome
6.7% (27/406)
8.8% (5/57)
1.6% (8/502)
***b
Table 4. Proportion of proteins in three human testis protein categories with immunity-related functions
or encoded on the X chromosome. Numbers of proteins in each category which are involved in immune
system processes or encoded on the human X chromosome and total numbers of proteins per category are given
in parentheses; aPearson’s Chi2. ***Highlight significance at the 0.1% level; ns, nonsignificant. bp of Fisher’s exact
test. SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 www.nature.com/scientificreports/ Evolutionary constraints measured as node degree, multifunctionality, and tissue specificity
(τ) among three human testis protein groups categorized according to their essentiality. (a) Median nod (Fig. 2c) male sub-/infertility proteins probably tend to engage in more tissue- and especially testis-specific func-
tions. In combination with their relaxed evolutionary constraints, such testis- or even sperm-specific functions
might render sub-/infertility proteins prone to the impact of positive, possibly sexual selection, as described above
(see also ref. 8). In summary, indispensability and evolutionary constraints largely restrain sequence evolution of human testis
proteins potentially associated with prepubertal lethality. Nonessential testis proteins are significantly less con-
strained and should be widely unaffected by functional importance, which both may increase their evolutionary
rates. Finally, highest median dN/dS values of proteins linked to male sub- or infertility can be ascribed to the
low levels of constraints they evolve under. This relative relaxation of evolutionary constraints and the potential
involvement in reproductive functions of some members of the male sub-/infertility category might facilitate
adaptive changes in response to postmating sexual selection. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 2. Evolutionary constraints measured as node degree, multifunctionality, and tissue specificity
(τ) among three human testis protein groups categorized according to their essentiality. (a) Median node
degree of lethality proteins is significantly higher than that of male sub-/infertility and nonessential proteins. Nonessential proteins have more PPI partners than male sub-/infertility proteins, but significance is lost
after correction against multiple testing (see Materials and Methods). (b) Human testis proteins potentially
associated with prepubertal lethality are more multifunctional than nonessential and male sub-/infertility
proteins. However, only the MWU test contrasting lethality and nonessential proteins gives a significant result. (c) Among our three human testis protein categories, male sub-/infertility proteins are most tissue-specific in
their expression. Vertical bars define 95% confidence intervals calculated from 100,000 pseudo-replicates. ***,
**, and * highlight significance at the 0.1%, 1%, and 5% level, respectively. Significances are corrected against
multiple testing using Holm’s procedure (see Materials and Methods). The asterisk in parentheses indicates
significance lost after correction against multiple testing. If no asterisk is given, the result of the MWU test is
nonsignificant. Dashed lines indicate genome-wide median values of node degree, multifunctionality, and τ (see
Supplementary Materials and Methods). Figure 2. Evolutionary constraints measured as node degree, multifunctionality, and tissue specificity
(τ) among three human testis protein groups categorized according to their essentiality. (a) Median node
degree of lethality proteins is significantly higher than that of male sub-/infertility and nonessential proteins. Nonessential proteins have more PPI partners than male sub-/infertility proteins, but significance is lost
after correction against multiple testing (see Materials and Methods). (b) Human testis proteins potentially
associated with prepubertal lethality are more multifunctional than nonessential and male sub-/infertility
proteins. However, only the MWU test contrasting lethality and nonessential proteins gives a significant result. (c) Among our three human testis protein categories, male sub-/infertility proteins are most tissue-specific in
their expression. Vertical bars define 95% confidence intervals calculated from 100,000 pseudo-replicates. ***,
**, and * highlight significance at the 0.1%, 1%, and 5% level, respectively. Significances are corrected against
multiple testing using Holm’s procedure (see Materials and Methods). The asterisk in parentheses indicates
significance lost after correction against multiple testing. If no asterisk is given, the result of the MWU test is
nonsignificant. Dashed lines indicate genome-wide median values of node degree, multifunctionality, and τ (see
Supplementary Materials and Methods). Figure 2. www.nature.com/scientificreports/ Thus, in addition to their higher dispensability, lower levels of evolutionary constraints might have
further reduced purifying selection in sequence evolution of nonessential proteins. p
y
g
q
p
Median node degree and numbers of biological processes per protein of the (in terms of dN/dS) most rap-
idly evolving male sub-/infertility category lay below the respective levels in lethality and nonessential proteins,
though only the difference regarding node degree in comparison with lethality proteins was significant after
correction against multiple testing (Fig. 2a and b). But with respect to their higher tissue specificity, male sub-/
infertility proteins differed significantly from both lethality and nonessential categories (Fig. 2c), reflecting a
well-known phenomenon of higher evolutionary rates in proteins expressed with greater tissue bias (see, e.g. refs 3,79 and 80). Higher dispensability, however, should not have impacted the evolution of male sub-/infertility
compared to lethality proteins, since both categories are expected to be equally important for an individual’s fit-
ness8. Instead, the above findings suggest that relaxation of constraints contributed to the accelerated evolution
of male sub- or infertility proteins. Kim et al.27 described a preferential occurrence of adaptation in noncentral
nodes of the human PPI network. One of the explanations discussed by the authors was that more peripheral pro-
teins were less structurally constrained than more central proteins, which might make the former more suscep-
tible to positive selection27. A similar pattern might apply to our category of male sub-/infertility proteins since
they occupy rather peripheral positions in the human PPI network used herein as evidenced by overall low node
degree (Fig. 2a). Notably, their node degree was of the same level as the genome-wide median (see Supplementar
y Materials and Methods) while the median node degree of the other two groups was higher (Fig. 2a). Moreover,
due to their higher tissue specificity compared with the two remaining categories and the genome-wide median SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 6 Materials and Methods Dataset of proteins from human seminiferous tubules. Proteins expressed in the seminiferous ducts
of human testis under physiological conditions were extracted from “normal tissue” data of the Human Protein
Atlas84 (http://www.proteinatlas.org/) version 12. We chose proteins from seminiferous tubules for our analyses
as they constitute a large part of testes and are the location of spermatogenesis and spermiogenesis. For reasons
of simplicity, we call the members of our dataset “testis proteins”, though we are aware that testes consist of more
constituents. In order to ensure high quality of the data, we exclusively considered 2,986 proteins whose expres-
sion in seminiferous ducts was designated “supportive” in the Human Protein Atlas data. Most Ensembl IDs
(version 73) provided in the respective downloadable “normal tissue” data were also available in Ensembl ver-
sion 82, which we used for subsequent analyses (see below). Altogether 53 genes whose IDs were unavailable in
Ensembl version 82 due to differences between the two genome assemblies were mapped to new IDs either via
Ensembl Biomart or manually using their gene names or the Uniprot IDs specified in Ensembl version 73. To
match the respective proteins to Human Protein Atlas RNA sequencing data and expression values provided
in the supplementary data of Kryuchkova-Mostacci and Robinson-Rechavi85, we used their old IDs (Ensembl
version 73; see below). For each protein we determined the human chromosome on which the corresponding
gene is encoded using Ensembl Biomart (version 82) to distinguish between X-chromosomal and all other genes. Human protein-protein interaction (PPI) network. Node degree values for each protein were extracted
from a human PPI network. We used the network published as supplementary data of the article by Chapple et al.55,
comprising only experimentally verified binary interactions, as a starting point for our network construction. The interactions represented by this network were not confined to testes, but rather combined PPIs from diverse
tissues. The Uniprot ID mapping tool was employed to map the entry names to current Uniprot accession num-
bers (state: November 2015). Proteins which had been deleted from Uniprot since the study by Chapple et al.55
were removed from the network. If a Uniprot entry name from the dataset by Chapple et al.55 had been mapped
to another entry name, the old was replaced by the new one. Subsequently, we extracted corresponding Ensembl
Gene IDs for each protein, again using the Uniprot ID mapping tool. www.nature.com/scientificreports/ had strongly tissue-biased expression, and exhibited low connectivity and relatively reduced numbers of biolog-
ical processes. These results suggest that among the three categories studied, constraints are strongest in lethality
proteins, while most relaxed in male sub-/infertility proteins. Accelerated evolution of the latter protein category
might, moreover, partly be ascribed to impact of sexual selection. g
p
y
p
Noteworthy, we used a rather conservative approach by including only proteins whose expression in human
testis was reported to be supportive in the Human Protein Atlas (see Materials and Methods). Furthermore, inter-
actomes relying on our present knowledge about PPIs are largely incomplete81. Thus, some peripheral proteins
might have been left unconsidered in the present study. Additionally, it is possible that some proteins herein cate-
gorized as nonessential or lethal are associated with female sub- or infertility. Such proteins are expected to evolve
at similar rates as male sub- or infertility proteins8 and might consequently slightly have blurred our results. The
fact that we detected distinct patterns in our dataset despite the potential limitations of our approach evidences
the strength of the described relationships. g
p
In spite of the opposing evolutionary forces reported herein, several researchers combined proteins indispen-
sable for survival and those required for reproduction into one single essential category (see, e.g., refs 3 and 36). However, according to our results it is advisable to differentiate between sub-/infertility and lethality proteins in
future investigations on the evolution of essential proteins. g
p
Finally, our observations demonstrate that PPI network connectivity and dN/dS values may be useful tools to
identify proteins essential for male fertility. Also tissue specificity and less so multifunctionality inform about the
essentiality status of testis proteins but these measures alone are insufficient to discriminate differential essential-
ity statuses in testis proteins. This knowledge is relevant since infertility affects 10–15% of couples worldwide (see,
e.g., refs 82 and 83). Also, proteins with testis-specific expression and function might be prime targets for male
non-hormonal contraception, since side effects in other tissues than testis can largely be excluded. SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 Conclusion We showed that essentiality has a major impact on the evolution of human testis proteins. It became evident that
proteins associated with male sub- or infertility and those potentially related to the risk of prepubertal death dis-
play different patterns regarding their evolutionary conservation, network connectivity, multifunctionality, and
tissue-specificity. While proteins linked to prepubertal death were strongly conserved, more widely expressed,
highly connected and multifunctional, the category associated with male sub- or infertility evolved more rapidly, SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 7 www.nature.com/scientificreports/ dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) Identifying potential associations of proteins with human lethality and male sub- or infertility. The use of targeted KO mutants for assessing gene essentiality has been applied before (see, e.g., refs 34 and 52). We identified murine 1-to-1 orthologues of the human genes incorporated in this study and subsequently
matched them with their MGI (Mouse Genome Informatics) IDs via Ensembl Biomart (version 82). All informa-
tion concerning mouse KOs used in this study relied on files downloaded from MGI88 (http://www.informatics. jax.org/) on August 19th 2015 (MGI version 5.22). Known phenotypic alleles generated by homologous recombination (allele type “Targeted”) were extracted
from the file MGI_PhenotypicAllele.rpt. We exclusively considered alleles whose allele attributes contained the
term “Null/knockout” and which affected single genes. To ensure the latter, we solely included alleles with a single
MGI Marker Accession ID in MGI_PhenoGenoMP.rpt. Furthermore, if more than one gene name was specified
in an allele symbol (as indicated by a forward slash) we checked if in fact more than one gene was targeted and
excluded alleles from categorization in such cases. To classify genes we accepted homozygous or – in case of
X-chromosomal genes in male mice – hemizygous targeted null mutations.i If an MP (Mammalian Phenotype) ID was used for classification with all its subterms, we downloaded these
subterm IDs from MouseMine89 (state: September 2015; http://www.mousemine.org/mousemine/begin.do). Single MP IDs were extracted from the Mammalian Phenotype Ontology at MGI (http://www.informatics.jax. org/searches/MP_form.shtml). A list of all MP IDs used is provided in the supplementary data (Tables S5 and S6). g
y
Lethality genes were associated with any of the MP IDs subsumed under “preweaning lethality” (MP:0010770),
or “lethality at weaning” (MP:0008569). These MP IDs are assigned to alleles which decrease viability so that
lower than Mendelian ratios of individuals with homo- or hemizygous null mutations appear at some time point
from their prenatal period up to three to four weeks of age. They are thus linked to death before puberty, which
initiates in slightly older mice (see http://www.informatics.jax.org/mgihome/other/mouse_facts1.shtml90). Genes
with alleles designated with “complete lethality” (MP:0011400), “partial lethality” (MP:0010831), “decreased sur-
vivor rate” (MP:0008770), or “abnormal survival” (MP:0010769) – all as single MP IDs without subterms – were
integrated into the lethality category if these null mutations resulted in death of some or all individuals aged up
to four weeks leading to the appearance of lower than Mendelian ratios, as derived from the literature cited in
MGI_PhenoGenoMP.rpt. SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 www.nature.com/scientificreports/ testis proteins, we left them in the network as single proteins with their original Uniprot entry names, but without
related Ensembl IDs. The final network comprised 12,144 nodes (proteins) and 71,765 edges (interactions). hi
p
(p
)
g
(
)
Although the analyses presented within this article are based on the human PPI network which was built as
described above, we analyzed two additional interactomes, for which results are given in the supplementary data. In one alternative network, CSH_HUMAN and HSP71_HUMAN were split into the proteins CSH1_HUMAN
and CSH2_HUMAN as well as HS71A_HUMAN and HS71B_HUMAN, respectively, each adopting all interac-
tions of the original protein. Thereby, B1A4H9_HUMAN, which represented the TrEMBL entry version of CSH2,
was deleted from the network and its interactions were transferred to the Swiss-Prot entry CSH2_HUMAN (over-
all: 12,145 nodes, 71,842 edges). The second alternative network was an adapted version of the original interac-
tome by Chapple et al.55, with only the deletions and changes of Uniprot accession numbers since its publication
incorporated and each, CSH_HUMAN and HSP71_HUMAN, kept as single protein (overall: 12,595 nodes,
73,367 edges). ,
g )
We employed Cytoscape86 version 2.8.3 to edit the network and remove duplicated edges as well as
self-interactions and used the plugin NetworkAnalyzer87 to extract node degree values. Ensembl Gene IDs cor-
responding to proteins expressed in human seminiferous ducts according to Human Protein Atlas version 12
(see above) were mapped to the Ensembl Gene IDs representing the Uniprot IDs from our PPI network. If an
Ensembl Gene ID was linked to more than one Swiss-Prot ID in the network, we chose the one with highest node
degree for analysis and for extraction of gene ontology (GO) annotations (see below); if an Ensembl Gene ID
corresponding to a protein from our testis dataset was not found to be linked to any Uniprot ID in the network,
we excluded it from further analyses. Materials and Methods If this procedure failed, we obtained their
Ensembl Gene IDs via Ensembl Biomart version 82 using their Swiss-Prot/TrEMBL accession numbers or their
associated gene names if provided. In some cases, gene names first had to be assigned to their current gene
symbol or its synonyms via HGNC (HUGO Gene Nomenclature Committee; http://www.genenames.org/). As
we aimed to solely include functional PPIs, we ignored all proteins whose genes represented a biotype other
than “protein_coding” according to Ensembl. Thus, we excluded, inter alia, pseudogenes, antisense-genes, but
also immunoglobulin und T-cell receptor genes as they constitute gene segments rather than protein-coding
genes in a strict sense. Proteins with given gene names which could not be related to their Ensembl Gene IDs or
were denoted not to be protein-coding in Ensembl were left in the network if they were a “protein-coding gene”
according to HGNC. If one Uniprot accession number had several Ensembl Gene IDs on the primary assembly
of the human genome, it was also left in the network, but its Ensembl IDs were ignored in further analyses; if it
had several Ensembl Gene IDs, but only one on the primary assembly, all others which were not assigned to the
primary assembly were discarded. Two further proteins (LC7L2_HUMAN, STBD1_HUMAN) were kept without
Ensembl Gene IDs because they had been assigned to readthrough proteins via Ensembl Biomart version 82. Uniprot accession numbers which could not be matched to Ensembl Gene IDs, had no given or identifiable gene
name, or were not coding for a functional protein were deleted from the network. If one or more TrEMBL IDs
corresponded to an Ensembl Gene ID which was also assigned to a Swiss-Prot ID, the TrEMBL accession num-
bers were deleted and their interactions inherited by the Swiss-Prot protein. However, mapping of one Ensembl
Gene ID to two or more Swiss-Prot accession numbers was accepted (see below). Additionally, since the compi-
lation of the network data by Chapple et al.55 the Swiss-Prot entry names CSH_HUMAN and HSP71_HUMAN
each had been demerged into two proteins. As neither of these proteins was contained in our dataset of human 8 SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 www.nature.com/scientificreports/ dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) The selection of lethality phenotypes (see supplementary data, Table S5) agreed largely
with that in ref. 4. If the homo- or hemizygous targeted KO of a gene resulted in male in- or subfertility (MP:0001925 or
MP:0001922), it was categorized as male sub-/infertility gene. Thereby, we accepted all causes of male in-/subfertil-
ity (see below). If quoted, these alleles were checked in the original literature specified in MGI_PhenoGenoMP.rpt. Genes for which targeted null mutations were reported to cause other male reproductive abnormalities – for
instance in spermatogenesis, male reproductive system morphology or physiology – were also included in the
group of male sub-/infertility genes if inspection of the literature given in MGI files confirmed male sub-/infer-
tility or noticeable reduction of male fertility in terms of pregnancy frequencies (for all MP IDs used, see supple-
mentary data, Table S6). For instance, this was the case in mice with arrest of male meiosis before its completion
(e.g., Brdt91) or sex reversal (e.g., Ar, see, e.g., ref. 92). Moreover, the human orthologue of the protein encoded by
murine Zbtb16 was included in the male sub-/infertility group although male KO mice were neither azoospermic
nor described as infertile; yet, Costoya et al.93 stated that the low numbers of viable sperm from these mice made
in vitro fertilization impossible, indicating serious fertility dysfunction. Applying the outlined criteria, KOs of
murine orthologues of most proteins in the male sub-/infertility category led to sterility or fertility issues due
to defects manifesting in testis or sperm. However, two members of this category were linked to male sub- or
infertility in mice due to erectile (Stam94) or ejaculatory (Etv4 or Pea395) dysfunction. The latter protein was also
part of the fertility sample studied by Torgerson et al.8. Despite different underlying causes of male reproductive
disturbances, all members of the male sub-/infertility category were essential for full fertility in male mice. If
the available null mutations of a gene were exclusively associated with fertility issues via “decreased litter size”
(MP:0001935) and this phenotype occurred in homozygous couples or could possibly be attributed to male KO
mutants, the gene was excluded from further analyses due to the problematical transferability of this phenotype SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 9 www.nature.com/scientificreports/ to human reproduction. Generally, we removed genes from the dataset if their ablation only resulted in repro-
ductive abnormalities in homozygous couples, while mice were basically fertile. dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) For one gene excluded due to such double essentiality, XRCC5, description of
reduced litter size was not found in the article quoted in MGI files100, but was instead confirmed via the original
publication101, which was cited in Henrie et al.100. All protein-coding genes for which phenotypic homo- or hemizygous KO alleles were available in the used
MGI data, but which were neither associated with prepubertal lethality nor with male sub- or infertility as
described above and which were not excluded from the dataset due to the aforementioned reasons, were catego-
rized as nonessential. Assessing selection and tissue specificity. In order to determine the extent and direction of selection
acting on each protein, we extracted dN/dS estimates from ENSEMBL version 82, which had been calculated
using CodeML as implemented in the PAML package102. We exclusively collected dN/dS values derived from
1-to-1 orthologues of human (Homo sapiens; genome assembly GRCh38.p3) and mouse (Mus musculus; genome
assembly GRCm38.p4). Since we considered only dN/dS estimates reported on the orthologues view pages of the
genes included, we avoided values potentially biased by saturation of dS, which are masked out on these pages
(http://sep2015.archive.ensembl.org/info/genome/compara/homology_method.html). For all proteins for which
dN/dS values could be extracted from their Ensembl pages, we additionally derived dN and dS values via Ensembl
Biomart (version 82).ii We investigated expression specificity of each protein using the tissue specificity index τ. Values of τ vary
between 0 for genes expressed at similar levels in all examined tissues and 1 for genes expressed in only one
tissue20. Following Kaiser et al.103 τ was based on FPKM (fragments per kilobase of transcript per million frag-
ments mapped) values which we downloaded from Human Protein Atlas version 12104. Excluding data referring
to three female-specific tissues (ovary, placenta, and uterus), we used FPKM values from altogether 24 tissues
to calculate τ. In supplementary data (Tables S2–S4, Fig. S1), we also report analyses including this variable in
which τ is based on all 27 tissues from Human Protein Atlas version 12 and with τ extracted from supplementary
data by Kryuchkova-Mostacci and Robinson-Rechavi85, which was calculated with the same data, but different
methodology. In analyses including the latter values of τ, one gene RECQL4, a member of the lethality category,
was missing from the dataset since its tissue specificity index had not been calculated by Kryuchkova-Mostacci
and Robinson-Rechavi85. Gene ontology (GO) categorization. dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) Moreover, genes were eliminated
from the dataset if they were associated with any of the male reproduction-related phenotype IDs used (see
Supplementary Table S6), but males were described as fertile in the quoted literature or the given information
were insufficient to evaluate fertility. Thereby, we also excluded genes associated with phenotypes potentially
increasing fertility, such as elevated sperm counts or enlarged testes, to avoid that the nonessential or lethality
(see below) categories contain genes linked to any male reproductive abnormalities. We furthermore left genes
with KO phenotypes only emerging upon manipulation of additional factors such as food composition out of the
final dataset. Three more proteins were removed due to the following reasons: First, NRIP1 was excluded since
male mice with a null mutation of this gene were fertile, but produced fewer homozygous offspring than expected,
which questions the viability assumed for these null mutants96. Second, Cesari et al.97 reported that the infertility
of hemizygous male Elk1-KO mice was probably due to aberrant expression of the HygTk fusion gene which had
been used to replace the coding sequence. Thus, we left ELK1 unconsidered in statistical analyses. Third, male
mice lacking functional Rad18 protein initially exhibited normal fertility comparable to wild-type littermates,
which, however, was reduced at 12 months of age98. Although fertility issues in men with advanced age are indeed
an important factor in andrology (see, e.g., ref. 99), we removed RAD18 from our dataset because mice younger
than 12 months appeared fertile. If a gene was connected to MP IDs potentially applying to both sexes (such as the
single MP ID “infertility” (MP:0001924)), but was found to be related to female-specific reproductive phenotypes
in the cited articles, it was classified as non-associated with male sub- or infertility, thus being nonessential or
lethal (see below). The same applies to female-specific subterms of “abnormal sex determination” (MP:0002210;
see also Supplementary Table S6). pp
y
Finally, we excluded genes from analyses which were related to both prepubertal death and male reproductive
anomalies, also if these were evoked by different alleles. Thereby, any of the reproductive phenotypes listed in
Supplementary Table S6 including “decreased litter size” was taken into account, unless they exclusively described
female abnormalities (see above). dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) The level of multifunctionality was defined as the number of biolog-
ical processes in which a protein is involved in21. To infer protein-specific values, we mapped biological process
annotations from the human GOA (Gene Ontology Annotation) file (state: November 9th 2015; http://www. ebi.ac.uk/GOA/downloads) to GOSlim generic terms using map2slim. Numbers of nonredundant biological
processes per protein were counted, thereby ignoring the term “biological process” (GO:0008150) if combined
with the evidence code “ND”, as it indicates unavailability of information (https://www.ebi.ac.uk/QuickGO/
GTerm?id=GO:0008150). Proteins involved in immune system processes (GO:0002376) were also identified
from these GOSlim annotations. Statistical analyses. All statistical analyses were conducted in SPSS version 22 (IBM) unless stated other-
wise. Analyses were based exclusively on proteins with all variables available and assigned to one of three catego-
ries according to their essentiality, i.e. lethality, male sub-/infertility, or nonessential proteins (see above). Overall,
965 proteins were considered. We performed Spearman’s rank correlations (two-tailed) between each possible
pair of the following four variables to study potential relationships among them: dN/dS, node degree, multi-
functionality, and tissue specificity (τ). In order to further disentangle the contributory role of single variables in
sequence evolution of the sampled proteins, we conducted pairwise partial rank correlations (two-tailed) between SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 10 www.nature.com/scientificreports/ dN/dS estimates and the three other variables in Matlab R2015b, controlling for the remaining two variables. Potential differences regarding the mentioned four variables as well as dN and dS were investigated among the
three protein categories applying Kruskal-Wallis tests. We conducted post-hoc MWU tests (two-tailed) between
each pair of protein categories for each variable. Furthermore, MWU tests were employed to compare dN/dS,
dN, and dS values of X-chromosomal or immunity-related proteins with all other members of the dataset in each
case. Using Chi2 tests (two-tailed) we tested for differential distribution of X-chromosomal or immunity proteins
among our three testis protein categories. If the result of the Chi2 test was significant, we conducted pairwise
post-hoc Chi2 tests (two-tailed) between each pair of the three testis protein categories. Fisher’s exact test was
utilized if the expected values in any of the cells of the contingency table were below 5. To account for problems
of multiplicity in post-hoc tests we applied Holm’s procedure. Furthermore, p-values of correlations including
dN/dS were calculated for both zero-order and partial correlations and were thus also adjusted with Holm’s pro-
cedure. dentifying potential associations of proteins with human lethality and male sub- or infertility
Th
f
d
f
l
h
b
l d b f
(
f
d
) Finally, all analyses including tissue specificity (τ) and node degree were conducted repeatedly because
we additionally calculated these variables by alternative approaches (see above). Therefore, we adjusted their
p-values according to the total number of similar tests performed, also taking into account the aforementioned
multiplicity issues. 95% confidence intervals of medians were calculated with a bootstrap algorithm building
100,000 pseudo-replicates. References Evolution in the fast lane: rapidly evolving sex-rel 13. Haerty, W. et al. Evolution in the fast lane: rapidly evolving sex-related genes in Drosophila. Genetics 177, 1321–1335 (2007). 13. Haerty, W. et al. Evolution in the fast lane: rapidly evolving sex related genes in Drosophila. Genetics 177, 1321 1335 (2007). 14. Turner, L. M., Chuong, E. B. & Hoekstra, H. E. Comparative analysis of testis protein evolution in rodents. Genetics 179, 2075–
(2008).h 15. Swanson, W. J. & Vacquier, V. D. The rapid evolution of reproductive proteins. Nat Rev Genet 3, 137–144 (2002). 5. Swanson, W. J. & Vacquier, V. D. The rapid evolution of reprodu h
16. Dorus, S. et al. Genomic and functional evolution of the Drosophila melanogaster sperm proteome. Nat Genet 38, 1440
(2006). 17. Dean, M. D. et al. Proteomics and comparative genomic investigations reveal heterogeneity in evolutionary rate of male
reproductive proteins in mice (Mus domesticus). Mol Biol Evol 26, 1733–1743 (2009). 18. Vicens, A., Lüke, L. & Roldan, E. R. S. Proteins involved in motility and sperm-egg interaction evolve more rapidly in mouse
spermatozoa. PLoS One 9, e91302 (2014). 19. Schumacher, J., Rosenkranz, D. & Herlyn, H. Mating systems and protein-protein interactions determine evolutionary rates o
primate sperm proteins. Proc Biol Sci 281, 20132607 (2014).ii 20. Yanai, I. et al. Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification. Bioinformatics 21, 650–659 (2005).fhf f
21. Salathé, M., Ackermann, M. & Bonhoeffer, S. The effect of multifunctionality on the rate of evolution in yeast. Mol Biol Evo
721–722 (2006). (
)
22. He, X. & Zhang, J. Toward a molecular understanding of pleiotropy. Genetics 173, 1885–1891 (2006). 23. Su, Z., Zeng, Y. & Gu, X. A preliminary analysis of gene pleiotropy estimated from protein sequences. J Exp Zool B Mol Dev Evol
314, 115–122 (2010). 24. Mank, J. E., Hultin-Rosenberg, L., Zwahlen, M. & Ellegren, H. Pleiotropic constraint hampers the resolution of sexual antagonism
in vertebrate gene expression. Am Nat 171, 35–43 (2008).if g
p
25. Papakostas, S. et al. Gene pleiotropy constrains gene expression changes in fish adapted to different thermal conditions. Na
Commun 5, 4071 (2014). 26. Fraser, H. B., Hirsh, A. E., Steinmetz, L. M., Scharfe, C. & Feldman, M. W. Evolutionary rate in the protein interaction network
Science 296, 750–752 (2002). 27. Kim, P. M., Korbel, J. O. & Gerstein, M. B. References 2. Hurst, L. D. & Smith, N. G. Do essential genes evolve slowly? Curr Biol 9, 747–750 (1999). 3. Liao, B.-Y., Scott, N. M. & Zhang, J. Impacts of gene essentiality, expression pattern, and gene compactness on the evolutionary rate
of mammalian proteins. Mol Biol Evol 23, 2072–2080 (2006). 4. Georgi, B., Voight, B. F. & Bućan, M. From mouse to human: evolutionary genomics analysis of human orthologs of essential genes
PLoS Genet 9, e1003484 (2013).if 5. Yang, J., Gu, Z. & Li, W.-H. Rate of protein evolution versus fitness effect of gene deletion. Mol Biol Evol 20, 772–774 (2003). 6. Wall, D. P. et al. Functional genomic analysis of the rates of protein evolution. Proc Natl Acad Sci USA 102, 5483–5488 (2005). g, J ,
,
,
R
pif
g
,
(
6. Wall, D. P. et al. Functional genomic analysis of the rates of protein evolution. Proc Natl Acad Sci USA 102, 5483–5488 (2 g
y
7. Zhang, J. & He, X. Significant impact of protein dispensability on the instantaneous rate of protein evolution. Mol Biol Evol 22
1147–1155 (2005).i 8. Torgerson, D. G., Whitty, B. R. & Singh, R. S. Sex-specific functional specialization and the evolutionary rates of essential fertility
genes. J Mol Evol 61, 650–658 (2005).h g
9. Castillo-Davis, C. I., Kondrashov, F. A., Hartl, D. L. & Kulathinal, R. J. The functional genomic distribution of protein divergence
in two animal phyla: coevolution, genomic conflict, and constraint. Genome Res 14, 802–811 (2004). l
10. Swanson, W. J., Nielsen, R. & Yang, Q. Pervasive adaptive evolution in mammalian fertilization proteins. Mol Biol Evol 20, 18–20
(2003).f 11. Wyckoff, G. J., Wang, W. & Wu, C. I. Rapid evolution of male reproductive genes in the descent of man. Nature 403, 304–309
(2000).i Swanson, W. J., Clark, A. G., Waldrip-Dail, H. M., Wolfner, M. F. & Aquadro, C. F. Evolutionary EST analysis identifies rapidly
evolving male reproductive proteins in Drosophila Proc Natl Acad Sci USA 98 7375 7379 (2001) 12. Swanson, W. J., Clark, A. G., Waldrip-Dail, H. M., Wolfner, M. F. & Aquadro, C. F. Evolutionary EST analysis identifies rapidly
evolving male reproductive proteins in Drosophila. Proc Natl Acad Sci USA 98, 7375–7379 (2001). ,
J ,
k,
,
p
,
,
,
q
,
y
evolving male reproductive proteins in Drosophila. Proc Natl Acad Sci USA 98, 7375–7379 (2001). Haerty, W. et al. www.nature.com/scientificreports/ www.nature.com/scientificreports/ 35. Liao, B.-Y. & Zhang, J. Null mutations in human and mouse orthologs frequently result in different phenotypes. Proc Natl Acad Sc
USA 105, 6987–6992 (2008). 36. Kim, J., Kim, I., Han, S. K., Bowie, J. U. & Kim, S. Network rewiring is an important mechanism of gene essentiality change. Sci Rep
2, 900 (2012). (
)
37. Johnson, M. T., Yang, H. S., Magnuson, T. & Patel, M. S. Targeted disruption of the murine dihydrolipoamide dehydrogenase gene
(Dld) results in perigastrulation lethality. Proc Natl Acad Sci USA 94, 14512–14517 (1997). p
g
y
38. Johnson, M. T. et al. Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic
development. Mol Genet Metab 74, 293–302 (2001).h p
39. Zhang, P. et al. The PERK eukaryotic initiation factor 2α kinase is required for the development of the skeletal system, postnata
growth, and the function and viability of the pancreas. Mol Cell Biol 22, 3864–3874 (2002).h 40. Yuan, L. et al. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mo
Cell 5, 73–83 (2000). 40. Yuan, L. et al. The murine SCP3 gene is required for synaptonemal complex assembly, chromosome synapsis, and male fertility. Mo
Cell 5, 73–83 (2000). 41. Aarabi, M. et al. Testicular expression of synaptonemal complex protein 3 (SYCP3) messenger ribonucleic acid in 110 patients with 41. Aarabi, M. et al. Testicular expression of synaptonemal complex protein 3 (SYCP3) messenger ribonucleic acid in 110 patients with
nonobstructive azoospermia. Fertil Steril 86, 325–331 (2006). p
42. Miyamoto, T. et al. Azoospermia in patients heterozygous for a mutation in SYCP3. Lancet 362, 1714–1719 (2003). 2. Miyamoto, T. et al. Azoospermia in patients heterozygous for a m 43. Blendy, J. A., Kaestner, K. H., Weinbauer, G. F., Nieschlag, E. & Schütz, G. Severe impairment of spermatogenesis in mi
the CREM gene. Nature 380, 162–165 (1996).i g
44. Nantel, F. et al. Spermiogenesis deficiency and germ-cell apop g
44. Nantel, F. et al. Spermiogenesis deficiency and germ-cell apoptosis in CREM-mutant mice. Nature 380, 159–162 (1996).f 45. Yanagimachi, R. et al. Production of fertile offspring from genetically infertile male mice. Proc Natl Acad Sci USA 101, 169
(2004). 46. Weinbauer, G. F., Behr, R., Bergmann, M. & Nieschlag, E. www.nature.com/scientificreports/ Testicular cAMP responsive element modulator (CREM) protein is
expressed in round spermatids but is absent or reduced in men with round spermatid maturation arrest. Mol Hum Reprod 4, 9–15
(1998).i (
)
47. Steger, K. et al. Round spermatids show normal testis-specific H1t but reduced cAMP-responsive element modulator and transition
protein 1 expression in men with round-spermatid maturation arrest. J Androl 20, 747–754 (1999). 48. Hahn, M. W. & Kern, A. D. Comparative genomics of centrality and essentiality in three eukaryotic protein-interaction networks
Mol Biol Evol 22, 803–806 (2005). 49. Lemos, B., Bettencourt, B. R., Meiklejohn, C. D. & Hartl, D. L. Evolution of proteins and gene expression levels are coupled in
Drosophila and are independently associated with mRNA abundance, protein length, and number of protein-protein interactions. Mol Biol Evol 22, 1345–1354 (2005). 50. Jeong, H., Mason, S. P., Barabási, A. L. & Oltvai, Z. N. Lethality and centrality in protein networks. Nature 411, 41–42 (2001).h g
y
y
p
51. Raman, K., Damaraju, N. & Joshi, G. K. The organisational structure of protein networks: revisiting the centrality-lethality
hypothesis. Syst Synth Biol 8, 73–81 (2014). 52. Liang, H. & Li, W.-H. Gene essentiality, gene duplicability and protein connectivity in human and mouse. Trends Genet 23
375–378 (2007). 53. Promislow, D. E. L. Protein networks, pleiotropy and the evolution of senescence. Proc Biol Sci 271, 1225–1234 (2004). 54. Podder, S., Mukhopadhyay, P. & Ghosh, T. C. Multifunctionality dominantly determines the rate of human housekeep
tissue specific interacting protein evolution. Gene 439, 11–16 (2009).i pi
g p
55. Chapple, C. E. et al. Extreme multifunctional proteins identified from a human protein interaction network. Nat Commun 6, 7412
(2015). Bossi, A. & Lehner, B. Tissue specificity and the human protein int i
57. Lin, W.-H., Liu, W.-C. & Hwang, M.-J. Topological and organizational properties of the products of house-keeping and tissue
specific genes in protein-protein interaction networks. BMC Syst Biol 3, 32 (2009). i
58. Gibbs, R. A. et al. Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 4
(2004). (2004). 59. Zhang, J. & Yang, J.-R. Determinants of the rate of protein sequence evolution. Nat Rev Genet 16, 409–420 (2015). (
)
59. Zhang, J. & Yang, J.-R. Determinants of the rate of protein sequence evolution. Nat Rev Genet 16, 409–420 (2015).f 60. Chamary, J. V. & Hurst, L. D. www.nature.com/scientificreports/ Evidence for selection on synonymous mutations affecting stability of mRNA secondary structu
mammals. Genome Biol 6, R75 (2005). 61. Parmley, J. L., Chamary, J. V. & Hurst, L. D. Evidence for purifying selection against synonymous mutations in mammalian exonic
splicing enhancers. Mol Biol Evol 23, 301–309 (2006).h p
g
62. Sharp, P. M. & Li, W. H. The rate of synonymous substitution in enterobacterial genes is inversely related to codon usage bias. Mo
Biol Evol 4, 222–230 (1987). 63. Chamary, J. V., Parmley, J. L. & Hurst, L. D. Hearing silence: non-neutral evolution at synonymous sites in mammals. Nat Rev Gene
7, 98–108 (2006). 64. Torgerson, D. G. & Singh, R. S. Sex-linked mammalian sperm proteins evolve faster than autosomal ones. Mol Biol Evol 20,
1705–1709 (2003). 65. Jordan, I. K., Rogozin, I. B., Wolf, Y. I. & Koonin, E. V. Essential genes are more evolutionarily conserved than are nonessentia
genes in bacteria. Genome Res 12, 962–968 (2002).f g
66. Dorus, S., Evans, P. D., Wyckoff, G. J., Choi, S. S. & Lahn, B. T. Rate of molecular evolution of the seminal protein gene SEMG2
l
i h l
l
f f
l
i
i
N t G
t 36 1326 1329 (2004) g
66. Dorus, S., Evans, P. D., Wyckoff, G. J., Choi, S. S. & Lahn, B. T. Rate of molecular evolution
correlates with levels of female promiscuity. Nat Genet 36, 1326–1329 (2004). 66. Dorus, S., Evans, P. D., Wyckoff, G. J., Choi, S. S. & Lahn, B. T. Rate of molecular evolution of the seminal protein gene SEMG2
correlates with levels of female promiscuity. Nat Genet 36, 1326–1329 (2004). 67. Ramm, S. A., McDonald, L., Hurst, J. L., Beynon, R. J. & Stockley, P. Comparative proteomics reveals evidence for evolutionary
diversification of rodent seminal fluid and its functional significance in sperm competition. Mol Biol Evol 26, 189–198 (2009).l il
gi
p
p
68. Miki, K. et al. Targeted disruption of the Akap4 gene causes defects in sperm flagellum and motility. Dev Biol 248, 331–342 (2 l
69. Okunade, G. W. et al. Targeted ablation of plasma membrane Ca2+-ATPase (PMCA) 1 and 4 indicates a major housekeeping
function for PMCA1 and a critical role in hyperactivated sperm motility and male fertility for PMCA4. J Biol Chem 279,
33742–33750 (2004). 70. Schuh, K. et al. Plasma membrane Ca2+ ATPase 4 is required for sperm motility and male fertility. References Positive selection at the protein network periphery: evaluation in terms of structura
constraints and cellular context. Proc Natl Acad Sci USA 104, 20274–20279 (2007). 28. Worth, C. L., Gong, S. & Blundell, T. L. Structural and functional constraints in the evolution of protein families. Nat Rev Mol Cel
Biol 10, 709–720 (2009). 29. Carnahan-Craig, S. J. & Jensen-Seaman, M. I. Rates of evolution of hominoid seminal proteins are correlated with function and
expression, rather than mating system. J Mol Evol 78, 87–99 (2014). p
g y
30. Clark, N. L. & Swanson, W. J. Pervasive adaptive evolution in primate seminal proteins. PLoS Genet 1, e35 (2005). 30. Clark, N. L. & Swanson, W. J. Pervasive adaptive evolution in p p
p
p
31. Swanson, W. J., Wong, A., Wolfner, M. F. & Aquadro, C. F. Evolutionary expressed sequence tag analysis of Drosoph
reproductive tracts identifies genes subjected to positive selection. Genetics 168, 1457–1465 (2004).h reproductive tracts identifies genes subjected to positive selection. Genetics 168, 1457 1465 (2004). 32. Goh, K.-I. et al. The human disease network. Proc Natl Acad Sci USA 104, 8685–8690 (2007). i
32. Goh, K.-I. et al. The human disease network. Proc Natl Acad Sci USA 104, 8685–8690 (2007). i
32. Goh, K.-I. et al. The human disease network. Proc Natl Acad Sci USA 104, 8685–8690 (2007). i
32. Goh, K.-I. et al. The human disease network. Proc Natl Acad S h
33. Park, D., Park, J., Park, S. G., Park, T. & Choi, S. S. Analysis of human disease genes in the context of gene essentiality. Genomics 92
414–418 (2008).i 34. Dickerson, J. E., Zhu, A., Robertson, D. L. & Hentges, K. E. Defining the role of essential genes in human disease. PLoS One 6,
e27368 (2011). SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 11 Author Contributions J.S. collected and analyzed data, interpreted the results, and wrote the manuscript. H.Z. and H.H. contributed to
data interpretation and co-wrote the manuscript. All authors read and approved the final manuscript. www.nature.com/scientificreports/ Genome Biol Evol
3, 1329–1337 (2011). 103. Kaiser, V. B., Zhou, Q. & Bachtrog, D. Nonrandom gene loss from the Drosophila miranda neo-Y chromosome. Genome Biol Evol
3, 1329–1337 (2011).i 04. Fagerberg, L. et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody
based proteomics. Mol Cell Proteomics 13, 397–406 (2014). 104. Fagerberg, L. et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-
based proteomics. Mol Cell Proteomics 13, 397–406 (2014). Acknowledgementsh g
This work was supported by the German Research Foundation (DFG; HE 3487/3-1 to HH). We thank Dr. David
Rosenkranz for providing Perl scripts with which we calculated 95% confidence intervals of medians. g
This work was supported by the German Research Foundation (DFG; HE 3487/3-1 to HH). We thank Dr. David
Rosenkranz for providing Perl scripts with which we calculated 95% confidence intervals of medians g
This work was supported by the German Research Foundation (DFG; HE 3487/3-1 to HH). We thank Dr. David
R
k
f
idi
P l
i t
ith
hi h
l
l t d 95%
fid
i t
l
f
di www.nature.com/scientificreports/ www.nature.com/scientificreports/ 79. Duret, L. & Mouchiroud, D. Determinants of substitution rates in mammalian genes: expression pattern affects selection intensity
but not mutation rate. Mol Biol Evol 17, 68–74 (2000).i 79. Duret, L. & Mouchiroud, D. Determinants of substitution rates in mammalian genes: expression pattern affects selection intensity
but not mutation rate. Mol Biol Evol 17, 68–74 (2000). 80 Zhang, L & Li, W-H Mammalian housekeeping genes evolve more slowly than tissue-specific genes Mol Biol Evol 21, 236–239 80. Zhang, L. & Li, W.-H. Mammalian housekeeping genes evolve more slowly than tissue-specific genes. Mol Biol Evol 21, 236–239
(2004). (2004). 81. Menche, J. et al. Disease networks. Uncovering disease-disease relationships through the incomplete interactome. Science 347,
1257601 (2015). 82. de Kretser, D. M. Male infertility. Lancet 349, 787–790 (1997) ,
y
,
(
)
83. Jungwirth, A. et al. European Association of Urology guidelines on male infertility: the 2012 update. Eur Urol 62, 324–332 (2012). 84. Uhlén, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 4,
1920–1932 (2005). y
(
)
ungwirth, A. et al. European Association of Urology guidelines on male infertility: the 2012 update. Eur Urol 62, 324–332 (2012). Uhlén, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 4, y
(
)
83. Jungwirth, A. et al. European Association of Urology guidelines on male infertility: the 2012 update. Eur Urol 62, 324–332 (2012) 83. Jungwirth, A. et al. European Association of Urology guidelines on male infertility: the 2012 update. Eur Urol 62, 324 332 (2012). 84. Uhlén, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol Cell Proteomics 4,
1920–1932 (2005).i (
)
85. Kryuchkova-Mostacci, N. & Robinson-Rechavi, M. Tissue-specific evolution of protein coding genes in human and mouse. PLoS
One 10, e0131673 (2015).t 86. Shannon, P. et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 13
2498–2504 (2003). 87. Assenov, Y., Ramírez, F., Schelhorn, S.-E., Lengauer, T. & Albrecht, M. Computing topological parameters of biological networks. Bioinformatics 24, 282–284 (2008).h f
88. Eppig, J. T., Blake, J. A., Bult, C. J., Kadin, J. A. & Richardson, J. E. The Mouse Genome Database (MGD): facilitating mouse as a
model for human biology and disease. Nucleic Acids Res 43, D726–36 (2015). gy
89. www.nature.com/scientificreports/ J Biol Chem 279, 28220–28226
(2004). 71. Moretti, E., Scapigliati, G., Pascarelli, N. A., Baccetti, B. & Collodel, G. Localization of AKAP4 and tubulin proteins in sperm with
reduced motility. Asian J Androl 9, 641–649 (2007).h y
72. Ikawa, M. et al. The putative chaperone calmegin is required for sperm fertility. Nature 387, 607–611 (1997). 72. Ikawa, M. et al. The putative chaperone calmegin is h
73. Fard Jahromi, S. S. & Shamsir, M. S. Construction and analysis of the cell surface’s protein network for human sperm-egg
interaction. ISRN Bioinform 2013, 962760 (2013). f
74. Clark, N. L. et al. Coevolution of interacting fertilization proteins. PLoS Genet 5, e1000570 (2009). 75. Hughes, A. L. & Yeager, M. Molecular evolution of the vertebrate immune system. Bioessays 19, 777–786 (1997). 76. Schlenke, T. A. & Begun, D. J. Natural selection drives Drosop 76. Schlenke, T. A. & Begun, D. J. Natural selection drives Drosophila immune system evolution. Genetics 164, 1471–1480 (2003). nke, T. A. & Begun, D. J. Natural selection drives Drosophila immun 76. Schlenke, T. A. & Begun, D. J. Natural selection drives Drosophila immune system evolution. Genetics 164, 1471–1480 (2003). 77 Counterman B A Ortíz Barrientos D & Noor M A F Using comparative genomic data to test for fast X evolution Evolu 76. Schlenke, T. A. & Begun, D. J. Natural selection drives Drosophila immune system evolution. Genetics 164, 1471–1480 (2003). 77. Counterman, B. A., Ortíz-Barrientos, D. & Noor, M. A. F. Using comparative genomic data to test for fast-X evolution. Evolution
58 656 660 (2004) 76. Schlenke, T. A. & Begun, D. J. Natural selection drives Drosophila immune system evolution. Genetics 164, 1471–1480 (2003
77. Counterman, B. A., Ortíz-Barrientos, D. & Noor, M. A. F. Using comparative genomic data to test for fast-X evolution. Evol
58, 656–660 (2004). h
h
d d
l
f
d
h
l
h 78. Torgerson, D. G. & Singh, R. S. Enhanced adaptive evolution of sperm-expressed genes on the mammalian X chromosome. Heredity (Edinb) 96, 39–44 (2006). SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 12 www.nature.com/scientificreports/ Motenko, H., Neuhauser, S. B., O’Keefe, M. & Richardson, J. E. MouseMine: a new data warehouse for MGI. Mamm Genome 26
325–330 (2015). (
)
90. Silver, L. M. Mouse Genetics: Concepts and Applications (Oxford University Press, 1995).i Mouse Genetics: Concepts and Applications (Oxford University Pres 90. Silver, L. M. Mouse Genetics: Concepts and Applications (Oxfo p
pp
y
91. Gaucher, J. et al. Bromodomain-dependent stage-specific male genome programming by Brdt. EMBO J 31, 3809–3820 (2012). 91. Gaucher, J. et al. Bromodomain-dependent stage-specific male genome programming by gi
g
g
g y
92. De Gendt, K. et al. A Sertoli cell-selective knockout of the androgen receptor causes spermatogenic arrest in meiosis. Proc N
Acad Sci USA 101, 1327–1332 (2004). 93. Costoya, J. A. et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 36, 653–659 (2004). 94. Yamada, M. et al. Loss of hippocampal CA3 pyramidal neurons in mice lacking STAM1. Mol Cell Biol 21, 3807–3819 (2001 95. Laing, M. A. et al. Male sexual dysfunction in mice bearing targeted mutant alleles of the PEA3 ets gene. Mol Cell Bio
9337–9345 (2000).h (
)
96. White, R. et al. The nuclear receptor co-repressor nrip1 (RIP140) is essential for female fertility. Nat Med 6, 1368–1374 (2000 97. Cesari, F. et al. Mice deficient for the ets transcription factor elk-1 show normal immune responses and mildly impaired neu
gene activation. Mol Cell Biol 24, 294–305 (2004). g
98. Sun, J. et al. Rad18 is required for long-term maintenance of spermatogenesis in mouse testes. Mech Dev 126, 173–183 (2009 98. Sun, J. et al. Rad18 is required for long-term maintenance of s 99. Johnson, S. L., Dunleavy, J., Gemmell, N. J. & Nakagawa, S. Consistent age-dependent declines in human semen qual
systematic review and meta-analysis. Ageing Res Rev 19, 22–33 (2015). y
y
g
g
00. Henrie, M. S. et al. Lethality in PARP-1/Ku80 double mutant mice reveals physiological synergy during early embryogenesis. DNA
Repair (Amst) 2, 151–158 (2003). p
101. Nussenzweig, A. et al. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 382, 551–555 (1 102. Yang, Z. PAML: a program package for phylogenetic analysis by maximum likelihood. Comput Appl Biosci 13, 555–556 (1997
h
h
d
l
f
h
h l
d
h
l 103. Kaiser, V. B., Zhou, Q. & Bachtrog, D. Nonrandom gene loss from the Drosophila miranda neo-Y chromosome. Additional Information Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srepi Supplementary information accompanies this paper at http://www.nature.com/srep Competing financial interests: The authors declare no competing financial interests. 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: Schumacher, J. et al. Effects of different kinds of essentiality on sequence evolution of
human testis proteins. Sci. Rep. 7, 43534; doi: 10.1038/srep43534 (2017). How to cite this article: Schumacher, J. et al. Effects of different kinds of essentiality on sequence evolution of
human testis proteins. Sci. Rep. 7, 43534; doi: 10.1038/srep43534 (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 an
institutional affiliations. This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017 SCientifiC Reports | 7:43534 | DOI: 10.1038/srep43534 13
|
https://openalex.org/W2087582191
|
https://europepmc.org/articles/pmc4077029?pdf=render
|
English
| null |
Multifocal subdural hematomas as the presenting sign of acquired hemophilia A: a case report
|
BMC research notes
| 2,014
|
cc-by
| 2,903
|
* Correspondence: mark.burish@ucsf.edu
1Department of Neurology, University of California, San Francisco, USA
2505 Parnassus Ave, Room M-798, Box 0114, San Francisco, CA 94143-0114,
USA Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 CASE REPORT Open Access © 2014 Burish et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated. Mark J Burish1,2*, Aimee Aysenne1 and Vineeta Singh1 Mark J Burish1,2*, Aimee Aysenne1 and Vineeta Singh1 Abstract Background: Acquired hemophilia A (AHA) is a rare coagulopathy linked to a variety of etiologies including
autoimmune diseases, neoplasms, diabetes, respiratory diseases, and the post-partum state. While bleeding in AHA
is often seen in mucocutaneous or intramuscular locations, intracranial and intraspinal bleeds are exceedingly rare. Case presentation: We report an unusual case of spontaneous multifocal subdural hematomas in a 25 year old
Asian woman with lupus who presented with headache and backache, and was found to have an elevated partial
thromboplastin time (PTT) level and new diagnosis of AHA. Background: Acquired hemophilia A (AHA) is a rare coagulopathy linked to a variety of etiologies including
autoimmune diseases, neoplasms, diabetes, respiratory diseases, and the post-partum state. While bleeding in AHA
is often seen in mucocutaneous or intramuscular locations, intracranial and intraspinal bleeds are exceedingly rare. Case presentation: We report an unusual case of spontaneous multifocal subdural hematomas in a 25 year old
Asian woman with lupus who presented with headache and backache, and was found to have an elevated partial
thromboplastin time (PTT) level and new diagnosis of AHA. Conclusions: Subdural hematomas as the initial sign of AHA are all but unknown in the medical literature. We
bring this entity to the attention of the neurology community because lumbar puncture and/or conventional
angiogram are often indicated in the work-up of idiopathic multifocal subdural hematomas, but may be dangerous
in patients with AHA. Keywords: Acquired hemophilia A, Nontraumatic subdural hematoma, Intracranial hemorrhage, Secondary
headache syndrome, Lupus, Spinal cord, Critical care which mixes serial dilutions of the patient’s blood with
normal plasma [11,12]. The etiology is idiopathic in 50%
and post-partum in 10%, with the remaining 40% covering
a wide spectrum of autoimmune, allergic, neoplastic, dia-
betic, and respiratory diseases [11,13]. Treatment in part
depends on the underlying etiology of the AHA, with
pregnancy- or allergy-related AHA often responding to
steroids or cyclophosphamide and resolving spontan-
eously, tumor-related AHA often depending on treatment
of the malignancy, and autoimmune-related AHA often
requiring stronger immunosuppression and rarely resolv-
ing spontaneously [13]. Background Acquired hemophilia A (AHA) is a rare disorder (approxi-
mately 1 in 1 million persons per year) but carries a sig-
nificant mortality of 8–22% [1]. While bleeding in AHA
most commonly involves mucocutaneous or intramuscu-
lar locations, it can also involve the gastrointestinal or
genitourinary tracts [2]. Hemarthrosis, which is consid-
ered a hallmark of congenital Hemophilia, is rarely en-
countered in AHA. While intracranial hemorrhage has
been well documented in congenital hemophilia A [3-6],
there are only a limited number of publications on intra-
cranial bleeds in acquired hemophilia A [7-10], suggesting
that it is a very rare phenomenon. Diagnosis is made
based on the finding of 1) an elevated partial thrombo-
plastin time (PTT) which does not correct after 2 hours
of mixing with normal plasma (mixing study), 2) no ad-
ministration of heparin, which can be determined by a
prolonged thrombin time but normal reptilase time, 3)
negative testing for lupus anticoagulants, and 4) the pres-
ence of an inhibitor as shown on the Bethesda assay, Case presentation A 25 year old Asian woman with a history of lupus pre-
sented to the emergency room with headaches not
responding to over the counter analgesics. The head-
aches started 6 days prior, with no reported history of
trauma. She described the headaches as a gradual onset
of intermittent 10/10 sharp right-sided head pain which
lasted for several seconds only, and occurred multiple
times per day. The pain occasionally spread to the right
neck with a pulsating quality but without tinnitus. There
was no postural component, no associated migrainous Page 2 of 4 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 history significant for a sister who died of anemia of an
unknown etiology at the age of 24. Our patient’s neuro-
logic examination was unremarkable. features (including nausea, vomiting, photophobia, pho-
nophobia, osmophobia, or vision changes), and no tri-
geminal autonomic symptoms (including facial pallor,
facial flushing, lacrimation, or rhinorrhea). In addition,
she occasionally had brief lower back spasmic-type pain
with no radiation. She did not have any symptoms of co-
agulopathy including easy bleeding, GI bleeds, epistaxis,
or hemoptysis. She carried a diagnosis of lupus with dis-
coid rash and arthralgia as predominant symptoms for
which she was taking prednisone 15 mg daily and myco-
phenylate 500 mg BID (she had self-discontinued the
mycophenylate 1 week prior to admission), and a family Given the new onset of severe unilateral headaches
without clear symptoms of a primary headache syn-
drome, head imaging was performed. Brain MRI re-
vealed bilateral infracerebellar and right pre-pontine
extra-axial collections most consistent with hematomas
(Figure 1A and B), and basic laboratory studies were sig-
nificant for an isolated elevation of PTT at 74 with a
normal CBC, chemistry panel, liver panel, INR, and
urine toxicology screen. Additional imaging included a Figure 1 Diffuse spontaneous subdural hematoma associated with acquired hemophilia A. MRI showing coronal brain FLAIR sequence
(A and B), sagittal T1 sequence of the cervical spine (C) and sagittal T1 sequence of the lumbar spine (D) demonstrating multiple subdural
hematomas indicated by arrows. The patient had subdural hematomas located in the bilateral infracerebellar, cervical (C1-C7, maximal at C6-7),
and lumbar (L4-S1) regions. Figure 1 Diffuse spontaneous subdural hematoma associated with acquired hemophilia A. Case presentation MRI showing coronal brain FLAIR sequence
(A and B), sagittal T1 sequence of the cervical spine (C) and sagittal T1 sequence of the lumbar spine (D) demonstrating multiple subdural
hematomas indicated by arrows. The patient had subdural hematomas located in the bilateral infracerebellar, cervical (C1-C7, maximal at C6-7),
and lumbar (L4-S1) regions. Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Page 3 of 4 factor 7a and rituximab, with repeat imaging showing
resolving hematomas. brain MR venogram with no venous thrombosis, a CT
angiogram with no vasculitis or aneurysms, and a spinal
MRI with additional subdural hematomas in her cervical
and lumbar regions (Figure 1C and D). Work-up of her
elevated PTT revealed a clotting mixing study that failed
to correct with a 1:1 ratio to plasma (suggestive of
the presence of an inhibitor) and an undetectable factor
8 level, consistent with AHA (one month after treat-
ment the factor 8 activity level was 36% with a normal
range of 56–191%). Lupus anticoagulant testing had
previously been performed twice over 6 months, with
an initial indeterminate anti-cardiolipin IgG of 17 that
was normal at 10 on rechecking, and normal levels of
anti-cardiolipin IgM, anti-beta-2-glycoprotein IgG, anti-
beta-2-glycoprotein IgM, Russell Viper Venom Test, and
Russell Viper Venom confirmation testing. Urine preg-
nancy testing was negative and she was not post-partum. Infectious labs revealed a positive beta-D-glucan and
negative galactomannan, but a brochoalveolar lavage
that was positive only for candida and rare gram-positive
cocci and was negative for acid fast bacilli, other fungi,
and viral cultures. Other infectious testing was negative
including a nasopharyngeal swab for influenza A and B,
urine cultures, blood cultures, histoplasma antigen,
and a hepatitis panel. She was started on recombinant Conclusions Of the multiple etiologies of AHA [11,13], our patient’s
disease was most likely related to her systemic lupus ery-
thematosus. Our patient’s headache and backache de-
scription, specifically the very brief and intermittent
nature of her symptoms, is unusual for pain related to
mass lesions. One previous case report of subdural he-
matomas and AHA described more classic symptoms of
headache and drowsiness [7], but given the paucity of
cases in the literature, it is difficult to say if there is any
characteristic headache associated with AHA. On initial work-up of our patient, a variety of diagnos-
tic tests were discussed. The differential diagnosis for
non-traumatic intracranial and intraspinal subdural he-
matomas is broad, and etiologies include neoplasms
such as myelodysplastic disorders [14] or meningiomas
[15], infections such as mycotic aneurysms [16], vascular
diseases such as arteriovenous malformations [17], aneu-
rysms [18], or primary CNS vasculitis [19], and coagu-
lopathies such as idiopathic thrombocytopenic purpura
[20]. Thus a thorough investigation for non-traumatic
subdural hematoma may lead to a lumbar puncture or a Figure 2 Algorithm for the diagnosis of acquired hemophilia A. Based on the response to mixing tests and phospholipid tests, acquired
hemophilia A can be distinguished from other disorders causing an isolated elevation of aPTT. Figure 2 Algorithm for the diagnosis of acquired hemophilia A. Based on the response to mixing tests and phospholipid tests, acquired
hemophilia A can be distinguished from other disorders causing an isolated elevation of aPTT. Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Burish et al. BMC Research Notes 2014, 7:134
http://www.biomedcentral.com/1756-0500/7/134 Page 4 of 4 Page 4 of 4 conventional angiogram. The risk of bleeding after lum-
bar puncture is not known in patients with AHA, or
even for similar procedures such as epidural injections
in more common coagulopathies [21]. However the risk
of bleeding after lumbar puncture is presumably higher
for patients with untreated AHA than for the general
population. Before performing a lumbar puncture on pa-
tients who are otherwise stable, providers might consider
initiating treatment or awaiting results of the AHA test-
ing. Similarly conventional angiogram may pose un-
necessary risks in patients with AHA, and deferral or
avoidance of angiogram should also be considered, espe-
cially if other vessel imaging such as MRA or CTA is
not suggestive of vascular malformations. 3. Philipp C: The aging patient with hemophilia: complications, comorbidities,
and management issues. Conclusions Hematology Am Soc Hematol Educ Program 2010,
2010:191–196. 4. Witmer C, Presley R, Kulkarni R, Soucie JM, Manno CS, Raffini L: Associations
between intracranial haemorrhage and prescribed prophylaxis in a large
cohort of haemophilia patients in the United States. Br J Haematol 2011,
152:211–216. 5. Nakar C, Cooper DL, DiMichele D: Recombinant activated factor VII safety
and efficacy in the treatment of cranial haemorrhage in patients with
congenital haemophilia with inhibitors: an analysis of the Hemophilia
and Thrombosis Research Society Registry (2004–2008). Haemophilia
2010, 16:625–631. 6. Ljung RCR: Intracranial haemorrhage in haemophilia A and B. Br J
Haematol 2008, 140:378–384. 7. Bonnaud I, Saudeau D, de Toffol B, Autret A: Recurrence of spontaneous
subdural haematoma revealing acquired haemophilia. Eur Neurol 2003,
49:253–254. 8. Micic D, Williams EC, Medow JE: Cerebellar hemorrhage as a first
presentation of acquired hemophilia A. Neurocrit Care 2011, 15:170–174. The findings of subdural hematoma with an unex-
plained elevated PTT should prompt further work-up for
AHA with mixing studies at 0.1 and 2 hours, phospholipid
studies for lupus anticoagulant, and the Bethesda assay for
the quantification of inhibitors (Figure 2) [11,12,22]. In pa-
tients with suspected AHA with any pain complaint, such
as lower back pain in our case, imaging should be consid-
ered to look for additional bleeds and to lend stronger
support for starting immediate factor replacement ther-
apy. In the acute setting, if emergent neurosurgical evacu-
ation of these patients is required, factor 7a should be
considered for uncontrolled intra-operative bleeding. 9. Mashiko R, Yamamoto T, Sato M, Noguchi S, Matsumura A: Acquired
hemophilia first manifesting as life-threatening intracranial hemorrhage:
case report. Neurol Med Chir (Tokyo) 2009, 49:93–95. 10. Saito R, Takahashi T, Endo H, Kimura N, Kaneko U: [A case of subarachnoid
hemorrhage complicated by acquired hemophilia]. No Shinkei Geka 2009,
37:1215–1219. 11. Shetty S, Bhave M, Ghosh K: Acquired hemophilia a: diagnosis, aetiology,
clinical spectrum and treatment options. Autoimmun Rev 2011, 10:311–316. 11. Shetty S, Bhave M, Ghosh K: Acquired hemophilia a: diagnosis, aetiology,
clinical spectrum and treatment options. Autoimmun Rev 2011, 10:311–316. 12. Collins P, Baudo F, Huth-Kühne A, Ingerslev J, Kessler CM, Castellano MEM, Shima M,
St-Louis J, Lévesque H: Consensus recommendations for the diagnosis
and treatment of acquired hemophilia A. BMC Res Notes 2010, 3:161. 12. Consent 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 of this journal. 16. Barami K, Ko K: Ruptured mycotic aneurysm presenting as an
intraparenchymal hemorrhage and nonadjacent acute subdural
hematoma: case report and review of the literature. Surg Neurol 1994,
41:290–293. 17. Kominato Y, Matsui K, Hata Y, Matsui K, Kuwayama N, Ishizawa S, Takizawa
H: Acute subdural hematoma due to arteriovenous malformation
primarily in dura mater: a case report. Leg Med (Tokyo) 2004, 6:256–260. 17. Kominato Y, Matsui K, Hata Y, Matsui K, Kuwayama N, Ishizawa S, Takizawa
H: Acute subdural hematoma due to arteriovenous malformation
primarily in dura mater: a case report. Leg Med (Tokyo) 2004, 6:256–260. Conclusions Collins P, Baudo F, Huth-Kühne A, Ingerslev J, Kessler CM, Castellano MEM, Shima M
St-Louis J, Lévesque H: Consensus recommendations for the diagnosis
and treatment of acquired hemophilia A. BMC Res Notes 2010, 3:161. q
p
13. Franchini M, Gandini G, Di Paolantonio T, Mariani G: Acquired hemophilia
A: a concise review. Am J Hematol 2005, 80:55–63. 13. Franchini M, Gandini G, Di Paolantonio T, Mariani G: Acquired hemophilia
A: a concise review. Am J Hematol 2005, 80:55–63. 14. Ichimura S, Horiguchi T, Inoue S, Yoshida K: Nontraumatic acute subdural
hematoma associated with the myelodysplastic/myeloproliferative
neoplasms. J Neurosci Rural Pract 2012, 3:98–99. 14. Ichimura S, Horiguchi T, Inoue S, Yoshida K: Nontraumatic acute subdural
hematoma associated with the myelodysplastic/myeloproliferative
neoplasms. J Neurosci Rural Pract 2012, 3:98–99. 15. Da Rocha AJ, Saade N, da Silva AZ: Meningioma associated with non-traumatic
subdural hematoma: an outstanding appearance of this common intracranial
tumor. Arq Neuropsiquiatr 2013, 71:417. 15. Da Rocha AJ, Saade N, da Silva AZ: Meningioma associated with non-traumatic
subdural hematoma: an outstanding appearance of this common intracranial
tumor. Arq Neuropsiquiatr 2013, 71:417. Competing interests Competing interests
The authors (M.J. Burish, A. Aysenne, and V. Singh) declare that they have no
competing interests. y
g
y
18. Ishikawa E, Sugimoto K, Yanaka K, Ayuzawa S, Iguchi M, Moritake T,
Kobayashi E, Nose T: Interhemispheric subdural hematoma caused by a
ruptured internal carotid artery aneurysm: case report. Surg Neurol 2000,
54:82–86. 18. Ishikawa E, Sugimoto K, Yanaka K, Ayuzawa S, Iguchi M, Moritake T,
Kobayashi E, Nose T: Interhemispheric subdural hematoma caused by a
ruptured internal carotid artery aneurysm: case report. Surg Neurol 2000,
54:82–86. Authors’ contributions 19. Fu M, Omay SB, Morgan J, Kelley B, Abbed K, Bulsara KR: Primary central
nervous system vasculitis presenting as spinal subdural hematoma. World Neurosurg 2012, 78:192.E5–192.E8. 19. Fu M, Omay SB, Morgan J, Kelley B, Abbed K, Bulsara KR: Primary central
nervous system vasculitis presenting as spinal subdural hematoma. World Neurosurg 2012, 78:192.E5–192.E8. MJB contributed by drafting and revising the manuscript for content, and
analysis/interpretation of data. He also assisted in acquisition of data. AA
contributed by revising the manuscript for content, and analysis/interpretation
of data. She also assisted in acquisition of data. VS contributed by revising the
manuscript for content, and analysis/interpretation of data. She also assisted in
acquisition of data. All authors read and approved the final manuscript. 20. Lee MS, Kim WC: Intracranial hemorrhage associated with idiopathic
thrombocytopenic purpura: report of seven patients and a meta-analysis. Neurology 1998, 50:1160–1163. 20. Lee MS, Kim WC: Intracranial hemorrhage associated with idiopathic
thrombocytopenic purpura: report of seven patients and a meta-analysis. Neurology 1998, 50:1160–1163. 21. Choi S, Brull R: Neuraxial techniques in obstetric and non-obstetric patients
with common bleeding diatheses. Anesth Analg 2009, 109:648–660. Acknowledgments 22. Lossing S, Kasper K, Feinstein I: Detection of factor VIII inhibitors with the
partial thromboplastin time. Blood 1977, 49:793–797. 22. Lossing S, Kasper K, Feinstein I: Detection of factor VIII inhibitors with the
partial thromboplastin time. Blood 1977, 49:793–797. We thank our patient who was incredibly generous with her time after her
hospital discharge. doi:10.1186/1756-0500-7-134
Cite this article as: Burish et al.: Multifocal subdural hematomas as the
presenting sign of acquired hemophilia A: a case report. BMC Research
Notes 2014 7:134. Received: 2 March 2014 Accepted: 4 March 2014
Published: 8 March 2014 References 1. Collins PW, Hirsch S, Baglin TP, Dolan G, Hanley J, Makris M, Keeling DM,
Liesner R, Brown SA, Hay CRM: Acquired hemophilia A in the United
Kingdom: a 2-year national surveillance study by the United Kingdom
Haemophilia Centre Doctors’ Organisation. Blood 2007 109:1870–1877 1. Collins PW, Hirsch S, Baglin TP, Dolan G, Hanley J, Makris M, Keeling DM,
Liesner R, Brown SA, Hay CRM: Acquired hemophilia A in the United
Kingdom: a 2-year national surveillance study by the United Kingdom
Haemophilia Centre Doctors’ Organisation. Blood 2007, 109:1870–1877. p
g
2. Yee TT, Taher A, Pasi KJ, Lee CA: A survey of patients with acquired
haemophilia in a haemophilia centre over a 28-year period. Clin Lab
Haematol 2000, 22:275–278.
|
https://openalex.org/W1987370905
|
https://www.phcogres.com/sites/default/files/PharmacognRes-5-2-71.pdf
|
English
| null |
Immune-stimulatory and anti-inflammatory activities of Curcuma longa extract and its polysaccharide fraction
|
Pharmacognosy research
| 2,013
|
cc-by
| 7,811
|
Published: 15-04-2013 Revised: 05‑01‑2013 Submitted: 09‑12‑2012 Submitted: 09‑12‑2012 Access this article online
Website:
www.phcogres.com
DOI: 10.4103/0974-8490.110527
Quick Response Code: Background: While curcuminoids have been reported to possess diverse biological activities,
the anti‑inflammatory activity of polar extracts (devoid of curcuminoids) of Curcuma longa
(C. longa) has seldom been studied. In this study, we have investigated immune‑stimulatory
and anti‑inflammatory activities of an aqueous based extract of C. longa (NR‑INF‑02)
and its fractions in presence and absence of mitogens. Materials and Methods: Effects
of NR‑INF‑02 (TurmacinTM, Natural Remedies Pvt. Ltd., Bangalore, India) on proliferation,
nitric oxide (NO), monocyte chemotactic protein‑1 (MCP‑1), interleukins (ILs) and
prostaglandin (PGE2) levels of mouse splenocytes and mouse macrophage (RAW264.7) cells
were determined. Results: NR‑INF‑02 increased splenocytes number in presence and absence
of lipopolysaccharide (LPS) or concanavalin A. Treatment of NR‑INF‑02 showed a significant
increase of NO, IL‑2, IL‑6, IL‑10, IL‑12, interferon (IFN) gamma, tumor necrosis factor (TNF)
alpha and MCP‑1 production in unstimulated mouse splenocytes and mouse macrophages. Interestingly, NR‑INF‑02 showed potent inhibitory effect towards release of PGE2 and IL‑12
levels in LPS stimulated mouse splenocytes. Further, NR‑INF‑02 was fractionated into
polysaccharide fraction (F1) and mother liquor (F2) to study their immune‑modulatory effects. F1 was found to be more potent than F2 toward inhibiting PGE2 and IL‑12 in LPS stimulated
splenocytes. Conclusion: Present findings revealed the novel anti‑inflammatory property of
NR‑INF‑02 and its polysaccharide fraction by inhibiting the secretion of IL‑12 and PGE2 in vitro. Access this article online
Website:
www.phcogres.com
DOI: 10.4103/0974-8490.110527
Quick Response Code: Key words: Curcuma longa, immunomodulation, inflammation, pain, polysaccharides,
T
i
TM Key words: Curcuma longa, immunomodulation, inflammation, pain, polysaccharides,
TurmacinTM O R I G I N A L A R T I C L E O R I G I N A L A R T I C L E P H C O G R E S . Chinampudur V. Chandrasekaran, Kannan Sundarajan, Jothie R. Edwin, Giligar M. Gururaja,
Deepak Mundkinajeddu, Amit Agarwal Published: 15-04-2013 Submitted: 09‑12‑2012
Revised: 05‑01‑2013 INTRODUCTION working through multiple mechanisms viz., suppression
of the activation of nuclear factor (NF)‑kappa B,
inhibition of cyclooxygenase (COX)‑2, down‑regulation
of the expression of cell proliferation, anti‑apoptotic, and
metastatic gene products.[2‑4] Curcuminoids have also been
demonstrated to modulate the proliferation and cellular
response of various immune cell types, such as T cells,
B cells, macrophages, neutrophils, natural killer NK cells
and dendritic cells.[5‑7] Curcuma longa (C. longa) Linn. commonly known as turmeric,
is a perennial plant belonging to the family Zingiberaceae. It is a common ingredient in many health supplements in
Asia, being used in various therapeutic applications such
as blood purifying, wound healing, and inflammatory
disorders and holds a prominent position in traditional
Indian medicinal system.[1] In addition to curcuminoids, turmerones and other
sesquiterpenoids from essential oil of C. longa have
been shown to possess various biological activities viz.,
anti‑inflammatory, antioxidant, and chemo‑preventive
properties.[8,9] Curcuminoids (mixture of curcumin, demethoxycurcumin,
and bisdemethoxycurcumin) are considered as key active
constituents of C. longa and are reported to possess several
biological activities. Numerous lines of evidence suggested,
that curcuminoids are potent anti‑inflammatory agents Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2
Address for correspondence:
Dr. Chandrasekaran CV, Plot No. 5B, Veerasandra Indl Area,
19th K.M. Stone, Hosur Road, Bangalore ‑ 560 100, Karnataka,
India. E‑mail: cvc@naturalremedy.com Among the polar constituents, polysaccharides viz.,
ukonan A, B, C, and D from rhizome of C. longa
were shown to have the activity on reticuloendothelial
system.[10‑14] Polysaccharides from medicinal plants have Address for correspondence:
Dr. Chandrasekaran CV, Plot No. 5B, Veerasandra Indl Area,
19th K.M. Stone, Hosur Road, Bangalore ‑ 560 100, Karnataka,
India. E‑mail: cvc@naturalremedy.com Address for correspondence:
Dr. Chandrasekaran CV, Plot No. 5B, Veerasandra Indl Area,
19th K.M. Stone, Hosur Road, Bangalore ‑ 560 100, Karnataka,
India. E‑mail: cvc@naturalremedy.com Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 71 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM attracted attention recently in the enhancement of host
defense mechanisms. Several polysaccharides isolated
from different medicinal plants, such as Lentinus edodes,
Schizophyllium commune, Angelica gigas, Phellinus linteus,
Platycodon grandiflorum have been shown to possess
immune‑stimulatory activity.[15‑17] Polysaccharides and
polysaccharide containing plant products have been
demonstrated for immune‑modulatory activity in
various pre‑clinical and human clinical models after
oral administration. Characterization of polysaccharides 13C Nuclear magnetic resonance spectrum (200 MHz,
Bruker, Switzerland) of F1 confirmed the presence of
polysaccharides as indicated by oxygen bearing carbons
at 70‑80 ppm (CHOH), 60‑62 ppm (CH2OH); anomeric
carbons in the range of 95‑111 ppm; rhamnosyl methyl
carbon (20‑22 ppm) and the carboxylic carbons at
170‑175 ppm [Figure 1]. MATERIALS AND METHODS Source of materials Source of materials
Lipopolysaccharide (LPS), concanavalin A (Con A),
3‑(4,5‑dimethyl‑2‑thiazolyl)‑2,5‑diphenyl‑2H tetrazolium
bromide (MTT), 1400 W dihydrochloride, dexamethasone,
beta‑mercaptoethanol (Beta‑ME), histopaque 1077
and Tween‑20 were purchased from Sigma‑Aldrich,
Inc. (St. Louis, MO, USA). Roswell Park Memorial
Institute‑1640 (RPMI‑1640) and Dulbecco’s modified Eagle’s
medium were supplied by Gibco Life Technologies (Grand
Island, NY, USA). Fetal bovine serum (FBS) was purchased
from Hyclone Laboratories, Inc. (Logan, UT, USA). Preparation of splenocyte culture Swiss albino male mice 8‑10 weeks old (25‑30 g body weight)
were taken from our experimental animal house (Natural
Remedies Pvt. Ltd., Bangalore). Mice were killed by cervical
dislocation and the spleen was removed from animals
under aseptic conditions. This work was approved by
Institutional Animal Ethics Committee (IAEC) held on
12.03.2011 (Approval Number: IAEC/CA/01/03.11). INTRODUCTION In addition, polysaccharides have
been shown to be bioavailable in various in vivo and
human clinical models after oral administration.[18,19]
Additionally, polysaccharides containing C. longa extracts
have been shown to have anti‑diabetic, anti‑tumor,
anti‑depressant, anti‑oxidant, anti‑microbial,
anti‑fertility, immune‑modulatory, and hepato protective
properties.[9,20‑26] at National Institute of Science Communication and
Information Resources. A voucher specimen (No. 653)
was deposited in our herbarium. Preparation of NR‑INF‑02 Coarsely powdered rhizomes of C. longa were subjected to
steam distillation and the oil was separated and collected. The rhizomes were further extracted by refluxing with
water in a commercial extraction facility. The liquid water
extract was concentrated by distillation under vacuum and
the resultant concentrated liquid was spray dried to obtain a
free flowing powder. NR‑INF‑02 was prepared by blending
the spray dried water extract and the turmeric oil at a
proportion of 99:1 (w/w) followed by sieving. NR‑INF‑02
was manufactured and registered as TurmacinTM by
Natural Remedies Pvt. Ltd., Bangalore, India. Content
of polysaccharides in NR‑INF‑02 were determined as
12.6% w/w by high‑performance liquid chromatography
as per the method described by Gomis et al.[28] Content of
curcuminoids in NR‑INF‑02 were found to be negligible
as determined by a modified USP method.[29] While anti‑inflammatory and immune‑modulatory activities
of curcuminoids have been studied extensively, very limited
reports on polar extracts (containing polysaccharides) of
C. longa are available. In this context, we developed an
aqueous based extract of C. longa, devoid of curcuminoids,
standardized to polysaccharides (NR‑INF‑02) and
evaluated for inflammation related health conditions. Preparation of polysaccharide fraction from NR‑INF‑02
Polysaccharide fraction was isolated from NR‑INF‑02 by
precipitation with alcohol as described earlier.[10] Briefly, the
extract was dissolved in water and added to 5 volumes of
ethanol. The above contents were centrifuged at 2000 rpm
for 20 min. The supernatant obtained was concentrated
under vacuum to get mother liquor (F2). The precipitate
obtained after centrifugation was stirred with 5 volumes of
ethanol at room temperature for 10 min and filtered. The
retentate obtained after filtration was dried under vacuum
at <70°C to obtain polysaccharide fraction (F1). A randomized placebo controlled study on
120 patients (37 males and 83 females) with primary
osteoarthritis demonstrated the efficacy of NR‑INF‑02
and it could possibly be a safer and effective option
for the management of primary painful knee and joint
pain.[27] In the present study, we have evaluated the potential
anti‑inflammatory and immune‑stimulatory activities of
NR‑INF‑02 in presence and absence of mitogens in cell
based systems (in vitro). Statistical analysis y
Pooled data are represented as mean ± standard
deviation (S.D) from three independent experiments with
three replicates in each experiment. Statistical significance
between groups was arrived using one‑way analysis of
variance (ANOVA) followed by Bonferroni’s multiple
comparisons using GraphPad Prism 5.0 (GraphPad
Software Inc., San Diego CA). In all data analysis, P < 0.05
was considered as significant. Dose response curves were
constructed manually to determine EC50, the effective
concentration of test material causing 50% inhibition or
activation was estimated from these dose response curves
by fixing untreated control, mitogen (LPS) values as 0 and
100%, respectively. (a) significant (P < 0.05) difference
between zero control and NR‑INF‑02 treated cells;
(b) significant (P < 0.05) difference between LPS control
and NR‑INF‑02 or reference standard (if applicable)
+ LPS treated cells; (c) significant (P < 0.05) difference
between NR‑INF‑02 treated cells and NR‑INF‑02 + LPS
treated cells; (d) significant (P < 0.05) difference between
Con A control and NR‑INF‑02 + Con A treated cells;
(e) significant (P < 0.05) difference between NR‑INF‑02
treated cells and NR‑INF‑02 + Con A treated cells. Lymphocyte proliferation assay Splenocytes were seeded in 48‑well plates containing
growth media and beta‑ME (50 μM). Cells were treated
with NR‑INF‑02 (0.8‑500 µg/mL) at the indicated
concentrations with or without mitogens (LPS [5 µg/mL] or
Con A [2.5 µg/mL]) and further incubated for 48 h at 37°C
under 5% CO2 humidified atmosphere. Post‑incubation,
the plates were centrifuged and cell culture supernatants
were collected for quantification of cytokines (IL‑2,
IL‑6, IL‑10, IL‑12, TNF alpha, interferon IFN gamma)
and prostaglandin E2 (PGE2). Proliferative response of
NR‑INF‑02 on splenic lymphocytes in presence and
absence of mitogens was determined by MTT assay. Dexamethasone and celecoxib were used as an inhibitor
of IL‑12 and PGE2 respectively. Interleukins and PGE2 quantification Plant material The immune cells were isolated from the spleens by
passing through 40 µm cell strainer and the resultant The rhizomes of C. longa Linn. were collected from
different parts of Tamil Nadu State, India and authenticated Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 72 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Figure 1: 13C NMR spectrum of polysaccharide fraction (F1) of
NR‑INF‑02 in D2O FBS at 37°C using a mixture of 95% air and 5% CO2. RAW264.7 cells were adjusted in cell culture medium to
a density of 1 × 105 cells per well in a 96 well plate. The
cells were pre incubated with indicated concentrations of
test substances for 30 min and treated with and without
LPS (1 µg/mL) for 24 h. 1400 W dihydrochloride (iNOS
inhibitor) and dexamethasone were used as reference
standards for NO and MCP‑1 assays respectively. Post
treatment, cell culture supernatant was quantified for NO
and MCP‑1 levels using Griess reaction and ELISA (ELISA
Kit, OptEIA™ from BD biosciences, USA) method,
respectively. All the above experiments were standardized using
respective positive and negative controls. Assay performance
measures like Z’, S/N ratio and % CV were calculated and
fulfilled to the prescribed limits.[31] Figure 1: 13C NMR spectrum of polysaccharide fraction (F1) of
NR‑INF‑02 in D2O cell suspension was collected and centrifuged. Single
splenocyte suspension was obtained by density‑gradient
centrifugation (Histopaque 1077). Cells were re‑suspended
in RPMI‑1640 medium containing 10% heat inactivated
FBS, and 1% antibiotics.[30] The above prepared splenocyte
single cell suspension was used for further experiments. NR‑INF‑02, F1 and F2 was dissolved in phosphate buffered
saline and filtered sterilized through 0.2 µm positively charged
nylon filter before adding to cell culture treatment media. cell suspension was collected and centrifuged. Single
splenocyte suspension was obtained by density‑gradient
centrifugation (Histopaque 1077). Cells were re‑suspended
in RPMI‑1640 medium containing 10% heat inactivated
FBS, and 1% antibiotics.[30] The above prepared splenocyte
single cell suspension was used for further experiments. NR‑INF‑02, F1 and F2 was dissolved in phosphate buffered
saline and filtered sterilized through 0.2 µm positively charged
nylon filter before adding to cell culture treatment media. Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 RESULTS Supernatant collected from above experiments was used
to determine IL‑2, IL‑6, IL‑10, IL‑12, TNF alpha and IFN
gamma by enzyme linked immunosorbent assay (ELISA Kit,
OptEIA™ from Becton, Dickinson (BD) biosciences, USA). PGE2 levels were quantified by Homogenous Time Resolved
Fluorescence (HTRF) method (HTRF kit, CisBio, France). Effect of NR‑INF‑02 on splenocyte proliferation
In this study, we have focused on cellular basis of
the immune‑modulating property of NR‑INF‑02. To
study this, the effect of NR‑INF‑02 on unstimulated
murine splenocytes was investigated. NR‑INF‑02 alone
showed significant concentration dependent increase on
proliferation of murine splenocytes within the experimental
concentration range of 0.8‑500 µg/mL [Figure 2a]. The
next step was to examine whether the association of
NR‑INF‑02 and mitogens influence the proliferation of Nitric oxide (NO) assay and monocyte chemotactic
protein‑1 (MCP‑1) assay Nitric oxide (NO) assay and monocyte chemotactic
protein‑1 (MCP‑1) assay RAW264.7 macrophages, obtained from American
Type Culture Collection were cultured in Dulbecco’s
Modified Eagle Medium DMEM supplemented with 10% Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 73 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM of IL‑2, IL‑6, IL‑10, IL‑12, IFN gamma, TNF alpha
and PGE2 levels [Figures 2b‑e, and 3a‑c]. Treatment with
LPS resulted in a significant increase of IL‑6 [Figure 3a],
IL‑10 [Figure 2d], IL‑12 [Figure 3b], TNF alpha [Figure 2e]
and PGE2 [Figure 3c]. NR‑INF‑02 produced distinct
concentration dependent decrease of IL‑12 [Figure 3b]
and PGE2 [Figure 3c] levels in LPS stimulated splenocytes. NR‑INF‑02 at the indicated concentrations exhibited
a mild significant decrease of IL‑6 [Figure 3a] levels
however, it did not alter the levels of IL‑10 [Figure 2d]
and TNF alpha [Figure 2e] in LPS stimulated murine
splenocytes. Con A treatment produced significant
increase of IL‑2 [Figure 2b], IL‑10 [Figure 2d] and IFN
gamma [Figure 2c]. NR‑INF‑02 produced significant
increase of IL‑2 and IFN gamma in Con A stimulated
splenocytes [Figure 2b and c]. NR‑INF‑02 at a concentration splenocytes. LPS was used at 5 µg/mL as B cell mitogen,
and Con A was used at 2.5 µg/mL as T cell mitogen. For splenic lymphocytes, the two mitogens produced
a significant increase in cell proliferation. NR‑INF‑02
produced statistically significant dose dependent increase
in both LPS and Con A stimulated splenocyte proliferation
at the indicated concentration range of 0.8‑500 µg/mL
[Figure 2a]. RESULTS Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l |
|
|
g
[
g
]
however, it did not alter the levels of IL‑10 [Figure 2d]
and TNF alpha [Figure 2e] in LPS stimulated murine
splenocytes. Con A treatment produced significant
increase of IL‑2 [Figure 2b], IL‑10 [Figure 2d] and IFN
gamma [Figure 2c]. NR‑INF‑02 produced significant
increase of IL‑2 and IFN gamma in Con A stimulated
splenocytes [Figure 2b and c]. NR‑INF‑02 at a concentration
Figure 2a: Effect of NR‑INF‑02 on lymphocyte proliferation in
unstimulated, concanavalin A (Con A) and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. Values
are expressed as cell number)
Figure 2c: Effect of NR‑INF‑02 on interferon (IFN) gamma release
in unstimulated and concanavalin A (Con A) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without Con
A and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for IFN gamma estimation and
the values are expressed as pg/mL)
Figure 2b: Effect of NR‑INF‑02 on interleukin (IL)‑2 release in unstimulated
and concanavalin A (Con A) stimulated murine splenocytes at 48 h of
cell treatment (Mouse splenocytes were treated with NR‑INF‑02 at the
indicated concentrations with and without Con A and further incubated
for 48 h. After 48 h, the cells were centrifuged and the supernatants were
collected for IL‑2 estimation and the values are expressed as pg/mL)
Figure 2d: Effect of NR‑INF‑02 on interleukin (IL)‑10 release in
unstimulated, concanavalin A (Con A), and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. After
48 h, the cells were centrifuged and the supernatants were collected
for IL‑10 estimation and the values are expressed as pg/mL)
Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2
As reflected in graphical representation, production of Th1
and Th2 related cytokines were found to be very low in
untreated splenic lymphocytes culture supernatants after
48 h culture. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l As reflected in graphical representation, production of Th1
and Th2 related cytokines were found to be very low in
untreated splenic lymphocytes culture supernatants after
48 h culture. However, NR‑INF‑02 treated splenocytes
produced significant concentration dependent increase Figure 2a: Effect of NR‑INF‑02 on lymphocyte proliferation in
unstimulated, concanavalin A (Con A) and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. Values
are expressed as cell number) Figure 2b: Effect of NR‑INF‑02 on interleukin (IL)‑2 release in unstimulated
and concanavalin A (Con A) stimulated murine splenocytes at 48 h of
cell treatment (Mouse splenocytes were treated with NR‑INF‑02 at the
indicated concentrations with and without Con A and further incubated
for 48 h. After 48 h, the cells were centrifuged and the supernatants were
collected for IL‑2 estimation and the values are expressed as pg/mL) Figure 2b: Effect of NR‑INF‑02 on interleukin (IL)‑2 release in unstimulated
and concanavalin A (Con A) stimulated murine splenocytes at 48 h of
cell treatment (Mouse splenocytes were treated with NR‑INF‑02 at the
indicated concentrations with and without Con A and further incubated
for 48 h. After 48 h, the cells were centrifuged and the supernatants were
collected for IL‑2 estimation and the values are expressed as pg/mL) Figure 2b: Effect of NR‑INF‑02 on interleukin (IL)‑2 release in unstimulated
and concanavalin A (Con A) stimulated murine splenocytes at 48 h of
cell treatment (Mouse splenocytes were treated with NR‑INF‑02 at the
indicated concentrations with and without Con A and further incubated
for 48 h. After 48 h, the cells were centrifuged and the supernatants were
collected for IL‑2 estimation and the values are expressed as pg/mL) Figure 2a: Effect of NR‑INF‑02 on lymphocyte proliferation in
unstimulated, concanavalin A (Con A) and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. Values
are expressed as cell number) Figure 2a: Effect of NR‑INF‑02 on lymphocyte proliferation in
unstimulated, concanavalin A (Con A) and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. RESULTS However, NR‑INF‑02 treated splenocytes
produced significant concentration dependent increase Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l Values
are expressed as cell number) Pharmacognosy Research | April June 2013 | Vol 5 | Issue 2
Figure 2d: Effect of NR‑INF‑02 on interleukin (IL)‑10 release in
unstimulated, concanavalin A (Con A), and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. After
48 h, the cells were centrifuged and the supernatants were collected
for IL‑10 estimation and the values are expressed as pg/mL) Figure 2d: Effect of NR‑INF‑02 on interleukin (IL)‑10 release in
unstimulated, concanavalin A (Con A), and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. After
48 h, the cells were centrifuged and the supernatants were collected
for IL‑10 estimation and the values are expressed as pg/mL) Figure 2c: Effect of NR‑INF‑02 on interferon (IFN) gamma release
in unstimulated and concanavalin A (Con A) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without Con
A and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for IFN gamma estimation and
the values are expressed as pg/mL) Figure 2d: Effect of NR‑INF‑02 on interleukin (IL)‑10 release in
unstimulated, concanavalin A (Con A), and lipopolysaccharide (LPS)
stimulated murine splenocytes at 48 h of cell treatment (Mouse
splenocytes were treated with NR‑INF‑02 at the indicated concentrations
with and without Con A or LPS and further incubated for 48 h. After
48 h, the cells were centrifuged and the supernatants were collected
for IL‑10 estimation and the values are expressed as pg/mL) Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 74 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Effect of NR‑INF‑02 fractions, F1 and F2 on
lymphocyte proliferation, interleukins and PGE2 of 500 µg/mL, exhibited mild significant decrease of IL‑10
production in Con A stimulated splenocytes [Figure 2d]. F1 and F2 significantly stimulated the proliferation
of mouse splenocytes after 48 h of treatment. The
relative EC50 of NR‑INF‑02 and F1 were found to be
18.6 and 9 µg/mL respectively. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l After 48 h, the cells were centrifuged
and the supernatants were collected for IL‑6 estimation and the values
are expressed as pg/mL. Dexamethasone (140 nM) was used as a
reference control)
Figure 3c: Effect of NR‑INF‑02 on prostaglandin E2 (PGE2) release
in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for PGE2 quantification and the
values are expressed as pg/mL. Celecoxib (33.3 nM) was used as a
reference control) Figure 2e: Effect of NR‑INF‑02 on tumor necrosis factor (TNF) alpha
release in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for TNF‑alpha estimation and the
values are expressed as pg/mL. Dexamethasone (55 nM) was used
as a reference control) Figure 3a: Effect of NR‑INF‑02 on interleukin (IL)‑6 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for IL‑6 estimation and the values
are expressed as pg/mL. Dexamethasone (140 nM) was used as a
reference control) Figure 3a: Effect of NR‑INF‑02 on interleukin (IL)‑6 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for IL‑6 estimation and the values
are expressed as pg/mL. Dexamethasone (140 nM) was used as a
reference control) Figure 2e: Effect of NR‑INF‑02 on tumor necrosis factor (TNF) alpha
release in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for TNF‑alpha estimation and the
values are expressed as pg/mL. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l F2 fraction showed less
potent (EC50 = 162 µg/mL) activity than the NR‑INF‑02
and F1. Polysaccharide fraction (F1) showed almost two
times more potent activity than NR‑INF‑02 [Figure 4a]. As shown in Figure 4b, F1 and F2 dose dependently
increased the cell number of LPS stimulated splenocytes. F1 showed very potent stimulatory activity with an EC50 of
0.072 µg/mL. F1 exhibited almost 10 times more potent Effect of NR‑INF‑02 on NO and MCP‑1 production
NR‑INF‑02 was found to significantly increase the
NO and MCP‑1 levels in RAW264.7 cells. A clear
concentration dependent increase in the levels of
NO and MCP‑1 was observed upon treatment with
NR‑INF‑02. Significant decrease of NO at lesser
concentrations (0.8‑20 µg/mL) and no decrease
or increase in MCP‑1 levels were observed in LPS
stimulated RAW264.7 cells at the tested concentrations
of NR‑INF‑02 [Figure 3d and e]. Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2
75
or increase in MCP 1 levels were observed in LPS
stimulated RAW264.7 cells at the tested concentrations
of NR‑INF‑02 [Figure 3d and e]. increased the cell number of LPS stimulated splenocytes. F1 showed very potent stimulatory activity with an EC50 of
0.072 µg/mL. F1 exhibited almost 10 times more potent
Figure 2e: Effect of NR‑INF‑02 on tumor necrosis factor (TNF) alpha
release in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for TNF‑alpha estimation and the
values are expressed as pg/mL. Dexamethasone (55 nM) was used
as a reference control)
Figure 3b: Effect of NR‑INF‑02 on interleukin (IL)‑12 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the tcells were centrifuged
and the supernatants were collected for IL‑12 estimation and the values
are expressed as pg/mL. Dexamethasone (132 nM) was used as a
reference control)
Figure 3a: Effect of NR‑INF‑02 on interleukin (IL)‑6 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l Dexamethasone (70 nM) was used as a reference control) Figure 3d: Effect of NR‑INF‑02 on nitric oxide release in unstimulated
and lipopolysaccharide (LPS) stimulated RAW264.7 cells at 24 h of
cell treatment (RAW264.7 cells were pre incubated at the indicated
concentrations of NR‑INF‑02 for 30 min and treated with and without
LPS for 24 h. After 24 h, the nitrite content in culture supernatant was
analyzed by Griess reaction assay and expressed in µM. 1400 W
dihydrochloride (12 µM) was used as reference control)
Figure 3e: Effect of NR‑INF‑02 on monocyte chemotactic
protein‑1 (MCP‑1) release in unstimulated and lipopolysaccharide (LPS)
stimulated RAW 264.7 cells at 24 h of cell treatment (RAW264.7 cells
were pre incubated at the indicated concentrations of NR‑INF‑02 for
30 min and treated with and without LPS for 24 h. After 24 h, the culture
supernatant was analyzed for MCP‑1 levels by ELISA and expressed
as pg/mL. Dexamethasone (70 nM) was used as a reference control)
Figure 4a: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of unstimulated mouse splenocytes
Figure 4b: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of lipopolysaccharide stimulated mouse splenocytes Figure 3e: Effect of NR‑INF‑02 on monocyte chemotactic
protein‑1 (MCP‑1) release in unstimulated and lipopolysaccharide (LPS)
stimulated RAW 264.7 cells at 24 h of cell treatment (RAW264.7 cells
were pre incubated at the indicated concentrations of NR‑INF‑02 for
30 min and treated with and without LPS for 24 h. After 24 h, the culture
supernatant was analyzed for MCP‑1 levels by ELISA and expressed
as pg/mL. Dexamethasone (70 nM) was used as a reference control) Figure 3d: Effect of NR‑INF‑02 on nitric oxide release in unstimulated
and lipopolysaccharide (LPS) stimulated RAW264.7 cells at 24 h of
cell treatment (RAW264.7 cells were pre incubated at the indicated
concentrations of NR‑INF‑02 for 30 min and treated with and without
LPS for 24 h. After 24 h, the nitrite content in culture supernatant was
analyzed by Griess reaction assay and expressed in µM. 1400 W
dihydrochloride (12 µM) was used as reference control) Figure 3e: Effect of NR‑INF‑02 on monocyte chemotactic
protein‑1 (MCP‑1) release in unstimulated and lipopolysaccharide (LPS)
stimulated RAW 264.7 cells at 24 h of cell treatment (RAW264.7 cells
were pre incubated at the indicated concentrations of NR‑INF‑02 for
30 min and treated with and without LPS for 24 h. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l Dexamethasone (55 nM) was used
as a reference control) Figure 3b: Effect of NR‑INF‑02 on interleukin (IL)‑12 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the tcells were centrifuged
and the supernatants were collected for IL‑12 estimation and the values
are expressed as pg/mL. Dexamethasone (132 nM) was used as a
reference control) Figure 3c: Effect of NR‑INF‑02 on prostaglandin E2 (PGE2) release
in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for PGE2 quantification and the
values are expressed as pg/mL. Celecoxib (33.3 nM) was used as a
reference control) Figure 3b: Effect of NR‑INF‑02 on interleukin (IL)‑12 release in
unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the tcells were centrifuged
and the supernatants were collected for IL‑12 estimation and the values
are expressed as pg/mL. Dexamethasone (132 nM) was used as a
reference control) Figure 3c: Effect of NR‑INF‑02 on prostaglandin E2 (PGE2) release
in unstimulated and lipopolysaccharide (LPS) stimulated murine
splenocytes at 48 h of cell treatment (Mouse splenocytes were treated
with NR‑INF‑02 at the indicated concentrations with and without LPS
and further incubated for 48 h. After 48 h, the cells were centrifuged
and the supernatants were collected for PGE2 quantification and the
values are expressed as pg/mL. Celecoxib (33.3 nM) was used as a
reference control) Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 75 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM Figure 3e: Effect of NR‑INF‑02 on monocyte chemotactic
protein‑1 (MCP‑1) release in unstimulated and lipopolysaccharide (LPS)
stimulated RAW 264.7 cells at 24 h of cell treatment (RAW264.7 cells
were pre incubated at the indicated concentrations of NR‑INF‑02 for
30 min and treated with and without LPS for 24 h. After 24 h, the culture
supernatant was analyzed for MCP‑1 levels by ELISA and expressed
as pg/mL. Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l After 24 h, the culture
supernatant was analyzed for MCP‑1 levels by ELISA and expressed
as pg/mL. Dexamethasone (70 nM) was used as a reference control) stimulatory activity than NR‑INF‑02. Furthermore, F1
could significantly stimulated the IL‑10 levels. NR‑INF‑02
and F1 showed equipotent activity towards production
of IL‑10 with an EC50 value of 42 and 36 µg/mL
Figure 4a: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of unstimulated mouse splenocytes
Figure 4b: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of lipopolysaccharide stimulated mouse splenocytes
Figure 4c: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on interleukin (IL)‑10 secretion in unstimulated mouse splenocytes
Figure 4d: Dose‑response curve generated for NR‑INF‑02, F1 and
F2 on interleukin (IL)‑12 secretion in lipopolysaccharide stimulated
mouse splenocytes Figure 4a: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of unstimulated mouse splenocytes Figure 4b: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of lipopolysaccharide stimulated mouse splenocytes Figure 4a: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of unstimulated mouse splenocytes Figure 4b: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on proliferation of lipopolysaccharide stimulated mouse splenocytes Figure 4d: Dose‑response curve generated for NR‑INF‑02, F1 and
F2 on interleukin (IL)‑12 secretion in lipopolysaccharide stimulated
mouse splenocytes Figure 4d: Dose‑response curve generated for NR‑INF‑02, F1 and
F2 on interleukin (IL)‑12 secretion in lipopolysaccharide stimulated
mouse splenocytes Figure 4c: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on interleukin (IL)‑10 secretion in unstimulated mouse splenocytes and F1 showed equipotent activity towards production
of IL‑10 with an EC50 value of 42 and 36 µg/mL stimulatory activity than NR‑INF‑02. Furthermore, F1
could significantly stimulated the IL‑10 levels. NR‑INF‑02 stimulatory activity than NR‑INF‑02. Furthermore, F1
could significantly stimulated the IL‑10 levels. NR‑INF‑02 76 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM respectively. However, F2 significantly stimulated IL‑10
production only at the highest concentration of 500 µg/
mL [Figure 4c]. The present results showed mitogenic activity of
NR‑INF‑02 which was comparable to classical mitogens
like LPS and Con A. Also, the effects of NR‑INF‑02 on
production of T cell cytokines including Th1 (IL‑2 and IFN
gamma) and Th2 cytokines (IL‑10) were measured. Results
showed that NR‑INF‑02 increased both Th1 (IL‑2 and
IFN gamma) and Th2 (IL‑10) cytokines indicating its dual
immune functions. NR‑INF‑02 significantly increased the
IL‑2 and IFN gamma levels in Con A stimulated splenic
lymphocytes. Effect of NR‑INF‑02 on production of Th1, Th2
cytokines and PGE2l The above results indicated that NR‑INF‑02
showed a specific immunity response by stimulating both
Th1 and Th2 cells. Results also indicated that NR‑INF‑02, F1 and F2
concentration dependently decreased the production
of IL‑12 in LPS stimulated splenocytes. F1 showed
remarkable inhibitory effect (EC50 = 0.1 µg/mL)
towards LPS stimulated IL‑12 production after 48 h of
treatment. NR‑INF‑02 and F2 showed dose dependent
inhibition of IL‑12 with an EC50 of 2 and 54 µg/mL
respectively [Figure 4d]. Similar trend was observed on
inhibition of LPS stimulated PGE2 levels. NR‑INF‑02, F1
and F2 significantly ameliorated the PGE2 production in
LPS stimulated mouse splenocytes [Figure 4e]. However,
F1 showed stronger inhibitory potential than NR‑INF‑02
and F2. F1 showed almost 20 times more potent activity
than NR‑INF‑02 towards inhibition of PGE2 and IL‑12
in LPS treated splenocytes [Figure 4d and e]. Polysaccharide fraction (F1) derived from NR‑INF‑02
showed potent immune stimulatory activity towards
proliferation of splenocytes cell number and IL‑10 secretion
than F2. In concordance to our results, the recent report
on polysaccharide fraction of the rhizome of C. longa
revealed the proliferative response and cytokine production
in peripheral blood mononuclear cells (PBMC) in vitro.[9]
Hence, we hypothesize that polysaccharides present in
this extract might be contributing to this proliferative and
cytokine release property in murine splenocytes. REFERENCES 1. Khanna NM. Turmeric, Nature’s precious gift. Curr Sci
1999;76:1351‑6. However, inflammatory processes subsequently
need to be down regulated to allow healing. These
divergent and at times seemingly contradictory effects
reflect the dichotomy of macrophages as both pro
and anti‑inflammatory effectors in response to host
environmental changes.[37] 2. Singh S, Aggarwal BB. Activation of transcription factor
NF‑kappa B is suppressed by curcumin (diferuloylmethane). J Biol Chem 1995;270:24995‑5000. 3. Plummer SM, Holloway KA, Manson MM, Munks RJ, Kaptein A,
Farrow S, et al. Inhibition of cyclo‑oxygenase 2 expression in
colon cells by the chemopreventive agent curcumin involves
inhibition of NF‑kappaB activation via the NIK/IKK signalling
complex. Oncogene 1999;18:6013‑20. NR‑INF‑02 exerted strong inhibition on LPS stimulated
PGE2 and IL‑12 production by macrophages. Furthermore,
polysaccharide fraction derived from NR‑INF‑02 showed
potent inhibitory effect toward LPS stimulated IL‑12 and
PGE2 secretion. 4. Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S,
Aggarwal BB. Curcumin (diferuloylmethane) down‑regulates
expression of cell proliferation and antiapoptotic and metastatic
gene products through suppression of IkappaBalpha kinase and
Akt activation. Mol Pharmacol 2006;69:195‑206. 5. Bhaumik S, Jyothi MD, Khar A. Differential modulation of nitric
oxide production by curcumin in host macrophages and NK
cells. FEBS Lett 2000;483:78‑82. NR‑INF‑02 showed mild inhibitory effect towards NO,
and IL‑6 production in the presence of LPS. Interestingly,
NR‑INF‑02 neither increased nor decreased the levels of
IL‑10, TNF alpha, and MCP‑1 cytokines in the presence
of LPS. NR‑INF‑02 showed mild inhibitory effect towards NO,
and IL‑6 production in the presence of LPS. Interestingly,
NR‑INF‑02 neither increased nor decreased the levels of
IL‑10, TNF alpha, and MCP‑1 cytokines in the presence
of LPS. NR‑INF‑02 showed mild inhibitory effect towards NO,
and IL‑6 production in the presence of LPS. Interestingly,
NR‑INF‑02 neither increased nor decreased the levels of
IL‑10, TNF alpha, and MCP‑1 cytokines in the presence
of LPS. 6. Churchill M, Chadburn A, Bilinski RT, Bertagnolli MM. Inhibition
of intestinal tumors by curcumin is associated with changes in
the intestinal immune cell profile. J Surg Res 2000;89:169‑75. 7. Jagetia GC, Aggarwal BB. Spicing up of the immune system by
curcumin. J Clin Immunol 2007;27:19‑35. According to our knowledge, for the first time, we
have demonstrated the anti‑inflammatory activity of
polysaccharide containing C. longa extract by down
regulating the PGE2 and IL‑12 levels in LPS stimulated
mouse splenocytes. DISCUSSION Number of medicinal plant extracts and their constituents
are known to alter immune function and display array of
immune‑modulatory effects. In various in vitro and in vivo
studies, herbal medicines have been reported to modulate
cytokine secretion, immunoglobulin secretion, lymphocyte
expression and phagocytosis.[32] In the present study, the
immune stimulatory and anti‑inflammatory properties of
an aqueous based extract of C. longa (NR‑INF‑02) were
evaluated in vitro. Macrophages are important as a first line of defense against
infections. NR‑INF‑02 increased the NO levels in mouse
macrophages (RAW264.7) which plays an important role
as cytotoxic agents against invading pathogens. Increased
synthesis of NO can induce immune‑stimulating activity
of macrophages. NR‑INF‑02 increases the release of MCP‑1 from murine
macrophages in a concentration dependent manner
indicating its role in the recruitment of monocytes to
sites of injury and infection. Four polysaccharides namely
ukonan A, ukonan B, ukonan C and ukonan D have been
isolated from C. longa and proved to have remarkable
reticulo endothelial system potentiating activity in carbon
clearance test.[10‑14] Macrophage activation of NR‑INF‑02
might be due to presence of these ukonan polysaccharides. Lymphocytes are the key constituents of the immune system
as they recognize the foreign antigens and mount an immune
response; a rise or fall in the concentration of these cells
affects the health/immune constitution of the body.[33] Figure 4e: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on prostaglandin E2 (PGE2) secretion in lipopolysaccharide stimulated
mouse splenocytes Activated macrophages in turn inhibit the invasion of
microorganisms by releasing cytokines. As a result of
direct macrophage activation by NR‑INF‑02, PGE2 levels
from macrophages of murine splenocytes were increased
as that of LPS stimulation. PGE2 is involved in diverse
functions, including nerve growth, wound healing, and the
immune response. NR‑INF‑02 activated the macrophages which secreted
many inflammatory mediators such as PGE2, IL‑6, IL‑12
and TNF alpha. The present findings are in concordance
with previous findings of Kim et al. who demonstrated
enhanced phagocytic activity in Gram positive and Figure 4e: Dose‑response curve generated for NR‑INF‑02, F1 and F2
on prostaglandin E2 (PGE2) secretion in lipopolysaccharide stimulated
mouse splenocytes Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 77 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM CONCLUSION negative bacteria and also augmented the oxygen burst
response with purified polysaccharide fraction of one
another species, Curcuma zeodoaria.[34] Additionally,
macrophage stimulating activity of C. zeodoaria was
confirmed by a significant increase of H2O2, NO, and
TNF alpha production in RAW264.7 cells. Similar to
this report, crude polysaccharide extract prepared from
rhizome of Curcuma xanthorrhiza significantly increased
the phagocytosis activity of macrophages, release of
NO, H2O2, TNF alpha and PGE2 in a dose dependent
manner. Increase of NO and PGE2 was mediated in part
by specific activation of NF‑kappa B.[35] Enhanced release
of pro‑inflammatory cytokines, including IFN gamma
and TNF alpha by aqueous extract of C. longa treatment
led to an expression of cell adhesion molecule such as
inter‑cellular adhesion molecule‑1, vascular‑cell adhesion
molecule‑1 and E‑selectin mediate the extravasation of
leukocytes from blood vessels to the sites of injury or
infection.[36] In conclusion, the present experiment shows that
NR‑INF‑02 exhibits potent in vitro immune‑stimulatory
activity by macrophage activation, splenocytes proliferation
and cytokine release. Interestingly, NR‑INF‑02
and polysaccharide fraction showed prominent
anti‑inflammatory activity by down regulating PGE2 and
IL‑12 secretion. Based on the data presented here on the
polysaccharide fraction of NR‑INF‑02, we hypothesize
that polysaccharides of C. longa contribute to the
anti‑inflammatory and immune‑stimulatory activities of
NR‑INF‑02. Further studies are directed to understand
the molecular mechanism of action of NR‑INF‑02 and
polysaccharide fraction towards inhibition of LPS induced
IL‑12 and PGE2 secretion. REFERENCES Furthermore, NR‑INF‑02 increased
IL‑10 in unstimulated splenocytes and is one of the
best‑known cytokine that inhibit various types of
inflammatory responses. An in vivo study by Yegnarayan
et al. proved that water extract of C. longa had potent
anti‑inflammatory activity in carrageenan induced oedema,
granuloma pouch and cotton pellet implantation method
in male albino mice.[38] The above study concluded that
water extract of C. longa is superior than curcuminoids
containing C. longa extract. 8. He XG, Lin LZ, Lian LZ, Lindenmaier M. Liquid chromatography-
electrospray mass spectrometric analysis of curcuminoids and
sesquiterpenoids in turmeric (Curcuma longa). J Chromatogr
1998;818:127‑32. 9. Yue GG, Chan BC, Hon PM, Kennelly EJ, Yeung SK, Cassileth BR,
et al. Immunostimulatory activities of polysaccharide extract
isolated from Curcuma longa. Int J Biol Macromol 2010;47:342‑7. 10. Gonda R, Tomoda M, Shimizu N, Kanari M. Characterization of
polysaccharides having activity on the reticuloendothelial system
from the rhizome of Curcuma longa. Chem Pharm Bull (Tokyo)
1990;38:482‑6. 11. Gonda R, Takeda K, Shimizu N, Tomoda M. Characterization of a
neutral polysaccharide having activity on the reticuloendothelial
system from the rhizome of Curcuma longa. Chem Pharm
Bull (Tokyo) 1992;40:185‑8. 12. Gonda R, Tomoda M, Takada K, Ohara N, Shimizu N. The core
structure of ukonan A, a phagocytosis‑activating polysaccharide Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2 78 Chandrasekaran, et al.: Anti‑inflammatory activity of TurmacinTM 26. Subramanian L, Selvam R. Prevention of C4‑induced
hepatotoxicity by aqueous extract of turmeric. Nutr Res
1999;19:429‑41. from the rhizome of Curcuma longa, and immunological activities
of degradation products. Chem Pharm Bull (Tokyo) 1992;40:990‑3. 13. Gonda R, Tomoda M, Ohara N, Takada K. Arabinogalactan core
structure and immunological activities of ukonan C, an acidic
polysaccharide from the rhizome of Curcuma longa. Biol Pharm
Bull 1993;16:235‑8. 27. Madhu K, Chanda K, Saji MJ. Safety and efficacy of Curcuma
longa extract in the treatment of painful knee osteoarthritis:
A randomized placebo‑controlled trial. Inflammopharmacology
2013;21:129-36. 14. Tomoda M, Gonda R, Shimizu N, Kanari M, Kimura M. A reticuloendothelial system activating glycan from the rhizomes
of Curcuma longa. Phytochemistry 1990;29:1083‑6. 28. Gomis DB, Tamayo DM, Alonso JM. Determination of
monosaccharides
in
cider
by
reversed‑phase
liquid
chromatography. Anal Chim Acta 2001;436:173‑80. 15. Wasser SP. Medicinal mushrooms as a source of antitumor and
immunomodulating polysaccharides. Appl Microbiol Biotechnol
2002;60:258‑74. 29. United States Pharmacopeia (USP) 35‑NF30, Vol. 1. 2012. p. 957. 16. Jeon YJ, Kim HM. REFERENCES Experimental evidences and signal
transduction pathways involved in the activation of NF‑kappa B/
Rel by angelan in murine macrophages. Int Immunopharmacol
2001;1:1331‑9. 30. Chandrasekaran CV, Sundarajan K, David K, Agarwal A. In vitro
efficacy and safety of poly‑herbal formulations. Toxicol in vitro
2010;24:885‑97. 31. Chandrasekaran CV, Edwin Jothie R, Kapoor P, Gupta A,
Agarwal A. Optimization of cell‑based assays to quantify the
anti‑inflammatory/allergic potential of test substances in 96‑well
format. Inflammopharmacology 2011;19:169‑81. 17. Yoon YD, Kang JS, Han SB, Park SK, Lee HS, Kang JS, et al. Activation of mitogen‑activated protein kinases and AP‑1 by
polysaccharide isolated from the radix of Platycodon grandiflorum
in RAW 264.7 cells. Int Immunopharmacol 2004;4:1477‑87. 32. Plaeger SF. Clinical immunology and traditional herbal
medicines. Clin Diagn Lab Immunol 2003;10:337‑8. 18. Ramberg JE, Nelson ED, Sinnott RA. Immunomodulatory
dietary polysaccharides: A systematic review of the literature. Nutr J 2010;9:54. 33. Jayathirtha MG, Mishra SH. Preliminary immunomodulatory
activities of methanol extracts of Eclipta alba and Centella
asiatica. Phytomedicine 2004;11:361‑5. 19. Trnovec T, Hrmová M. Immunomodulator polysaccharides:
Chemistry, disposition and metabolism. Biopharm Drug Dispos
1993;14:187‑98. 34. Kim KI, Shin KS, Jun WJ, Hong BS, Shin DH, Cho HY,
et al. Effects of polysaccharides from rhizomes of Curcuma
zedoaria on macrophage functions. Biosci Biotechnol Biochem
2001;65:2369‑77. 20. Mohankumar S, McFarlane JR. An aqueous extract of Curcuma
longa (turmeric) rhizomes stimulates insulin release and mimics
insulin action on tissues involved in glucose homeostasis in vitro. Phytother Res 2011;25:396‑401. 35. Kim AJ, Kim YO, Shim JS, Hwang JK. Immunostimulating
activity of crude polysaccharide extract isolated from Curcuma
xanthorrhiza Roxb. Biosci Biotechnol Biochem 2007;71:1428‑38. 21. Deshpande SS, Ingle AD, Maru GB. Chemopreventive efficacy
of curcumin‑free aqueous turmeric extract in 7,12‑dimethylbenz
a anthracene‑induced rat mammary tumorigenesis. Cancer Lett
1998;123:35‑40. 36. Madan B, Gade WN, Ghosh B. Curcuma longa activates
NF‑kappaB
and
promotes
adhesion
of
neutrophils
to
human umbilical vein endothelial cells. J Ethnopharmacol
2001;75:25‑32. 22. Yu ZF, Kong LD, Chen Y. Antidepressant activity of aqueous
extracts of Curcuma longa in mice. J Ethnopharmacol
2002;83:161‑5. 37. Zhai Z, Liu Y, Wu L, Senchina DS, Wurtele ES, Murphy PA,
et al. Enhancement of innate and adaptive immune functions by
multiple Echinacea species. J Med Food 2007;10:423‑34. 23. Selvam R, Subramanian L, Gayathri R, Angayarkanni N. The anti‑oxidant activity of turmeric (Curcuma longa). J Ethnopharmacol 1995;47:59‑67. 38. Yegnanarayan R, Saraf AP, Balwani JH. Comparison of
anti‑inflammatory activity of various extracts of Curcuma
longa (Linn.). Cite this article as: Chandrasekaran CV, Sundarajan K, Edwin JR, Gururaja
GM, Mundkinajeddu D, Agarwal A. Immune-stimulatory and anti-inflammatory
activities of Curcuma longa extract and its polysaccharide fraction. Phcog
Res 2013;5:71-9. Cite this article as: Chandrasekaran CV, Sundarajan K, Edwin JR, Gururaja
GM, Mundkinajeddu D, Agarwal A. Immune-stimulatory and anti-inflammatory
activities of Curcuma longa extract and its polysaccharide fraction. Phcog
Res 2013;5:71-9. Source of Support: Nil, Conflict of Interest: None declared. REFERENCES Indian J Med Res 1976;64:601‑8. 24. Anbu Jeba Sunilson J, Suraj R, Rejitha G, Anandarajagopal K,
Anita Gnana Kumari, AV, Promwichit P. In vitro antimicrobial
evaluation of Zingiber officinale, Curcuma longa and Alpinia
galanga extracts as natural food preservatives. Am J Food
Technol 2009;4:192‑200. Cite this article as: Chandrasekaran CV, Sundarajan K, Edwin JR, Gururaja
GM, Mundkinajeddu D, Agarwal A. Immune-stimulatory and anti-inflammatory
activities of Curcuma longa extract and its polysaccharide fraction. Phcog
Res 2013;5:71-9. 25. Mishra RK, Singh SK. Reversible antifertility effect of aqueous
rhizome extract of Curcuma longa L. in male laboratory mice. Contraception 2009;79:479‑87. Source of Support: Nil, Conflict of Interest: None declared. 79 Pharmacognosy Research | April-June 2013 | Vol 5 | Issue 2
|
https://openalex.org/W3199498712
|
https://bmcmededuc.biomedcentral.com/track/pdf/10.1186/s12909-020-02240-z
|
English
| null |
Learning Outcomes of a Flipped Classroom Teaching Approach in an Adult-health Nursing Course: A Quasi-Experimental Study
|
Research Square (Research Square)
| 2,020
|
cc-by
| 7,774
|
© The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Fan et al. BMC Medical Education (2020) 20:317
https://doi.org/10.1186/s12909-020-02240-z Fan et al. BMC Medical Education (2020) 20:317
https://doi.org/10.1186/s12909-020-02240-z Open Access Abstract Background: New teaching strategies must be developed not only to enhance nurse’s competence but also to
allow nurses to respond to the complex health care needs of today’s society. The purpose of this study was to
explore the learning outcomes of a flipped classroom teaching approach in an adult-health nursing course for
students in a two-year Bachelor of Science in Nursing program. Methods: The study had a quasi-experimental design. An 18-week flipped classroom teaching approach was
applied in an adult-health nursing course. In total, 485 nursing students enrolled in the study, with 287 in the
experimental group and 198 in the control group. The Self-Evaluated Core Competencies Scale, Metacognitive
Inventory for Nursing Students, Self-Directed Learning Readiness Scale, and self-designed learning satisfaction
questionnaire were used to evaluate the students’ learning outcomes. Results: The experimental group showed a statistically significant increase in the overall scores for self-evaluated
core competencies, the “self-modification” subscale of the Metacognitive Inventory for Nursing Students, and in
overall self-directed learning readiness; further, they also showed high levels of course satisfaction. Conclusions: A flipped classroom teaching approach had a positive impact on student’s learning motivation and
contributed to better learning outcomes in an adult-health nursing course. The flipped classroom combined with
hybrid teaching methods is a suitable and effective learning strategy for a registered nurse (RN) to Bachelor of
Science in Nursing (BSN) program to tackle today’s complex revolution in nursing curricula, and may enhance
nursing students’ abilities to address numerous challenges. Keywords: Flipped classroom, Adult-health nursing, Bachelor of science in nursing performing basic clinical tasks [2, 3]. Thus, new ap-
proaches and educational models must be developed to
allow nurses to respond rapidly to the changes in the
medical
field
[1]. Past
educational
methods
(e.g.,
teacher-centered lecturing) for nurses are no longer ad-
equate for addressing the complex health care needs of
today’s society [4, 5]. Therefore, many nursing schools
have become aware of this transition and begun to re-
view their missions, core competencies, and competency
indicators, while also initiating a shift from training stu-
dents in task-based proficiencies to providing education Learning outcomes of a flipped classroom
teaching approach in an adult-health
nursing course: a quasi-experimental study Jun-Yu Fan1*, Ying-Jung Tseng2, Li-Fen Chao2, Shiah-Lian Chen3 and Sui-Whi Jane4 Jun-Yu Fan1*, Ying-Jung Tseng2, Li-Fen Chao2, Shiah-Lian Chen3 and Sui-Whi Jane4 * Correspondence: jyfan@gw.cgust.edu.tw; jyfan0921@gmail.com
1Department of Nursing & Graduate Institute of Nursing, Chang Gung
University of Science and Technology, Division of Nursing, Chang Gung
Memorial Hospital, Linkou Branch, 261, Wen-Hua 1st Road, Kwei-Shan,
Tao-Yuan 33303, Taiwan (R.O.C.)
Full list of author information is available at the end of the article Background Health professional education is expected to produce
graduates who are proficient in core competencies and
have the ability to provide safe, high-quality, patient-
centered care [1]. However, many employers find that
recent nursing graduates are not competent enough at * Correspondence: jyfan@gw.cgust.edu.tw; jyfan0921@gmail.com
1Department of Nursing & Graduate Institute of Nursing, Chang Gung
University of Science and Technology, Division of Nursing, Chang Gung
Memorial Hospital, Linkou Branch, 261, Wen-Hua 1st Road, Kwei-Shan,
Tao-Yuan 33303, Taiwan (R.O.C.)
Full list of author information is available at the end of the article Flipped classrooms and hybrid teaching methods Flipped classrooms and hybrid teaching methods
A BL method known as the “flipped classroom” ap-
proach is rapidly growing in popularity in health care
educational disciplines with the purpose of activating or
facilitating students’ engagement in the learning process
[8–10]. Many approaches such as PBL, TBL, simulated-
based learning, role play, or web-based learning are used
to flip students’ learning style from teacher-centered
(passive) to learner-centered (active), as well as to in-
crease student engagement, enhance the development of
critical thinking, and improve learning outcomes [8, 11–
13]. The key elements of the flipped classroom include
pre-class content, in-class activities, post-class assess-
ment, and student-triggered inquiry [8, 14, 15]. To create a successful flipped classroom, students’ intrinsic
motivations are a key element in achieving desired learning
outcomes. In the digital era, instructors or teachers often com-
bine computer-mediated technology and face-to-face in-class
activities to enhance students’ engagement in pre-class work,
in-class learning, and post-class assignments, as well as to help
students achieve their goals during the flipped classroom pro-
cessing period [8, 12, 13]. Several studies have shown that stu-
dents prefer a hybrid course structure and their learning
outcomes can improve in knowledge, decision-making skills,
satisfaction, thinking abilities, and reflections concerning clin-
ical practice [16–26]. Most of these studies also demonstrated
the mastery learning behavior suggested by Bloom [27], which
posits that learning activities applied in class, such as profes-
sional knowledge, relevant skills, and scholarly inquiry, might
transfer to real clinical practice [8]. To help further ADN nurses’ education, an RN-to-BSN
program was developed, and by Fall 2019, there were 17
accredited RN-to-BSN programs offered by public or pri-
vate universities, colleges, universities of technology, or
technical colleges throughout Taiwan. All the RN-to-BSN
programs are classroom-based, and off-campus online
programs are not permitted by the Ministry of Education. Our university is a private university with two cam-
puses, 235 km apart: the Linkou (L) campus in northern
Taiwan and the Chiayi (C) campus in southern Taiwan,
with a total of approximately 6400 students, as of 2020. Our RN-to-BSN program is a two-year program, offering
a total of 72 credits, with an average of 18 credits per se-
mester, including 450 h of hospital-based clinical practi-
cum. There are approximately 300 and 200 students per
year in L and C campuses, respectively. Both campuses
share the same course list and conduct the same core
courses simultaneously. Page 2 of 11 Page 2 of 11 Fan et al. BMC Medical Education (2020) 20:317 in higher-level competencies, such as decision-making,
quality improvement, systems thinking, evidence-based
practice, and inter-professional teamwork and collabor-
ation [1]. thinking and analysis, evidence-based practice, problem-
solving skills, communication and informatics, decision-
making and clinical judgment, teamwork and collabor-
ation, and life-long self-directed learning. To meet our
desired learning outcomes, we have applied various new
teaching strategies, such as scenario simulation, problem-
based learning (PBL), team-based learning (TBL), blended
learning (BL), and objective simulation clinical examination
to equip students with the competencies and skills needed to
deliver quality care in today’s complex health care system. Two-year RN-to-BSN program in Taiwan The universal goal of educating nursing professionals is
to produce graduates who can meet patients’ needs and
deliver safe, quality patient care. In Taiwan, to become a
registered nurse (RN), students must graduate from an
accredited program. The two options available are an as-
sociate degree or a bachelor’s degree in nursing. The As-
sociate Degree in Nursing (ADN) is a five-year program
which is offered by junior colleges and admits graduates
from junior high schools. The Bachelor of Science in
Nursing (BSN) degree program, which is offered through
the university educational system (including universities,
colleges, universities of technology, and technical col-
leges), is usually 4 years in duration and requires 12
years of prior education [6]. Both BSN and ADN gradu-
ates must pass professional and technical personnel ex-
aminations before becoming RNs. These examinations
are administered by the Examination Yuan. In Taiwan,
an RN with an ADN can provide the same level of care
as an RN with a BSN. Regarding long-term career mobil-
ity, however, nurses with BSNs tend to have more op-
tions for professional development and advancement
(such as administrative and leadership positions or vari-
ous
nursing
specialties),
skill-building,
and
cultural
awareness, as well as obtain higher salaries [7]. Research design and samples This study was a quasi-experimental design and was
conducted at a private university with two campuses as
previously described in the “Two-year RN-to-BSN pro-
gram in Taiwan” section of this paper. The study was
conducted from September 2015 to February 2016. There were 504 total students enrolled in the two-year
RN-to-BSN program with 304 and 200 students at the L
and C campuses, respectively. All 504 RN-to-BSN stu-
dents were potential participants. To minimize interven-
tion “contamination” between experimental and control
participants, the students from L campus belonged to
the experimental group (EG) and those from C campus
belonged to the control group (CG). Flipped classrooms and hybrid teaching methods The faculty members from both
campuses shared input in terms of course design, revi-
sion, and evaluation via an internet meeting before
course start and after course end, but faculty members
and students do not cross campus due to distance in-
convenience. Our curriculum was designed to help ADN
nurses reach advanced levels of competencies in critical The flipped classroom combined with a hybrid teach-
ing course structure provides students with not only a
flexible way to learn materials (mostly online) at their
own pace before class (autonomy) but also reinforces
students’ in-class discussion with peers (relatedness),
and
reevaluates students’ strengths
and weaknesses
(competence). Ultimately, the flipped classroom may
help students to develop self-directed learning skills and
cultivate life-long learning habits [20]. Thus, the purpose
of the present study was to explore students’ learning
outcomes of a flipped classroom teaching approach in
adult-health nursing course in a two-year RN-to-BSN
program. Page 3 of 11 Page 3 of 11 Fan et al. BMC Medical Education (2020) 20:317 Traditional teaching
h
f
h In the first phase, 2 weeks before the course began, it
was optional for the instructor to upload adult-health
nursing course teaching materials or videos online and
the students were not required to review materials or
videos before the course began. The second phase was
face-to-face traditional classroom teaching during which
the instructor delivered the knowledge mostly using
slides. The third phase was a face-to-face simulation ac-
tivity (at least 1 simulation activity). The students partic-
ipated in the classroom and laboratory activities in both
face-to-face phases. The last phase, the instructor com-
peted a written evaluation of the course in the form of a
written report and/or revised the course design for next
semester. The students were asked to submit homework
reports and evaluate the course. Figure 1 shows the
flipped classroom and traditional teaching procedures. In the present study, a flipped classroom was implemented
in an adult-health nursing course on L campus that incor-
porated face-to-face TBL and simulation activity and on-
line self-directed learning (via the “e-campus” platform). The adult-health nursing course is one of the core nursing
courses taught in the ADN program. The reasons why the
adult-health nursing course was chosen in our two-year
RN-to-BSN program curriculum was because its applica-
tion and utilization play an important role in clinical prac-
tice, and the majority of our graduates’ work in medical or
surgical wards. Thus, more in-depth course work from an
ADN level is necessary, and this may help students have a
better understanding of the cultural, economic, and social
issues affecting patients. Before the flipped classroom was implemented, we
held several faculty-training sessions on L campus to en-
sure consistency in content and teaching materials, in-
cluding quiz questions used in TBL (in-class), the
simulation scenario (in-class), the assignment format
(post-class), and reading materials (pre-class). The adult-
health nursing course is a 36-h, two-credit course taught
during the first semester of the first academic year at
both campuses. In terms of the content of the adult-
health nursing course, we divided 36 h into five blocks
for five topics based on the leading causes of death in
Taiwan: diabetes mellitus, chronic obstructive pulmon-
ary disease, acute coronary syndrome, stroke, and can-
cer. We
adapted
an
in-depth
approach
to
course
contents by integrating pathophysiology, physical assess-
ments, nursing care, and psychosocial issues. Each topic
was addressed in four phases. Methods assignments, and reflection and evaluation forms) on e-
campus. The second phase was a face-to-face TBL in-
class activity. The two- to four-hour TBL process began
with an individual quiz, followed by a group discussion,
and ended with an appeal or argument process. The
third phase was a face-to-face simulation activity in a la-
boratory. There was a two- to four-hour simulation ex-
ercise related to the TBL content, with a case based on
actual clinical practice. The last phase involved complet-
ing a post-class assignment on e-campus. The students
were asked to submit their completed assignments, re-
flection reports, and course evaluations, as well as any
comments they had concerning the e-campus platform. The e-campus platform also facilitated interaction, dis-
cussion, and announcements. Instruments Students’ demographic data, including age and gender
were collected. To understand the learning outcomes of
the flipped classroom, particularly regarding the stu-
dents’ mental self-evaluation processes (also referred to
as metacognitive ability) and self-directed learning skills
the following instruments were used [3]. Self-evaluated Core competencies scale The Self-Evaluated Core Competencies Scale (SECC) in-
cludes eight core competencies stipulated by the Taiwan
Nursing Accreditation Council [28]. The SECC contains
55 items, grouped into two sections and eight subscales. The humanity/responsibility section includes four sub-
scales: caring (6 items), ethics (9 items), accountability (7
items), and life-long learning (5 items). The cognitive/
performance section also includes four subscales: com-
munication and teamwork capability (6 items), critical
thinking and reasoning (5 items), general clinical skills
(9 items), and basic biomedical science (5 items). An The first phase was an online self-directed learning
pre-class phase in which students reviewed assigned
reading materials via the online e-campus platform. Two
weeks before the course began, the instructor was asked
to upload reading materials (e.g., syllabus, related papers
or videos, case descriptions for simulation exercises, Page 4 of 11 Fan et al. BMC Medical Education Fan et al. BMC Medical Education (2020) 20:317 Fig. 1 Flipped classroom and traditional teaching procedures g. 1 Flipped classroom and traditional teaching procedures Fig. 1 Flipped classroom and traditional teaching procedures Fig. 1 Flipped classroom and traditional teaching procedures awareness of their own thoughts and behaviors. The
MINS
includes
28
items
and
five
subscales:
self-
monitoring (7 items), self-modification (7 items), self-
awareness (6 items), effective learning (3 items), and
problem solving (5 items). Scores are measured using a
5-point Likert scale ranging from 1 (never) to 5 (always),
with higher scores indicating higher metacognitive abil-
ity (ranging from 28 to 140). The MINS demonstrated
good internal consistency, and Cronbach’s alpha was .94
for the total scale and ranged from .73 to.90 for the five
subscales, explaining 53.09% of the variance [29]. additional three items measure overall competence, con-
fidence conducting clinical practice, and the ability to
adapt after graduation. An 8-point Likert scale, ranging
from 0 (cannot assess) to 7 (excellent competence), is
used to indicate the level of competency. Higher scores
indicate higher levels of competency, ranging from 0 to
385 points. The SECC showed a good Cronbach’s alpha
of .80. Cronbach’s alphas for the humanity/responsibility
section (.81), cognitive/performance sections (.63), and
eight subscales (ranging from .63 to .81) all demon-
strated good internal consistency as well [28]. Metacognitive inventory for nursing students
The
Metacognitive
Inventory
for
Nursing
Students
(MINS) developed by Hsu [29] was used to measure the
association between participants’ knowledge and their Flipped classroom satisfaction questionnaire A satisfaction questionnaire was developed exclusively for
this study. This questionnaire has 35 items grouped into
four subscales: teacher’s teaching (14 items), course con-
tent (8 items), learning environment (10 items), and ad-
ministrative service (3 items). The 35 items measuring
satisfaction were scored using a scale ranging from 1 (to-
tally disagree) to 5 (totally agree), with higher scores
representing higher levels of satisfaction. In current study,
the satisfaction questionnaire demonstrated a good in-
ternal consistency with Cronbach’s alphas of .98, .95, .94,
.95, and .82 for the total scale, teacher’s teaching subscale,
course content subscale, learning environment subscale,
and administrative service subscale, respectively. Participants’ characteristics Participants’ characteristics A total of 504 nursing students who were in the RN-to-
BSN program were our potential participants. Seven stu-
dents refused to participate, and we ultimately obtained
497 written informed consent forms. Twelve participants
were excluded as a result of incomplete data. A total of
485 nursing students participated (mean age 20.18 ± .59),
and these individuals were assigned to either the EG
(n = 287) or the CG (n = 198). The majority were female
(465; 95.90%). In terms of homogeneity between groups,
there were no significant differences in age and gender
but the EG scored significantly higher on one subscale
(estimated competence after graduation) of the SECC
and
on
two
subscales
(self-monitoring
and
self-
awareness), as well as on the mean overall score, of the
MINS in CG. The overall pre-test and post-test scores,
scores for the SECC, MINS, SDLRS, and flipped class-
room satisfaction subscales are shown in Table 1. Statistics We used the generalized estimating equation (GEE)
model to evaluate the differences between pre- and post-
intervention’s scores on the SECC, MINS, and SDLRS
scores. Each GEE model included a main effect of group
(EG vs. CG), a main effect of time (post-test vs. pre-
test), and a two-way interaction effect of group by time. The parameter estimate of the two-way interaction effect
indicates group differences concerning the change from
the pretest to the post-test. Data analysis was performed
using SPSS 22 (IBM SPSS, Armonk, NY: IBM Corp). Procedure This study was approved by an institutional review board
(IRB No. 104-5709C) before data collection. During the first
week of the adult-health nursing course, either the principal
investigator or co-investigator explained the study’s purpose,
as well as the procedures regarding the distribution of the
questionnaires, to potential participants. For the entire se-
mester, the EG students on L campus received the flipped
classroom method, as shown in Fig. 1, while those in the CG
received traditional teaching methods (see Fig. 1). Before
(pre-test) and after (post-test) the adult-health nursing
course, students in both groups completed the SECC, MINS,
and SDLRS questionnaires. The flipped classroom satisfac-
tion questionnaire was administered only to those in the EG. Evaluation of the differences between pre- and post-
intervention scores on the SECC Table 2 summarizes the results of GEE regarding the
pre- and post- intervention scores on the SECC. The re-
sults showed that, in most subscales, participants in the
EG demonstrated greater improvements than those in
the CG (p < 0.01; Fig. 2); however, group differences re-
garding changes in basic biomedical science, life-long
learning, and estimated competence after graduation
were not significant. Metacognitive inventory for nursing students The Self-Directed Learning Readiness Scale (SDLRS)
used in this study was adapted from Tang [30] to exam-
ine
participants’
readiness
to
perform
self-directed Page 5 of 11 Page 5 of 11 Fan et al. BMC Medical Education (2020) 20:317 Fan et al. BMC Medical Education (2020) 20:317 learning. The SDLRS contains 36 items grouped into six
subscales: effective learning (6 items), love of learning (7
items), learning motivation (5 items), active learning (9
items), independent learning (5 items), and creative
learning (4 items). A 5-point Likert scale ranging from 1
(never) to 5 (most of the time) was used, with higher
scores indicating higher trends of self-directed learning
(total scores range from 0 to 180 points). Cronbach’s
alpha was .92 for the total scale, explaining 54.33% of
the variance. For each subscale, Cronbach’s alphas
ranged from .70 to .88, for this study. Evaluation of the differences between pre- and post-
intervention scores on the MINS Table 3 lists the results of GEE regarding the pre- and
post-intervention MINS scores. Specifically, GEE analyses
showed that the improvement from pre-test to post-test
in the mean scores of self-modification in the EG were
greater than that in the CG (B = 0.12, p < 0.05). However,
the results regarding self-awareness were the opposite; the
CG showed greater improvement in self-awareness from
the pre-test to the post-test (B = −0.18, p < 0.001). Evaluation of the differences between pre- and post-
intervention scores on SDLRS Table 4 shows the results of GEE regarding the pre- and
post-intervention
SDLRS
scores
(B = 0.07,
p = 0.039;
Fig. 3). The intervention was found to improve the mean
scores of active learning (B = 0.13, p < 0.01) and desired
learning (B = 0.10, p < 0.05). However, no significant dif-
ferences between pre- and post- intervention scores
were observed for the other SDLRS subscales. Fan et al. Evaluation of the differences between pre- and post-
intervention scores on SDLRS BMC Medical Education (2020) 20:317 Page 6 of 11 Table 1 Descriptive statistics of each outcome measure in the pre-test and post-test
Variable
CG (n = 198)
EG (n = 287)
Pre-test
Post-test
Pre-test
Post-test
Age (years)
20.24 ± 0.81
20.14 ± 0.37
Gender (female) n (%)
279 (57.5%)
186 (38.4%)
SECC
Basic biomedical science
4.60 ± 0.75
4.86 ± 0.73
4.62 ± 0.69
5.00 ± 0.59
General clinical skills
5.05 ± 0.70
5.21 ± 0.71
5.07 ± 0.68
5.47 ± 0.62
Communication and teamwork capability
5.55 ± 0.73
5.61 ± 0.80
5.58 ± 0.75
5.85 ± 0.73
Critical thinking and reasoning
4.84 ± 0.82
5.12 ± 0.73
4.85 ± 0.80
5.33 ± 0.70
Caring
5.73 ± 0.76
5.68 ± 0.84
5.69 ± 0.79
5.98 ± 0.72
Ethics
5.97 ± 0.73
5.85 ± 0.82
5.90 ± 0.72
6.17 ± 0.66
Accountability
5.78 ± 0.79
5.81 ± 0.82
5.77 ± 0.75
6.05 ± 0.66
Life-long learning
5.40 ± 0.87
5.54 ± 0.84
5.44 ± 0.81
5.72 ± 0.71
Estimated competence after graduation*
4.86 ± 0.97
5.22 ± 0.93
5.03 ± 0.96
5.34 ± 0.81
Overall
5.38 ± 0.66
5.48 ± 0.63
5.39 ± 0.64
5.71 ± 0.56
MINS
Self-monitoring**
3.20 ± 0.64
3.44 ± 0.59
3.39 ± 0.63
3.55 ± 0.63
Self-modification
3.65 ± 0.58
3.75 ± 0.60
3.65 ± 0.56
3.87 ± 0.57
Self-awareness***
2.93 ± 0.58
3.29 ± 0.60
3.12 ± 0.59
3.30 ± 0.61
Effective learning
3.48 ± 0.56
3.71 ± 0.58
3.50 ± 0.59
3.68 ± 0.60
Problem solving
3.30 ± 0.55
3.47 ± 0.58
3.39 ± 0.57
3.56 ± 0.57
Overall*
3.30 ± 0.49
3.52 ± 0.52
3.41 ± 0.51
3.59 ± 0.52
SDLRS
Learning motivation
3.47 ± 0.63
3.58 ± 0.63
3.46 ± 0.58
3.65 ± 0.59
Active learning
3.72 ± 0.47
3.73 ± 0.56
3.66 ± 0.51
3.81 ± 0.48
Love of learning
3.48 ± 0.58
3.55 ± 0.56
3.43 ± 0.55
3.61 ± 0.55
Independent learning
3.46 ± 0.44
3.37 ± 0.53
3.39 ± 0.51
3.30 ± 0.58
Creative learning
3.51 ± 0.63
3.57 ± 0.61
3.46 ± 0.62
3.60 ± 0.62
Effective learning
3.04 ± 0.70
3.29 ± 0.68
3.11 ± 0.63
3.31 ± 0.68
Overall
3.45 ± 0.43
3.53 ± 0.46
3.42 ± 0.43
3.57 ± 0.43
Flipped classroom Satisfaction
Teacher’s teaching***
3.82 ± 0.55
4.13 ± 0.51
Course content***
3.72 ± 0. 61
3.97 ± 0. Evaluation of the differences between pre- and post-
intervention scores on SDLRS 63
Learning environment***
3.97 ± 0.61
4.20 ± 0.57
Administration service***
3.73 ± 0.66
4.03 ± 0.61
Overall***
3.83 ± 0.55
4.11 ± 0.47
CG Control group, EG Experimental group, SECC Self-Evaluated Core Competencies Scale, MINS Metacognitive Inventory for Nursing Students, SDLRS Self-Directed
Learning Readiness Scale *p < 0 05 **p < 0 01 ***p < 0 001 (Independent samples t-test was used to identify the statistical significance in the pre-test between Overall*** CG Control group, EG Experimental group, SECC Self-Evaluated Core Competencies Scale, MINS Metacognitive Inventory for Nursing Students, SDLRS Self-Directed
Learning Readiness Scale. *p < 0.05, **p < 0.01, ***p < 0.001. (Independent samples t-test was used to identify the statistical significance in the pre-test between
the control and experimental groups) g
the control and experimental groups) Discussion Discussion Evaluation of the differences between pre- and post-
i t
ti
th
SECC Evaluation of the differences between pre- and post-
i t
ti
th
SECC The results showed that the overall level of satis-
faction was 3.99 ± 0.50, approaching a score of 4
(agree). The
EG
demonstrated
high
satisfaction
scores overall and for all four subscales (all p
values < .001). Evaluation of the differences bet
intervention scores on the SECC This study showed that our flipped classroom teaching
approach has a potential to create positive learning out-
comes in terms of clinical skills, communication, and Fan et al. BMC Medical Education (2020) 20:317 Page 7 of 11 Table 2 Summary of GEE analysis results regarding the pre- and post-intervention SECC scores
Parameter
Intercept
Group (E vs. C)
Time (Post vs. Pre)
Interaction between groups by time
Variable
B
P
B
P
B
P
B
P
Basic biomedical Science
4.33
< .001
−0.10
.393
0.27
< .001
0.12
.054
General clinical skills
4.89
< .001
−0.23
.039
0.16
.001
0.24
< .001
Communication and teamwork capability
5.50
< .001
−0.20
.092
0.05
.323
0.22
.001
Critical thinking and Reasoning
4.55
< .001
−0.17
.192
0.29
< .001
0.19
.009
Caring
5.77
< .001
−0.37
.003
−0.04
.451
0.33
< .001
Ethics
6.09
< .001
−0.46
<.001
−0.12
.024
0.39
< .001
Accountability
5.75
< .001
−0.26
.035
0.03
.579
0.25
< .001
Life-long learning
5.26
< .001
−0.10
.480
0.14
.019
0.14
.073
Estimated competence after graduation
4.49
< .001
0.23
.139
0.37
< .001
−0.06
.526
Overall
5.29
< .001
−0.23
.020
0.09*
.023
0.23
< .001
GEE Generalized estimating equation, SECC Self-Evaluated Core Competencies Scale, E Experimental group, C Control group; B indicates the estimated parameter
derived from GEE analysis Table 2 Summary of GEE analysis results regarding the pre- and post-intervention SECC scores students who enter the RN-to-BSN program [1]. These
results may have been driven by the advantages of the
flipped classroom approach, which provides students
with more peer communication, knowledge validation
(during TBL and simulation activities), use of real clin-
ical cases to engage student’s visually, and flexible access
to materials [8, 20, 33]. teamwork capabilities, as well as the competencies of
critical thinking, caring, work ethic, and accountability. Our findings were partially consistent with those of Jang
and Hong [31] (critical thinking), Durmaz et al. Discussion [17] (ad-
mission skills), Kim and Kim [23] (fundamental nursing
practice course), Gerdsprasert et al. [19] (intrapartum
care competency), McMullan et al. [24] (drug calculation
ability), Bloomfield et al. [16] (handwashing skills), Sas-
sen et al. [32] (shared decision making), and Kaveevi-
vitchai et al. [21] (vital signs assessment skill). Our
findings also provided evidence that flipped classrooms
not only improve students’ clinical skills but also en-
hance their higher-level competencies (communication
and teamwork capabilities, critical thinking, caring, work
ethic, and accountability), which are required by ADN Evaluation of the differences between pre- and post-
intervention scores on the MINS To develop higher-level nursing competencies, such as
critical
thinking
and
analysis,
the
integration
of
evidence-based
practice,
problem-solving
skills,
etc.,
which are influenced by nursing students’ levels of com-
fort, confidence, and self-efficacy, is necessary [34, 35]. A Fig. 2 Results of the GEE analysis regarding the pre- and post-intervention on overall SECC Fig. 2 Results of the GEE analysis regarding the pre- and post-intervention on overall SECC Fan et al. BMC Medical Education (2020) 20:317 Page 8 of 11 Table 3 Summary of GEE analysis results regarding the pre- and post-intervention MINS scores
Parameter
Intercept
Group (E vs. C)
Time (Post vs. Pre)
Interaction between group by time
Variable
B
P
B
P
B
P
B
P
Self-monitoring
2.96
<.001
0.27
.010
0.24
<.001
−0.08
.159
Self-modification
3.54
<.001
−0.11
.225
0.10
.015
0.12
.033
Self-awareness
2.57
<.001
0.37
<.001
0.36
<.001
−0.18
<.001
Effective learning
3.26
<.001
0.05
.584
0.22
<.001
−0.04
.507
Problem solving
3.13
<.001
0.10
.265
0.17
<.001
−0.01
.909
Overall
3.09
<.001
0.14
.058
0.22
<.001
−0.04
.399
GEE Generalized estimating equation, MINS Metacognitive Inventory for Nursing Students, E Experimental group, C Control group, B indicates the estimated
parameter derived from GEE analysis Table 3 Summary of GEE analysis results regarding the pre- and post-intervention MINS scores have been because they recognized their limitations and
changed their learning strategies to improve their adjustment
to the ever-changing health care environment [29, 42]. person’s understanding of his or her own learning pro-
cesses is known as metacognition [36], and this consists
of awareness, cognitive strategy, planning, and self-
checking, which are important for allowing nursing stu-
dents to engage in clinical learning and problem solving
[37, 38]. Our findings showed that our flipped classroom
teaching approach could be a beneficial strategy for the
development of metacognitive ability, and this method
may enhance students’ higher-level nursing competen-
cies and critical thinking, which was consistent with the
findings of Hsu and Hsieh [39] and Jang and Hong [31]. In particular, the simulation was the preferred activity of
the flipped classroom, which placed an emphasis on the
direct translation of knowledge to practice rather than
knowledge for its own sake [33, 40, 41]. Authors’ contributions Authors’ contributions
JYF and SWJ contributed in designing the study, YJT, LFC and SLC collected
the data, and analyzed by JYF. The final report and article were written by
JYF and SWJ and all authors were read and approved. Authors contributions
JYF and SWJ contributed in designing the study, YJT, LFC and SLC collected
the data, and analyzed by JYF. The final report and article were written by
JYF and SWJ and all authors were read and approved. Regarding future studies, factors that may influence
students’ learning outcomes, such as degree of motiv-
ation, learning style, and frequency and duration of Acknowledgements The authors wish to acknowledge all 485 participants for the time and effort
they contributed to this study. Flipped classroom satisfaction questionnaire Flipped classroom satisfaction questionnaire Flipped classroom satisfaction questionnaire Flipped classroom satisfaction questionnaire accessing online resources, must be considered. Further,
the application of a randomized clinical trial study de-
sign and rigorous control for heterogeneity factors dur-
ing the intervention period may confirm the flipped
classroom’s probable effects. Our finding that the flipped classroom produced high satis-
faction scores at the end of the semester is consistent with
those of several studies [18, 21, 24, 25, 45]. We believe the
flipped classroom teaching approach was highly accepted by
students for two reasons. First, the advantages of an asyn-
chronous e-learning portion, including its flexibility, conven-
tion, and the capacity for students to be self-paced, catered
to different learning style as well and met individual educa-
tional needs. Second, the face-to-face portion provided more
peer interaction and immediate support from teachers when
questions arose [9, 33, 46, 47]. Abbreviations
d RN: Registered nurse; ADN: Associate Degree in Nursing; BSN: Bachelor of
Science in Nursing; PBL: Problem-based learning; TBL: Team-based learning;
BL: Blended learning; L campus: Linkou; C campus: Chiayi; EG: Experimental
group; CG: Control group; SECC: Self-Evaluated Core Competencies Scale;
MINS: Metacognitive Inventory for Nursing Students; SDLRS: Self-Directed
Learning Readiness Scale; GEE: Generalized estimating equation Limitations and suggestions for future research This study is not without limitations. First, students were
self-report and not blinded to the purpose of the study and,
given that both groups were administered the same program
at the same institution, cross-contamination may have oc-
curred. While there were no significant differences in age
and gender, it is not possible to secure homogeneity for the
factors influencing the results of this study in addition to age
and gender. Thus, the generalizability of the findings is lim-
ited. Second, faculty members on both campuses could have
communicated or shared their teaching strategies, which
may have influenced the results. Third, the nursing faculty
experienced challenges in terms of producing online teach-
ing materials. Lastly, we did not monitor the students on
whether they familiarized themselves with material via the e-
campus platform, which is something to be considered in fu-
ture research as it may have affected our results. Conclusions This study showed that a flipped classroom combined
with hybrid teaching methods could be an effective learn-
ing strategy for an RN-to-BSN program. The students in
this program were satisfied the flipped classroom teaching
and demonstrated improvements in core competencies,
metacognitive abilities, and self-directed learning. Thus,
we feel the flipped classroom approach is one of the most
suitable teaching methods for today’s complex revolution
in nursing curricula, and may enhance nursing students’
abilities to address numerous challenges. Evaluation of the differences between pre- and post-
intervention scores on SDLRS Self-directed learning, a central element in e-learning,
has been widely used in professional health care disci-
plines; however, there is little or mixed evidence con-
cerning its impact on learning outcomes [43, 44]. Our
findings indicated that those in the EG showed a higher
interaction pattern in terms of overall SDL scores, active
learning, and love of learning than those in the CG. This
differed from the findings of Gagnon et al. [43], indicat-
ing that BL has no direct impact on knowledge acquisi-
tion, satisfaction, or self-learning readiness. This result
might come from increasing a student’s motivation to-
ward a self-directed learning habit. Specifically, the ac-
tive learning and love of learning sub-domains are two
crucial constructs in nursing students’ life-long learning
core competencies, and these skills may help them ad-
dress clinical problems of varying difficulties in a wide
range of situations. Our study also demonstrated that those in the EG had sig-
nificantly higher scores in the self-monitoring domain, which
plays a crucial role in problem-solving. This indicates that
students in the EG may have monitored their knowledge,
which increased their awareness of effective skills for moni-
toring their progress toward obtaining solutions for the is-
sues at hand [39]. However, the results regarding the self-
awareness subdomain were the opposite, with the CG show-
ing a significant improvement in self-awareness. This may Table 4 Summary of GEE analysis results regarding the regarding the pre- and post-intervention SDLRS scores
Parameter
Intercept
Group (E vs. C)
Time (Post vs. Pre)
Interaction
between group by
time
Variable
B
P
B
P
B
P
B
P
Learning motivation
3.36
<.001
−0.10
.246
0.11
.004
0.09
.065
Active learning
3.71
<.001
−0.19
.010
0.01
.688
0.13
.003
Love learning
3.40
<.001
−0.14
.090
0.08
.042
0.10
.032
Independent learning
3.54
<.001
−0.07
.311
−0.09
.014
0.00
.915
Creative learning
3.44
<.001
−0.13
.184
0.06
.154
0.08
.127
Effective learning
2.80
<.001
0.12
.213
0.24
<.001
−0.05
.334
Overall
3.40
<.001
−0.09
.115
0.07
.009
0.07
.039
GEE Generalized estimating equation, SDLRS Self-Directed Learning Readiness Scale. E Experimental group, C Control group, B indicates the estimated parameter
derived from GEE analysis Fan et al. BMC Medical Education (2020) 20:317 Page 9 of 11 Fig. 3 Results of the GEE analysis regarding the pre- and post-intervention on overall SDLRS References 27. Bloom BS. Recent developments in mastery learning. Educ Psychol. 1973;10:53. 1. Institute of Medicine. The future of nursing: Leading change, advancing
health. 2010. http://books.nap.edu/openbook.php?record_id=12956&page=
R1. Accessed 23 Feb 2018. 1. Institute of Medicine. The future of nursing: Leading change, advancing
health. 2010. http://books.nap.edu/openbook.php?record_id=12956&page=
R1. Accessed 23 Feb 2018. 28. Hsu LL, Hsieh SI. Testing of a measurement model for baccalaureate nursing
students’ self-evaluation of core competencies. J Adv Nurs. 2009;65:2454–63. https://doi.org/10.1111/j.1365-2648.2009.05124.x. 2. Chen YH, Duh YJ, Feng YF, Huang YP. Preceptors’ experiences training new
graduate nurses: a hermeneutic phenomenological approach. J Nurs Res. 2011;19:132–40. 29. Hsu LL. Metacognitive inventory for nursing students in Taiwan: instrument
development and testing. J Adv Nurs. 2010;66:2573–81. https://doi.org/10. 1111/j.1365-2648.2010.05427.x. 3. Fan JY, Wang YH, Chao LF, Jane SW, Hsu LL. Performance evaluation of
nursing students following competency-based education. Nurse Educ
Today. 2015;35:97–103. https://doi.org/10.1016/j.nedt.2014.07.002. 30. Tang Y-L. Adult teaching and self-directed learning (in Chinese). Wunan:
Taipei; 1995. 31. Jang H-J, Hong S-Y. The effects of blended learning in nursing education
on critical thinking and learning satisfaction of nursing students. Adv Sci
Technol Letters. 2016;122:100–3. https://doi.org/10.14257/astl.2016.122.19. 31. Jang H-J, Hong S-Y. The effects of blended learning in nursing education
on critical thinking and learning satisfaction of nursing students. Adv Sci
Technol Letters. 2016;122:100–3. https://doi.org/10.14257/astl.2016.122.19. 4. Oyelana O, Martin D, Scanlan J, Temple B. Learner-centred teaching in a
nonlearner-centred world: an interpretive phenomenological study of the lived
experience of clinical nursing faculty. Nurse Educ Today. 2018;67:118–23. experience of clinical nursing faculty. Nurse Educ Today. 2018;67:118–23 32. Sassen B, Kok G, Schepers J, Vanhees L. Supporting health care professionals
to improve the processes of shared decision making and self-management
in a web-based intervention: randomized controlled trial. J Med Internet
Res. 2014;16:e211. 5. Sittner BJ, Aebersold ML, Paige JB, Graham LL, Schram AP, Decker SI, Lioce
L. INACSL standards of best practice for simulation: past, present, and future. Nurs Educ Perspect. 2015;36:294–8. 6. Ministry of Education, Republic of China (Taiwan). Educational system:
introduction. 2017. http://english.moe.gov.tw/ct.asp?xItem=15742&CtNode=
11434&mp=1. Accessed 23 Feb 2018. 33. Lawn S, Zhi X, Morello A. An integrative review of e-learning in the delivery
of self-management support training for health professionals. BMC Med
Educ. 2017;17:183. https://doi.org/10.1186/s12909-017-1022-0. p
7. Nightingale College. RN-TO-BSN Program, vol. 2019; 2019. https://
i h i
l
d /
b
/
b
fi / 7. Nightingale College. RN-TO-BSN Program, vol. 2019; 2019. https:// 34. Bressington DT, Wong WK, Lam KKC, Chien WT. Funding
Th
k This work was supported by the Ministry of Science and Technology [MOST
104–2511-S-255-002]. This research was also supported by a grant from the Page 10 of 11 Page 10 of 11 Page 10 of 11 Fan et al. BMC Medical Education (2020) 20:317 Fan et al. BMC Medical Education (2020) 20:317 Administration Center of the Medical Research Department, Chang Gung
Memorial Hospital, Taiwan (BMRPB80), which was awarded to the first author
and corresponding author. 15. Schmidt SMP, Ralph DL. The flipped classroom: a twist on teaching. Contemp Issue Educ Res. 2016;9:1. 15. Schmidt SMP, Ralph DL. The flipped classroom: a twist on teaching. Contemp Issue Educ Res. 2016;9:1. 16. Bloomfield J, Roberts J, While A. The effect of computer-assisted learning
versus conventional teaching methods on the acquisition and retention of
handwashing theory and skills in pre-qualification nursing students: a
randomized controlled trial. Int J Nurs Stud. 2010;47:287–94. 16. Bloomfield J, Roberts J, While A. The effect of computer-assisted learning
versus conventional teaching methods on the acquisition and retention of
handwashing theory and skills in pre-qualification nursing students: a
randomized controlled trial. Int J Nurs Stud. 2010;47:287–94. Received: 1 April 2020 Accepted: 10 September 2020 26. Shiau S, Kahn LG, Platt J, Li C, Guzman JT, Kornhauser Z, et al. Evaluation of
a flipped classroom approach to learning introductory epidemiology. BMC
Med Educ. 2018;18:63. Availability of data and materials The datasets used and/or analysed during the current study are available
from the corresponding author on reasonable request. 17. Durmaz A, Dicle A, Cakan E, Cakir S. Effect of screen-based computer
simulation on knowledge and skill in nursing students’ learning of
preoperative and postoperative care management: a randomized controlled
study. Comput Inform Nurs. 2012;30:196–203. 17. Durmaz A, Dicle A, Cakan E, Cakir S. Effect of screen-based computer
simulation on knowledge and skill in nursing students’ learning of
preoperative and postoperative care management: a randomized controlled
study. Comput Inform Nurs. 2012;30:196–203. Competing interests Competing interests
The authors declare that they have no competing interests. 21. Kaveevivitchai C, Chuengkriankrai B, Luecha Y, Thanooruk R, Panijpan B,
Ruenwongsa P. Enhancing nursing students’ skills in vital signs assessment
by using multimedia computer-assisted learning with integrated content of
anatomy and physiology. Nurse Educ Today. 2009;29:65–72. Consent for publication
Not applicable. 20. Hsu LL. Qualitative assessment of a blended learning intervention in an
undergraduate nursing course. J Nurs Res. 2012;20:291–9. https://doi.org/10. 1097/jnr.0b013e31827363bc. Author details
1 1Department of Nursing & Graduate Institute of Nursing, Chang Gung
University of Science and Technology, Division of Nursing, Chang Gung
Memorial Hospital, Linkou Branch, 261, Wen-Hua 1st Road, Kwei-Shan,
Tao-Yuan 33303, Taiwan (R.O.C.). 2Department of Nursing, Chang Gung
University of Science and Technology, 261, Wen-Hua 1st Road, Kwei-Shan,
Tao-Yuan 33303, Taiwan (R.O.C.). 3Department of Nursing, National Taichung
University of Science and Technology, No.129, Sec. 3, Sanmin Rd., North Dist.,
Taichung City 40401, Taiwan (R.O.C.). 4Department of Nursing & Graduate
Institute of Nursing, Chang Gung University of Science and Technology, 261,
Wen-Hua 1st Road, Kwei-Shan, Tao-Yuan 33303, Taiwan (R.O.C.). 22. Keefe G, Wharrad H. Using e-learning to enhance nursing students’ pain
management education. Nurse Educ Today. 2012;32:66–72. 23. Kim H, Kim YH. An action research on flipped learning for fundamental
nursing practice courses. J Korean Acad Fundam Nurs. 2017;24:265–76. 24. McMullan M, Jones R, Lea S. The effect of an interactive e-drug calculations
package on nursing students’ drug calculation ability and self-efficacy. Int J
Med Inform. 2011;80:421–30. 24. McMullan M, Jones R, Lea S. The effect of an interactive e-drug calculations
package on nursing students’ drug calculation ability and self-efficacy. Int J
Med Inform. 2011;80:421–30. 25. Missildine K, Fountain R, Summers L, Gosselin K. Flipping the classroom to
improve student performance and satisfaction. J Nurs Educ. 2013;52:597–9. 25. Missildine K, Fountain R, Summers L, Gosselin K. Flipping the classroom to
improve student performance and satisfaction. J Nurs Educ. 2013;52:597–9. Ethics approval and consent to participate This study was approved by an institutional review board (IRB No. 104-
5709C) before data collection. All participants gave written informed consent
to participate. 18. Gerdsprasert S, Pruksacheva T, Panijpan B, Ruenwongsa P. Development of
a web-based learning medium on mechanism of labour for nursing
students. Nurse Educ Today. 2010;30:464–9. 18. Gerdsprasert S, Pruksacheva T, Panijpan B, Ruenwongsa P. Development of
a web-based learning medium on mechanism of labour for nursing
students. Nurse Educ Today. 2010;30:464–9. 19. Gerdsprasert S, Pruksacheva T, Panijpan B, Ruenwongsa P. An interactive
web-based learning unit to facilitate and improve intrapartum nursing care
of nursing students. Nurse Educ Today. 2011;31(5):531–5. 19. Gerdsprasert S, Pruksacheva T, Panijpan B, Ruenwongsa P. An interactive
web-based learning unit to facilitate and improve intrapartum nursing care
of nursing students. Nurse Educ Today. 2011;31(5):531–5. References BMC Medical Education (2020) 20:317 Fan et al. BMC Medical Education (2020) 20:317 40. Corbridge SJ, Robinson FP, Tiffen J, Corbridge TC. Online learning versus
simulation for teaching principles of mechanical ventilation to nurse
practitioner students. Int J Nurs Educ Scholarsh. 2010;7:12. 41. McGaghie WC, Siddall VJ, Mazmanian PE, Myers J. Lessons for continuing
medical education from simulation research in undergraduate and graduate
medical education: effectiveness of continuing medical education: American
College of Chest Physicians evidence-based educational guidelines. Chest. 2009;135:62S–8S. 42. Anema MG, McCoy J. Competency-based nursing education: guide to
achieving outstanding learner outcomes. New York: Springer Publishing
Company; 2010. 42. Anema MG, McCoy J. Competency-based nursing education: guide to
achieving outstanding learner outcomes. New York: Springer Publishing
Company; 2010. 43. Gagnon M, Gagnon J, Desmartis M, Njoya M. The impact of blended
teaching on knowledge, satisfaction, and self-directed learning in nursing
undergraduates: a randomized, controlled trial. Nurse Educ Perspect. 2013;
34:377–82. 44. Sinclair PM, Kable A, Levett-Jones T, Booth D. The effectiveness of internet-
based e-learning on clinician behaviour and patient outcomes: a systematic
review. Int J Nurs Stud. 2016;57:70–81. 45. Sowan AK, Jenkins LS. Use of the seven principles of effective teaching to
design and deliver an interactive hybrid nursing research course. Nurse
Educ Perspect. 2013;34:315–22. 46. Chiu Y-L, Tsai C-C, Fan Chiang C-Y. The relationships among nurses’ job
characteristics and attitudes toward web-based continuing learning. Nurs
Educ Today. 2013;33:327–33. 47. Fu XH, Zhang JL, Li RQ. Application of flipped classroom combined with
TBL teaching in surgical nursing teaching in higher vocational colleges. Health Vocational Education. 2017;35:73–75. References Concept mapping to
promote meaningful learning, help relate theory to practice and improve
learning self-efficacy in Asian mental health nursing students: a mixed-
methods pilot study. Nurse Educ Today. 2018;60:47–55. nightingale.edu/rn-to-bsn/program-benefits/. nightingale.edu/rn-to-bsn/program-benefits/. 8. Persky AM, McLaughlin JE. The flipped classroom—from theory to practice
in health professional education. Am J Pharm Educ. 2017;8:118. https://doi. org/10.5688/ajpe816118. 8. Persky AM, McLaughlin JE. The flipped classroom—from theory to practice
in health professional education. Am J Pharm Educ. 2017;8:118. https://doi. org/10.5688/ajpe816118. 35. Pijl-Zieber EM, Barton S, Konkin J, Awosoga O, Caineet V. Competence and
competency-based nursing education: finding our way through the issues. Nurse Educ Today. 2013. https://doi.org/10.1016/j.nedt.2013.09.007. 9. Sajid MR, Laheji AF, Abothenain F, Salam Y, AlJayar D, Obeidat A. Can
blended learning and the flipped classroom improve student learning and
satisfaction in Saudi Arabia? Int J Med Educ. 2016;7:281–5. 36. Ormrod JE. Human learning. 2nd ed. Englewood Cliffs: Prentice Hall; 1995. 10. Tugun V, Uzunboylu H, Ozdamli F. Coding education in a flipped classroom. TEM J. 2017;6:599–606. 37. Deng XB, Xiao YL, Yang HH, Wang XF, Wu CX, Xiong W. Application of
flipped classroom teaching model based on micro video in practical
nursing teaching of midwifery profession. Contemp Med Symposium. 2017;
15:143–4. 11. Kim H, Jang Y. Flipped learning with simulation in undergraduate nursing
education. J Nurs Educ. 2017;56:329–36. 12. McLaughlin JE, Roth MT, Glatt DM, Gharkholonarehe N, Davidson CA, Griffin
LM, et al. The flipped classroom: a course redesign to foster learning and
engagement in a health professions school. Acad Med. 2014;89:236–43. 12. McLaughlin JE, Roth MT, Glatt DM, Gharkholonarehe N, Davidson CA, Griffin
LM, et al. The flipped classroom: a course redesign to foster learning and
engagement in a health professions school. Acad Med. 2014;89:236–43. 38. Yan LX, Xie HY. Application of flipped classroom teaching model in nursing
comprehensive skill training. J Adv Nurs Educ. 2015;30:626–8. 39. Hsu L, Hsieh S. Effects of a blended learning module on self-reported
learning performances in baccalaureate nursing students. J Adv Nurs. 2011;
67:2435–44. https://doi.org/10.1111/j.1365-2648.2011.05684.x. 13. Persky AM, Pollack GM. A modified team-based learning physiology course. Am J Pharm Educ. 2011;75:204. 13. Persky AM, Pollack GM. A modified team-based learning physiology course. Am J Pharm Educ. 2011;75:204. 14. Mok HN. Teaching tip: the flipped classroom. J Inform Sys Educ. 2014;25:7. 14. Mok HN. Teaching tip: the flipped classroom. J Inform Sys Educ. 2014;25:7. Page 11 of 11 Fan et al. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations. Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
|
W2978060396.txt
| null |
en
|
Lying in Wait: Modeling the Control of Bacterial Infections via Antibiotic-Induced Proviruses
|
MSystems
| 2,019
|
cc-by
| 10,321
|
RESEARCH ARTICLE
Ecological and Evolutionary Science
Lying in Wait: Modeling the Control of Bacterial Infections via
Antibiotic-Induced Proviruses
Sara M. Clifton,a Ted Kim,b Jayadevi H. Chandrashekhar,b George A. O’Toole,c Zoi Rapti,a,d Rachel J. Whitakerb,d
a
Department of Mathematics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
b
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
c
Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, New Hampshire, USA
d
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA
ABSTRACT Most bacteria and archaea are infected by latent viruses that change
their physiology and responses to environmental stress. We use a population model
of the bacterium-phage relationship to examine the role that latent phage play in
the bacterial population over time in response to antibiotic treatment. We demonstrate that the stress induced by antibiotic administration, even if bacteria are resistant to killing by antibiotics, is sufficient to control the infection under certain conditions. This work expands the breadth of understanding of phage-antibiotic synergy
to include both temperate and chronic viruses persisting in their latent form in bacterial populations.
IMPORTANCE Antibiotic resistance is a growing concern for management of common
bacterial infections. Here, we show that antibiotics can be effective at subinhibitory levels when bacteria carry latent phage. Our findings suggest that specific treatment
strategies based on the identification of latent viruses in individual bacterial strains
may be an effective personalized medicine approach to antibiotic stewardship.
KEYWORDS bacteria, bacteriophage, temperate, phage, chronic, latent, lytic,
lysogenic, Pseudomonas aeruginosa, cystic fibrosis, resistance, population dynamics,
mathematical model, antibiotic resistance, latent infection, mathematical modeling
A
worldwide growth of antibiotic resistance threatens the efficacy of antibiotic
treatments for common infections, driving medical professionals to seek alternative treatments (1). Infections by Pseudomonas aeruginosa alone represent about 10%
of nosocomial infections, are a leading cause of death among patients with cystic
fibrosis (CF), and have been deemed a serious threat on the United States Centers for
Disease Control and Prevention watch list for antibiotic resistance (2–4). Despite the
increasing trend of multidrug resistance, antibiotic regimes remain the consensus first
treatment for P. aeruginosa infection (5). As a last resort and as an attempt to prevent
the evolution of resistance in P. aeruginosa, clinicians have turned to combination
therapies (6) with bacteriophage (viruses) and antibiotics to treat recalcitrant bacteria.
Synergy between phage and antibiotic treatment (PAS) is now rising in interest for
treatment of P. aeruginosa and other recalcitrant bacteria (7–9). Combination phage
therapy uses viruses that kill bacteria (often in phage cocktails) and different types of
antibiotics either at the same time or in series to clear bacteria and prevent the
evolution of new resistant phenotypes (10–18). Although preexisting proviruses are
highly prevalent in P. aeruginosa infections and appear to be induced by certain
antibiotic treatments, synergy has not been considered in the context of temperate
virus induction. Here, we investigate the role that phages play during antibiotic
treatment when they are already present in the system. We show that, even without
September/October 2019 Volume 4 Issue 5 e00221-19
Citation Clifton SM, Kim T, Chandrashekhar JH,
O’Toole GA, Rapti Z, Whitaker RJ. 2019. Lying in
wait: modeling the control of bacterial
infections via antibiotic-induced proviruses.
mSystems 4:e00221-19. https://doi.org/10
.1128/mSystems.00221-19.
Editor Katrine L. Whiteson, University of
California, Irvine
Copyright © 2019 Clifton et al. This is an openaccess article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Sara M. Clifton,
smc567@illinois.edu.
Received 1 April 2019
Accepted 30 August 2019
Published 1 October 2019
msystems.asm.org 1
Clifton et al.
deliberate phage therapy, phages may play a critical role in antibiotic treatment,
especially if the bacteria are antibiotic resistant.
Background. Bacteriophages are viruses that infect bacteria and hijack cell functions in order to reproduce. Just as bacteria have evolved many strategies to evade
infection, phages have developed multiple strategies to circumvent cell defenses.
Phages can be characterized by their lifestyles (obligately lytic, temperate, or chronic)
within the host (19). Lytic viruses replicate within the host and kill host cells by bursting
them open to release new particles. Temperate viruses have a lytic cycle but can also
integrate into host genomes, where they remain latent until they are induced to
replicate (19). In chronic infection, productive host cells shed new phages that bud from
the cell without killing the bacterium (20). Both temperate and chronic viruses have a
lysogenic (latent lytic or latent chronic) cycle in which phage DNA is incorporated into
the bacterium’s genome, and the cell transmits the phage’s genetic material (prophage) to daughter cells vertically (21).
Comparative genomics among closely related bacterial strains has uncovered a
plethora of proviruses of both temperate and chronic lifestyles (22–24). The large
genome of the opportunistic pathogen P. aeruginosa is no exception (25–27). Each
sequenced strain reveals multiple proviral genomes of both the temperate and chronic
lifestyles, each in both active and inactive (latent) forms (28). These proviruses change
bacterial fitness and environmental response, sometimes conferring competitive advantage, virulence, and antibiotic resistance (29–32).
Stressful environmental conditions (e.g., radiation, heat, and sublethal antibiotics)
may trigger the cell to induce latent prophage and begin phage production (33–37).
The induction of such latent phages is proposed to be one of the mechanisms behind
the synergistic effect of antibiotics and phage infection (37, 38). The environmental
conditions, especially dynamic antibiotic dosing regimes, under which these phage
types may coexist are not well understood. We therefore develop a population model
to understand the impact of antibiotics on the bacterium-phage system with multiple
phage strategies and antibiotic resistance. We address conditions under which the
bacterium-phage-antibiotic ecosystem results in control of the bacterial infection (14).
Previous work. Many mathematical models of bacterium-phage systems exist at
various levels of complexity. The simplest models include only one phage strategy
(lysis); in this simple scenario, either all bacteria are affected by the phage (39) or some
bacteria are resistant to infection (40). More complex models study the competition
between two different phage strategies, such as lysis and lysogeny (41) or lysis and
productive chronic infection (42). The scope of many studies is extended to also include
interactions among bacteria, phages, the host’s immune response, and/or antibiotic
treatment. The immune response and antibiotic agent have been modeled implicitly by
modifying the rates of change of bacteria and phages (40) or explicitly by adding
compartments governing antibiotic and immune response rate of change (43–45).
Other distinctions among models of bacterial infections can be made based on how
bacteria reproduce. Mechanistic models incorporate a limited nutrient as an additional
compartment (45–47), while more phenomenological models assume that bacteria
grow logistically (39, 41, 48, 49). Furthermore, many models are used to study bacterial
evolution of resistance to either phages (45, 47) or antibiotics (50). These models are
either deterministic (47) or stochastic (45, 50).
Phage and antibiotic synergy has been investigated experimentally using phage
isolated from wastewater or other sources. Attention has primarily been paid to the
breadth of killing that lytic phage exhibit on a diversity of P. aeruginosa strains, while
little attention has been given to other parts of the phage lifestyle. Accordingly, models
for phage-antibiotic synergy incorporate only the killing aspects of viruses (14). These
models suggest that pretreatment with phage decreases the bacteria to a low-enough
level that antibiotics can extinguish bacterial populations; they do not yet consider
potential for phage to spread within a population and be induced by antibiotic treatment
at a later time.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 2
Modeling Control of Bacterial Infection via Proviruses
Consideration has been given to the impact of antibiotic treatment on the mobilization of temperate phage genetic material (including antibiotic resistance genes)
between cells via transduction (51, 52). However, to our knowledge, no mathematical
models of bacterium-phage interaction have analyzed the competition between temperate and chronic phage strategies in an environment with pulses of antibiotic stress,
as would happen during treatment. Filling this knowledge gap is critical to understanding the impact of antibiotic treatment on a patient infected with the bacterium P.
aeruginosa.
RESULTS
First, we examine the model without antibiotic administration. Without external
stress, the bacterial population eventually stabilizes at carrying capacity, with doubly
infected productive bacteria dominating the population (Fig. 1). Because we have
assumed that infection by one phage type does not prevent infection by a different
type (i.e., no cross-infection exclusion) and that coinfection does not impose a fitness
cost on bacteria, eventually all bacteria are infected with both phages.
Productive bacteria dominate the population because, initially, populations of bacteria latently infected with temperate phage increase faster than those latently infected
with chronic phage due to the early rapid proliferation of temperate phage. Subsequently the productive strains dominate since they are formed at a much higher frequency
on secondary infection than either latent infection. With a substantial population of
chronically infected bacteria producing phage at steady state, the ratio of free chronic
phage to bacteria stabilizes at approximately 10:1. Although little is known about the
proportion of phage types seen in either clinical or wild settings, it is known that both
temperate and chronic strains are often found in the same environment (53). Figure 2
shows a visualization of the dominant path through the model system without antibiotics.
Antibiotic treatment. Next, we examine the model where all bacteria are sensitive
to antibiotics (i.e., bacteria are not resistant to the antibiotic’s intended killing mechanism, namely, inhibiting bacterial DNA replication [54]) using baseline parameter
values (see Table 2). For the purpose of illustration, we choose the period of antibiotic
treatment T ⫽ 7.3, which is one antibiotic dose every 24 h; this is a typical clinical
dosing protocol (55). When all bacteria are sensitive to antibiotics, periodic administration of antibiotic leads to periodic dips in bacterial populations and periodic spikes
in induced free phage (Fig. 3). During antibiotic treatment, the total bacterial population remains well below the carrying capacity, and the ratio of free phage to bacteria
is around 20:1 on average and about 30:1 at most. These values are consistent with
existing studies of bacterium-to-phage ratios (28, 56).
Figure 1 shows that without antibiotic administration, productive bacteria that are
latently carrying the temperate phage are the dominant bacterial strain due to their
high frequency of formation in early stages. With each antibiotic dose, the productive
bacteria are replaced with strains doubly infected by latent phage, which eventually
dominate the system (Fig. 3). This phenomenon occurs because most bacteria that are
共T兲
latently infected with temperate virus (including PCT
) respond to antibiotic stress by
inducing lysis, which brings the number of bacteria to a very low number. The drop in
bacterial population allows the doubly latently infected bacteria (unencumbered by
phage production) to grow slightly faster than productive bacteria and eventually
dominate the population. Antibiotic administration resets the population structure
from one set by initial relative frequencies of latent and active infection to one that is
set by relative fitness (growth rate). The number of free chronic phage decreases over
time because latently infected strains cannot become productive in this model.
To control an infection, there are two primary parameters that can be independently
varied: antibiotic administration period T and antibiotic efficacy . The antibiotic dosing
period and deadliness required to control an infection depend on other model parameters, especially the amplitude of stress caused by antibiotics and the metabolic decay
rate of the antibiotic (Fig. 4). Antibiotics must be administered more frequently if
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 3
Clifton et al.
( )
bacteria population size
4e7
CFU
mL
1
(a)
0.9
Susceptible S
Latent lytic L T
Productive PC
0.8
Latent chronic L C
T
Latent lytic/chronic L CT
0.7
T
Productive/latent lytic PCT
0.6
Latent lytic/chronic
LC
CT
Productive/latent lytic PC
CT
0.5
0.4
0.3
0.2
0.1
0
0
( )
4e8
10
PFU
mL
9
50
100
(b)
150
Free temperate phage V T
Free chronic phage VC
phage population size
8
7
6
5
4
3
2
1
0
0
50
(6.8 days)
100
(13.6 days)
150
(20.4 days)
time
FIG 1 Simulation of population dynamics with no antibiotic administration: bacterial population (a) and free phage population (b).
共T兲
Without antibiotics, the dominant bacterial strain is producing chronic virus while also latently infected with temperate phage 共PCT
兲, and
the only free phage are chronic (VC). All bacteria and phage types are described in Table 1. All parameter values are taken from the baselines
in Table 2, with h ⫽ 1/2, h ⫽ 1, h␥ ⫽ 1. Note that both axes are linear, not logarithmic. Initially, S(0) ⫽ 1e⫺3, VT(0) ⫽ VC(0) ⫽ 1e⫺7, according
to the work of Sinha et al. (41).
antibiotics are less effective at killing bacteria either directly or via induced lysis, or if
antibiotics are metabolized more quickly (Fig. 4a). On the other hand, antibiotics must
be more effective in order to control an infection if antibiotics are administered less
frequently, if antibiotic stress induces lysis less effectively, or if antibiotics are metabolized more quickly (Fig. 4b). See Text S2 in the supplemental material for technical
details on the sensitivity analysis.
Antibiotic resistance. If all bacteria are resistant to antibiotics ( ⫽ 0), then the
population dynamics are qualitatively similar to those when bacteria are sensitive to
antibiotics. In both cases, antibiotic administration causes doubly latently infected
bacteria to dominate the system. However, when all bacteria are antibiotic resistant, the
total bacterial population and phage populations are noticeably larger (Fig. 5).
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 4
Modeling Control of Bacterial Infection via Proviruses
infection
Susceptible (S)
transition
reproduction
stress response
Lytic (IT)
Pre-productive (IC)
Latent Lytic (LT)
Pre-productive
(ICT, T)
Latent Lytic and
Latent Chronic
(LCT, T)
Productive and
Latent Lytic (PCT, T)
dominant path
with antibiotics
dominant path
without antibiotics
Productive (PC)
Latent Chronic (LC)
Lytic (ICT, P)
Lytic (ICT, C)
Productive and
Latent Lytic (PCT, C)
Latent Lytic and
Latent Chronic
(LCT, C)
FIG 2 Full flowchart of bacterium-phage system, corresponding to model system (equations S1 to S15 in Text S1), with results superimposed.
The dominant path through the model compartments without antibiotics is shown in blue, while the dominant path with periodic antibiotic
dosing is shown in red. Skull sketch courtesy of Dawn Hudson (CC0).
Pharmacological implications with antibiotic resistance. The main concern when
treating an infection with antibiotics is the size of the bacterial population. Therefore,
we investigate the total bacterial population under a range of antibiotic dosing
frequencies (Fig. 6). We compute the average total bacterial population over the first
300 bacterial reproductive cycles (40.8 days), and we find that both antibiotics and
temperate phage are critical to controlling the infection and work synergistically even
when bacteria are antibiotic resistant. We define infection control to be an average
bacterial population less than 10% of carrying capacity (i.e., 1-log decrease in bacterial
levels compared with placebo).
If only chronic phage are present in the system (see Fig. S1a in the supplemental
material), effective antibiotics are required to control the infection. If all bacteria are
sensitive to antibiotics, the presence of chronic phage controls the infection slightly
better than if there are no chronic phage due to the cost of production during productive
infection.
If only temperate phage are present in the system (Fig. S1b), infection is controlled
even when bacteria are resistant. In fact, the efficacy of temperate phage alone is
similar to the efficacy of antibiotics alone. With both effective antibiotics and temperate
phage, the number of antibiotic doses required to keep the infection under control is
cut in half compared with antibiotics alone or temperate phage alone.
If both phages are present in the system (Fig. 6), infection control is marginally
better than if only temperate phage are present (Fig. S1b). These results demonstrate
the synergy between temperate phage and antibiotics even in resistant populations.
No deliberate combination therapy may be needed to treat these infections because
temperate phage are commonly found in natural populations of P. aeruginosa bacteria
(53).
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 5
Clifton et al.
( )
bacteria population size
1e7
0.25
CFU
mL
(a)
Susceptible S
Latent lytic L T
Productive PC
0.2
Latent chronic L C
T
Latent lytic/chronic L CT
T
Productive/latent lytic PCT
0.15
Latent lytic/chronic LC
CT
C
Productive/latent lytic PCT
0.1
0.05
0
0
( )
phage population size
2.8e7
PFU
mL
50
100
150
7
(b)
6
Free temperate phage V T
Free chronic phage VC
5
4
3
2
1
0
0
50
(6.8 days)
100
(13.6 days)
150
(20.4 days)
time
FIG 3 Simulation of population dynamics with no antibiotic resistance: bacterial population (a) and free phage population (b). All bacteria
and phage types are described in Table 1. All parameter values are taken from the baselines in Table 2, with h ⫽ 1/2, h ⫽ 1, h␥ ⫽ 1
(see Text S2 in the supplemental material for more details). Antibiotics are administered periodically every T ⫽ 7.3 bacterial reproductive
cycles (once-daily dose). Note that both axes are linear, not logarithmic. Initially, S(0) ⫽ 1e⫺3, VT(0) ⫽ VC(0) ⫽ 1e⫺7, according to the work
of Sinha et al. (41).
DISCUSSION
The model presented here shows that temperate phage infection makes antibiotic
treatment of bacterial infections both more effective and more efficient, whether or not
the bacteria are susceptible to the antibiotics. When bacteria are sensitive to antibiotics,
then antibiotic treatments need not be as frequent if temperate phage are present.
Even if some or all bacterial strains are antibiotic resistant, antibiotics may still be able
to control the infection in the presence of phages by triggering phage induction and
cell lysis. For the rest of the discussion, we will assume that an infection is controlled
if the average total bacterial population remains below 10% of carrying capacity over
300 bacterial reproductive cycles; in clinical terms, control is a 1-log difference between
P. aeruginosa density in sputum for patients given antibiotics versus placebo over
40.8 days.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 6
Modeling Control of Bacterial Infection via Proviruses
(a)
1
*** ***
sensitivity of kappa to model parameters
sensitivity of T to model parameters
1
0
(b)
***
***
0
***
−1
daetaT etaC kappa A
lamb
***
k delta fT
fC
bT
bC bMax d ratioIC
−1
daetaT etaC T
lamb
A
k delta fT
fC
bT
bC bMax d ratioIC
FIG 4 Sensitivity of the antibiotic dosing period T required to control the infection (a) and the antibiotic deadliness required to control the infection (b).
The sensitivity analyses use Latin hypercube sampling (LHS) of parameter space and partial rank correlation coefficients (PRCC) (92). Infection control is an
average total bacterial population below 10% of carrying capacity over 300 bacterial reproductive cycles. All parameter values are taken near the baselines
in Table 2, with h ⫽ 1/2, h ⫽ 1, h␥ ⫽ 1. Initially, S(0) ⫽ 1e⫺3, VT(0) ⫽ 1e⫺7, VC(0) ⫽ ratio IC ⫻ 1e⫺7. The number of simulations is n ⫽ 150. Asterisks indicate
significance (***, P ⬍ 0.001; no asterisks, P ⬎ 0.05). See Text S2 in the supplemental material for technical details.
For P. aeruginosa bacterial infections that respond to antibiotics, the model predicts
that standard antibiotic doses need to be administered approximately every 12.1 h if no
phage are present but only once every 25.1 h if temperate phage are present (Fig. 6).
If bacteria are all antibiotic resistant, then temperate phages are required to control the
infection, and antibiotic dosing is required every 12.6 h to sufficiently induce lysis.
These findings are consistent with clinical evidence; patients with cystic fibrosis (CF)
given aerosolized levofloxacin twice daily experienced a nearly 10-fold decrease in P.
aeruginosa density (our definition of infection control) over the treatment period
compared with the placebo group (57). The study did not investigate the presence of
phage but did note that approximately 60% of P. aeruginosa isolates showed resistance
to levofloxacin, supporting our prediction that dosing should fall between once and
twice daily depending on the susceptibility of the bacteria to antibiotics. Our findings
are also consistent with existing antibiotic dosing protocols; although aerosolized
quinolones are no longer approved for CF patients, intravenous (i.v.) and oral doses are
commonly recommended on a once-, twice-, or three-times-daily schedule (55, 58).
While chronic phages are marginally beneficial in controlling infections, they are not
able to control an infection without either temperate phages or effective antibiotics. In
fact, chronic phages may actually inhibit control of infections by disrupting the human
immune response (59, 60), a detail not yet incorporated into our model.
Like all models, our model has limitations. In the interest of simplicity, we have
ignored the possibility of multiple infections by the same phage type. However, many
phages that infect P. aeruginosa produce superinfection exclusion proteins that effectively prevent multiple infections by the same phage type (61, 62). We also do not
include the exclusion of one phage type by the other. Little is known about crossresistance to phage infection; it is often assumed to be uncommon, but including
cross-resistance may dramatically impact the model predictions. If cross-resistance is in
fact common, it is possible that phage-antibiotic synergy breaks down for some range
of model parameters; we leave this analysis for future study.
Also, our model assumes that antibiotics induce phage, so this model is applicable
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 7
Clifton et al.
( )
bacteria population size
3.2e7 0.8
CFU
mL
(a)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
T
Susceptible S
Latent lytic/chronic L CT
Latent lytic L T
Productive/latent lytic PCT
T
LC
CT
Productive PC
Latent lytic/chronic
Latent chronic L C
Productive/latent lytic P
C
CT
0
0
( )
1.4e9
PFU 35
mL
50
100
150
(b)
phage population size
30
25
20
15
10
Free temperate phage V T
5
Free chronic phage VC
0
0
50
(6.8 days)
100
(13.6 days)
150
(20.4 days)
time
FIG 5 Simulation of population dynamics with complete antibiotic resistance: bacterial population (a) and free phage population (b). All
bacteria and phage types are described in Table 1. All parameter values are taken from the baselines in Table 2, with h ⫽ 1/2, h ⫽ 1,
h␥ ⫽ 1, and ⫽ 0 for all bacteria (see supplemental material for more details). Antibiotics are administered periodically every T ⫽ 7.3
bacterial reproductive cycles (once-daily dose). Initially, S(0) ⫽ 1e⫺3, VT(0) ⫽ VC(0) ⫽ 1e⫺7, according to the work of Sinha et al. (41).
with only quinolone antibiotics like levofloxacin and ciprofloxacin (34). However, drugs
from this class of antibiotics are commonly used to treat P. aeruginosa infections (57, 63).
In addition, some phage are able to detect bacterial population density, which
appears to affect the frequency of lysogeny (64, 65). If this process applies to P. aeruginosa
and its phages, a more sophisticated model would incorporate a density-dependent
latency probability: fT(Btot) and fC(Btot).
The model additionally assumes that bacteria resistant to antibiotics are still susceptible to lysis via phage induction, but this phenomenon depends on the mechanism
of antibiotic resistance. There are many mechanisms of resistance to quinolones and
fluoroquinolones. However, subinhibitory concentrations of several antibiotics are
known to induce SOS but not result directly in cell death (34, 61, 66–68). Therefore, we
model the impact of phage induction on P. aeruginosa population size with and
without antibiotic resistance.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 8
Modeling Control of Bacterial Infection via Proviruses
( )
average bacteria population size
4e7
0
CFU 10
mL
( )
4e6
−1
CFU 10
mL
( )
(placebo)
(1-log decrease)
both phage − sensitive
no phage − sensitive
both phage − resistant
no phage − resistant
4e5
−2
CFU 10
(2-log decrease)
mL
0
0.05
0.15
0.1
(
less antibiotic needed
(infection easier to control)
dose every
25.1 hours
0.2
)
frequency (1/T) of antibiotic dosing
0.25
(
dose every
12.6 hours
0.3
)(
dose every
12.1 hours
)
more antibiotic needed
(infection harder to control)
FIG 6 Average total bacterial population for a range of periodic antibiotic dosing protocols. All parameter values are taken at the
baselines in Table 2, with h ⫽ 1/2, h ⫽ 1, h␥ ⫽ 1, tmax ⫽ 300 (see the supplemental material for more details). Solid lines indicate
that all bacteria are sensitive to antibiotics, and dashed lines indicate that all bacteria are resistant. Note that the vertical axis is
logarithmic, while the horizontal axis is linear. Nondimensional units are supplemented with standard units parenthetically.
Initially, S(0) ⫽ 1e⫺3, VT(0) ⫽ VC(0) ⫽ 1e⫺7, unless otherwise noted.
Because this model does not include an evolutionary dynamics component, the results
presented here are applicable only to acute exacerbations. If bacterium/phage evolution were integrated into this model, it might be able to explain longer-term dynamics
seen in chronic infections in humans (28).
Also, all latent chronic infection states are final such that virus production cannot be
induced by stress. We believe that changing the model structure to accommodate
chronic phage induction might change the number of productive bacteria but would
not change the overall impact of antibiotic synergy, which primarily occurs with
temperate infections.
Finally, the quantitative results presented in Fig. 6 depend significantly on how
effective antibiotics are at killing bacteria directly versus killing via phage induction (
in our model). To our knowledge, no study has experimentally measured the relative
number of bacteria killed by the intended antibiotic mechanism versus phage induction, so we assume that antibiotics kill via each method equally quickly. If antibiotics
directly kill bacteria much more quickly ( ⬎ 1), then antibiotic resistance is more
detrimental to infection control than lack of phages. If antibiotics trigger phage
induction much more quickly ( ⬍ 1), then a lack of phages is more detrimental to
infection control than antibiotic resistance. Experimental work is needed to determine
a reasonable range for and test whether it is an evolvable trait.
Conclusion. Antibiotic resistance threatens the efficacy of standard treatments for
many dangerous and common infections. Using P. aeruginosa infections as motivation,
we present a theoretical case for using antibiotics that trigger phage induction (e.g.,
quinolones) to treat bacterial infections. We show that if bacteria are antibiotic resistant, then using antibiotics in the presence of phages can still control the infection. If
bacteria are susceptible to antibiotics, then the presence of phages allows for lessfrequent antibiotic dosing, which reduces the risk for antibiotic resistance in the future.
In either case, the natural presence of phages in bacterial populations allows for more
effective treatment of common bacterial infections. These, strain-dependent responses
to antibiotics suggest the importance of personalized medicine approaches to treatment of infectious disease.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 9
Clifton et al.
FIG 7 Flowchart of bacterium-phage system with both temperate (orange) and chronic (blue) phages. Boxes
indicate a bacterial state, and arrows indicate an infection by phage. If a bacterium is infected by temperate phage,
the probability of going latent lytic is fT. If a bacterium is infected by chronic phage, the probability of becoming
latent chronic is fC. Skull sketch courtesy of Dawn Hudson (CC0).
As a final perspective, we remember that phage induction and bacterial death may
occur across the microbiome of individual hosts treated with antibiotics. The impact of
these dynamics in a community context must be considered carefully for the stability
of the microbiome ecosystem as a whole.
MATERIALS AND METHODS
Modeling framework. Consider a system of two competing types of bacteriophage (e.g., see
references 41 and 42): one temperate phage VT with lytic and latent lytic stages and one chronic phage
VC with productive and latent stages. During the productive phase of the chronic lifestyle, phage particles
are released through budding and do not kill the host bacterium. Each phage attacks one strain of
bacteria that is initially susceptible to infection by either phage type. Figure 7 shows an overview of the
process; Fig. 8 shows the complete modeling framework.
We assume the total bacterial population Btot grows logistically at a rate ␥ to a carrying capacity K
(69). Each phage infects susceptible bacteria S at a rate . Bacteria infected by the temperate phage VT
will either become latently infected LT with probability fT or will enter a lytic state IT with probability
(1 ⫺ fT). Bacteria in the lytic state produce phage and burst (with burst size T) at a rate ␦. (This modeling
choice circumvents the necessity of a delay differential equation.) While in the lytic state, the phage
hijacks cell functions, and the cell cannot reproduce (70, 71). Bacteria do not move between lytic and
latent states unless there is a perturbation or stress to the system where viruses are induced.
Bacteria infected by the chronic phage VC will either become latently infected LC with probability fC
or will enter a preproductive state IC with probability (1 ⫺ fC). Bacteria in the preproductive state stop
reproducing and prepare to manufacture phage with delay rate ␦. After the production delay, the
preproductive bacteria enter the productive state PC, continue reproducing at a potentially reduced rate
␥, and begin producing phage at a rate C without cell death (72). As above, after chronic phage enter
the latent or productive state in a cell, they will not change state. Latent chronic phage cannot be
induced by stress to become productive; however, productively infected strains produce more phage
under stress and reproduce more slowly. We note that biologically, productively infected strains can
revert to latent infection and latent hosts can induce chronic virus production.
Once a bacterium is infected, we assume that it will exclude superinfection by the same phages but may
be infected by phages of the other type (73). If a bacterium that is latently infected by the temperate phage
is additionally infected with the chronic phage, the bacterium will either become latently infected
共T兲
共T兲
兲 with probability fC or will enter a preproductive state ICT
with probability
with both phages 共LCT
(1 ⫺ fC). Bacteria in the preproductive state stop reproducing and prepare to manufacture phage with
共T兲
delay rate ␦. After the production delay, the infected bacteria enter the productive state PCT
, continue
reproducing at a potentially reduced rate ␥, and begin producing phage at a rate C without cell death
(72).
Similarly, if a bacterium that is latently infected with a chronic phage is infected with the temperate
共C兲
共C兲
兲 with probability fT or will enter a lytic state ICT
with
phage, it will either become latently infected 共LCT
probability (1 ⫺ fT). Bacteria in the lytic state produce phage and burst (with burst size T) at a rate ␦.
While in the lytic state, the phage hijacks cell functions, and the cell cannot reproduce.
If a productive bacterium is then infected with the temperate phage, the bacterium will become
共C兲
兲 with probability fT. Otherwise, the productive bacterium will
latently infected with temperate phage 共PCT
共P兲
enter a lytic state ICT
with probability (1 ⫺ fT). Bacteria in the lytic state produce phage and burst (with
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 10
Modeling Control of Bacterial Infection via Proviruses
infection
Susceptible (S)
transition
reproduction
stress response
Lytic (IT)
Pre-productive (IC)
Latent Lytic (LT)
Pre-productive
(ICT, T)
Latent Lytic and
Latent Chronic
(LCT, T)
Productive (PC)
Latent Chronic (LC)
Lytic (ICT, P)
Lytic (ICT, C)
Latent Lytic and
Latent Chronic
(LCT, C)
Productive and
Latent Lytic (PCT, C)
Productive and
Latent Lytic (PCT, T)
FIG 8 Full flowchart of bacterium-phage system, corresponding to model system (see equations S1 to S15 in Text S1). Skull sketch courtesy of Dawn Hudson
(CC0).
burst size T) at a rate ␦. While in the lytic state, the phage hijacks cell functions, and the cell cannot
reproduce.
As shown in Fig. 7, without the addition of new susceptible bacteria, this infection process results
quickly in a population of cells that phenotypically are either doubly infected by both phages in the
latent state or producing the chronic virus and latently infected with temperate phage.
Infection. Many models of bacterium-phage interaction assume that a mass action process governs
infection (41, 44), but P. aeruginosa-phage infection rates are not well approximated by a mass action
process (74, 75). More realistically, infection rates decrease as population growth activates quorumsensing and biofilm formation (76). One way to accommodate this infection process is to replace a mass
action term with a Michaelis-Menten or Hollings type II functional response. In this case, all infection and
absorption rates are proportional to the nonlinear response
r(V, B) ⫽
VB
h ⫹ B
(1)
where V is the phage of interest, B is the bacterium of interest, and h is the bacterial population
at which the infection rate is half of the maximum. For small bacterial populations (B ⬇ 0), infection
is approximately a mass action process. As the bacterial population grows, the infection rate
saturates (Fig. 9a).
Antibiotics. Because patients infected with P. aeruginosa are typically treated with antibiotics at the
time of bacterial detection (77, 78), we must incorporate the effects of antibiotic doses administered at
times ti on the bacterium-phage ecosystem. We assume that system stress spikes at times ti (when
antibiotics become bioavailable) and decays exponentially, consistent with typical antibiotic metabolism
in the human system (79–81). The functional form of stress is then
s(t, 兵ti其) ⫽ A
冘
N
H(t ⫺ ti)exp(⫺k(t ⫺ ti))
(2)
i⫽1
where t is the current time, {ti} is a list of antibiotic dose times, A is the amplitude of stress due to one
antibiotic dose, N is the total number of antibiotic doses, H is the Heaviside function, and k is the decay
rate of antibiotics in the system. For inhaled or intravenous antibiotics, the dose times are the exact times
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 11
Clifton et al.
7
(a)
(b)
r(V,B)
s(t, {ti})
1
0
0
1
10
0
0
0
5
0
1
10
time t
15
20
(d)
g(s)
(c)
b(s)
100
bacteria B
stress s
8
0
0
stress s
8
FIG 9 Sketches of the functions for infection r(V,B) with phage density V ⫽ 10 (a), antibiotic stress s(t,{ti}) with
{ti} ⫽ {5,15} (b), phage production b(s) (c), and cell reproduction multiplier g(s) (d). Parameter values are taken
from the baselines in Table 2.
of antibiotic administration. For oral antibiotics, {ti} are the times at which the antibiotics become
bioavailable in the bloodstream (Fig. 9b).
When the system is stressed, the following three processes occur. (i) Bacteria that are susceptible to
the antibiotics die at a rate proportional to the amount of antibiotic in the system (82). If certain strains
of bacteria are resistant to antibiotics, then they will not be killed directly by antibiotics (83–85). (ii)
Bacteria that are infected by temperate phage induce phage production at a rate equal to the stress (34,
86, 87). In other words, stress measures the rate at which latent lytic bacteria induce phage. Note that
not all antibiotics induce phage (34), so we focus only on the types of antibiotics known to do so (e.g.,
quinolones like levofloxacin and ciprofloxacin) (8, 88). We assume that even antibiotic-resistant bacteria
induce viruses in the presence of antibiotics, which has been demonstrated for several classes of
antibiotics (34, 61, 66–68). (iii) Productive bacteria increase phage production and decrease cell reproduction (89, 90). A simple way to incorporate increased phage production during system stress is with
TABLE 1 Description of model variables in bacterium-phage systema
Variable
S
IT
IC
LT
PC
LC
共T兲
ICT
共P兲
ICT
共C兲
ICT
共T兲
LCT
共T兲
PCT
共C兲
LCT
共C兲
PCT
Btot
VT
VC
Vtot
t
Meaning
Density of susceptible bacteria
Density of lytic bacteria preparing to burst
Density of preproductive bacteria preparing to manufacture phage
Density of latent lytic bacteria
Density of productive bacteria
Density of latent chronic bacteria
Density of latent lytic bacteria that have entered preproductive state
Density of productive bacteria that have become lytic
Density of latent chronic bacteria that have become lytic
Density of latent chronic and latent lytic bacteria (first infection, VT; second infection, VC)
Density of productive and latent lytic bacteria (first infection, VT; second infection, VC)
Density of latent chronic and latent lytic bacteria (first infection, VC; second infection, VT)
Density of productive and latent lytic bacteria (first infection, VC; second infection, VT)
Density of all bacteria
Density of free temperate phage
Density of free chronic phage
Density of all free phage
Time normalized by bacterial reproduction rate
aSee
equations S1 to S15 in Text S1 in the supplemental material. Due to nondimensionalization of density and time, all variables and parameters are
nondimensional; all densities are relative to the bacterial carrying capacity, and all rates are relative to the growth rate of bacteria under ideal conditions.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 12
Modeling Control of Bacterial Infection via Proviruses
TABLE 2 Description of model parameters in bacterium-phage systemm
Parameter
␥
K
A
k
{ti}
␦
fT
fC
T
C
max
d
Meaning
Growth rate of bacteria under ideal conditions, normalized to 1a
Proportion growth rate change due to productive chronic infection
Carrying capacity of bacteria, normalized to 1c
Infection rate
Bacterial death rate due to antibiotic, relative to antibiotic lysis induction rate
Amplitude of stress (rate at which antibiotic induces lysis) introduced with
one antibiotic dose
Metabolic decay rate of antibiotic within the system
Vector of antibiotic administration times
Rate at which infection leads to phage production (eclipse and rise phase)
Fraction of bacteria infected with VT that become latently infected
Fraction of bacteria infected with VC that become latently infected
Burst size for bacteria infected with VT
Phage production rate for bacteria infected with VC
Maximum phage production rate for bacteria infected with VC under
maximum stress
Rate of free phage degradation
Range
1
(0.5, 3)b
1
(0, 40)
(0, 3.5)e
(0, 2)
Baseline
1 (5.1e⫺3 min⫺1)
1
1 (4e7 CFU/ml)
20 (0.10 min⫺1)d
1
1.1 (5.6e⫺3 min⫺1)f
(1e⫺3, 0.6)g 0.3 (1.7e⫺3
min⫺1)h
(1.5, 7.3)i
(0, 1)
(0, 1)
(10, 1,000)
(5, 200)
(10, 10,000)
4 (2.0e⫺2 min⫺1)
0.01
0.01j
100
10 (5.1e⫺2 min⫺1)k
100 (0.51 min⫺1)
(0.9, 3.6)l
1 (5.1e⫺3 min⫺1)
Reference(s)
93, 94
72
95, 96
41
93, 97
93, 98
93, 99, 100
55
101, 102
103, 104
101, 102, 105–109
34
110
rate is approximately 5.1e⫺3 min⫺1 for P. aeruginosa grown in vitro but is highly variable in cystic fibrosis patients.
bEstimates based on Escherichia coli and M13 phage.
cStable bacterial density in sputum is highly variable in patients with cystic fibrosis; a study of viable P. aeruginosa densities in sputum of 12 patients not undergoing
treatment ranged from 5.3e3 CFU/ml to 1.8e11 CFU/ml; log differences between control/placebo and treatment are more commonly reported. We select a carrying
capacity near the geometric mean of that range; see the supplemental material for details.
dEstimate based on E. coli and phage; see the supplemental material for details.
eEstimate for antibiotic levofloxacin (upper limit on death rate may include death by phage induction).
fEstimated from in vitro experiment using antimicrobial peptides and meropenem; see the supplemental material for details.
gLow estimate is for meropenem in vitro; high estimate is for ciprofloxacin in vivo (human).
hAntibiotic is levofloxacin (half-life approximately 6.9 h); see the supplemental material for details.
iLow estimate is for PAXYB1 phage and PAO1 host, and high estimate is for PAK_P3 phage and PAO1 host; see the supplemental material for details.
jGuess based on temperate phage.
kGuess based on author experience.
lLow estimate is for phage extracted from Raunefjorden, and high estimate is for phage extracted from Bergen Harbor (strains unknown).
mSee equations S1 to S15 in Text S1 in the supplemental material. Due to nondimensionalization of density and time, all variables and parameters are
nondimensional; all densities are relative to the bacterial carrying capacity, and all rates are relative to the growth rate of bacteria under ideal conditions. Commonly
used density and time units are noted in parentheses for baseline rates.
aGrowth
a Hollings-like functional response. With no system stress, the phage production rate is C, and with
increasing system stress, the phage production rate saturates at max:
b(s) ⫽ C ⫹
s
( ⫺ C)
h ⫹ s max
(3)
where s is the time-dependent stress level (equation 2) in the system, h is the stress level at which the
production rate is halfway between the minimum and maximum, and max is the maximum production
rate when stress is maximal (Fig. 9c). We assume that bacteria that are latently infected with the chronic
virus do not induce phage production, although there is evidence that this occurs in real-world systems.
Similarly, a simple way to incorporate decreased cell reproduction during system stress is with a
Hollings-like functional response. With no system stress, the cell reproduction rate is ␥, and with
increasing system stress, reproduction slows by a factor of g(s), and the cell eventually stops reproducing:
g(s) ⫽ 1 ⫺
s
h␥ ⫹ s
(4)
where s is the time-dependent stress level (equation 2) in the system and h␥ is the stress level at which the
growth rate is half the maximum. As stress increases, the bacterium eventually stops reproducing (Fig. 9d).
Tables 1 and 2 show variable and parameter definitions, respectively. See Text S2 in the supplemental
material for a discussion on parameter selection. See equations S1 to S15 in Text S1 for the dynamical
systems model.
Data availability. All software (Matlab.m files) is publicly available from the Illinois Data Bank at
https://databank.illinois.edu/datasets/IDB-9721455 (91).
SUPPLEMENTAL MATERIAL
Supplemental material for this article may be found at https://doi.org/10.1128/
mSystems.00221-19.
TEXT S1, PDF file, 0.1 MB.
TEXT S2, PDF file, 0.2 MB.
FIG S1, PDF file, 0.1 MB.
September/October 2019 Volume 4 Issue 5 e00221-19
msystems.asm.org 13
Clifton et al.
ACKNOWLEDGMENTS
This work was funded in part by the National Science Foundation grant DMS1815764 (Z.R.), the Cystic Fibrosis Foundation grant WHITAK16PO (R.J.W.), an Allen
Distinguished Investigator Award (R.J.W.), and National Institutes of Health grant R37
AI83256-06 (G.A.O.). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
The authors declare no competing interests.
REFERENCES
1. Ventola CL. 2015. The antibiotic resistance crisis: part 1: causes and
threats. P T 40:277–283.
2. Jarvis WR, Martone WJ. 1992. Predominant pathogens in hospital infections. J Antimicrob Chemother 29:19 –24. https://doi.org/10.1093/
jac/29.suppl_A.19.
3. Hancock RE, Speert DP. 2000. Antibiotic resistance in Pseudomonas
aeruginosa: mechanisms and impact on treatment. Drug Resist Updat
3:247–255. https://doi.org/10.1054/drup.2000.0152.
4. Centers for Disease Control and Prevention. 2013. Antibiotic resistance
threats in the United States, 2013. Centers for Disease Control and
Prevention, Atlanta, GA.
5. Driscoll JA, Brody SL, Kollef MH. 2007. The epidemiology, pathogenesis
and treatment of Pseudomonas aeruginosa infections. Drugs 67:
351–368. https://doi.org/10.2165/00003495-200767030-00003.
6. Kanj SS, Sexton DJ. 2019. Principles of antimicrobial therapy of Pseudomonas aeruginosa infections. UpToDate Inc, Waltham, MA.
7. Lin Y, Chang RYK, Britton WJ, Morales S, Kutter E, Chan HK. 2018.
Synergy of nebulized phage PEV20 and ciprofloxacin combination
against Pseudomonas aeruginosa. Int J Pharm 551:158 –165. https://doi
.org/10.1016/j.ijpharm.2018.09.024.
8. Comeau AM, Tétart F, Trojet SN, Prere MF, Krisch H. 2007. Phageantibiotic synergy (PAS): -lactam and quinolone antibiotics stimulate
virulent phage growth. PLoS One 2:e799. https://doi.org/10.1371/
journal.pone.0000799.
9. Kutter E, De Vos D, Gvasalia G, Alavidze Z, Gogokhia L, Kuhl S,
Abedon ST. 2010. Phage therapy in clinical practice: treatment of
human infections. Curr Pharm Biotechnol 11:69 – 86. https://doi.org/
10.2174/138920110790725401.
10. Jansen M, Wahida A, Latz S, Krüttgen A, Häfner H, Buhl EM, Ritter K,
Horz HP. 2018. Enhanced antibacterial effect of the novel T4-like bacteriophage KARL-1 in combination with antibiotics against multi-drug
resistant Acinetobacter baumannii. Sci Rep 8:14140. https://doi.org/10
.1038/s41598-018-32344-y.
11. Valério N, Oliveira C, Jesus V, Branco T, Pereira C, Moreirinha C, Almeida
A. 2017. Effects of single and combined use of bacteriophages and
antibiotics to inactivate Escherichia coli. Virus Res 240:8 –17. https://doi
.org/10.1016/j.virusres.2017.07.015.
12. Moulton-Brown CE, Friman VP. 2018. Rapid evolution of generalized resistance mechanisms can constrain the efficacy of phage-antibiotic treatments. Evol Appl 11:1630–1641. https://doi.org/10.1111/eva.12653.
13. Torres-Barceló C, Hochberg ME. 2016. Evolutionary rationale for phages
as complements of antibiotics. Trends Microbiol 24:249 –256. https://
doi.org/10.1016/j.tim.2015.12.011.
14. Chaudhry WN, Concepción-Acevedo J, Park T, Andleeb S, Bull JJ, Levin
BR. 2017. Synergy and order effects of antibiotics and phages in killing
Pseudomonas aeruginosa biofilms. PLoS One 12:e0168615. https://doi
.org/10.1371/journal.pone.0168615.
15. Knezevic P, Curcin S, Aleksic V, Petrusic M, Vlaski L. 2013. Phageantibiotic synergism: a possible approach to combatting Pseudomonas
aeruginosa. Res Microbiol 164:55– 60. https://doi.org/10.1016/j.resmic
.2012.08.008.
16. Torres-Barceló C, Arias-Sánchez FI, Vasse M, Ramsayer J, Kaltz O, Hochberg ME. 2014. A window of opportunity to control the bacterial
pathogen Pseudomonas aeruginosa combining antibiotics and phages.
PLoS One 9:e106628. https://doi.org/10.1371/journal.pone.0106628.
17. Oechslin F, Piccardi P, Mancini S, Gabard J, Moreillon P, Entenza JM,
Resch G, Que YA. 2017. Synergistic interaction between phage therapy
and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence. J Infect Dis 215:703–712. https://doi.org/
10.1093/infdis/jiw632.
September/October 2019 Volume 4 Issue 5 e00221-19
18. Torres-Barceló C, Franzon B, Vasse M, Hochberg ME. 2016. Long-term
effects of single and combined introductions of antibiotics and bacteriophages on populations of Pseudomonas aeruginosa. Evol Appl
9:583–595. https://doi.org/10.1111/eva.12364.
19. Weinbauer MG. 2004. Ecology of prokaryotic viruses. FEMS Microbiol
Rev 28:127–181. https://doi.org/10.1016/j.femsre.2003.08.001.
20. Rakonjac J. 2012. Filamentous bacteriophages: biology and applications. In eLS. Wiley Online Library. John Wiley & Sons, Inc, Hoboken, NJ.
21. Lwoff A. 1953. Lysogeny. Bacteriol Rev 17:269.
22. Davies EV, Winstanley C, Fothergill JL, James CE. 2016. The role of
temperate bacteriophages in bacterial infection. FEMS Microbiol Lett
363:fnw015. https://doi.org/10.1093/femsle/fnw015.
23. Roux S, Hallam SJ, Woyke T, Sullivan MB. 2015. Viral dark matter and
virus– host interactions resolved from publicly available microbial genomes. Elife 4:e08490. https://doi.org/10.7554/eLife.08490.
24. Mathee K, Narasimhan G, Valdes C, Qiu X, Matewish JM, Koehrsen M,
Rokas A, Yandava CN, Engels R, Zeng E, Olavarietta R, Doud M, Smith
RS, Montgomery P, White JR, Godfrey PA, Kodira C, Birren B, Galagan JE,
Lory S. 2008. Dynamics of Pseudomonas aeruginosa genome evolution.
Proc Natl Acad Sci U S A 105:3100 –3105. https://doi.org/10.1073/pnas
.0711982105.
25. Mosquera-Rendón J, Rada-Bravo AM, Cárdenas-Brito S, Corredor M,
Restrepo-Pineda E, Benítez-Páez A. 2016. Pangenome-wide and molecular evolution analyses of the Pseudomonas aeruginosa species. BMC
Genomics 17:45. https://doi.org/10.1186/s12864-016-2364-4.
26. Spencer DH, Kas A, Smith EE, Raymond CK, Sims EH, Hastings M, Burns
JL, Kaul R, Olson MV. 2003. Whole-genome sequence variation among
multiple isolates of Pseudomonas aeruginosa. J Bacteriol 185:
1316 –1325. https://doi.org/10.1128/JB.185.4.1316-1325.2003.
27. Kung VL, Ozer EA, Hauser AR. 2010. The accessory genome of Pseudomonas aeruginosa. Microbiol Mol Biol Rev 74:621– 641. https://doi
.org/10.1128/MMBR.00027-10.
28. James CE, Davies EV, Fothergill JL, Walshaw MJ, Beale CM, Brockhurst
MA, Winstanley C. 2015. Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections. ISME J 9:1391. https://doi.org/10.1038/ismej.2014.223.
29. Torres-Barceló C. 2018. The disparate effects of bacteriophages on
antibiotic-resistant bacteria. Emerg Microbes Infect 7:168. https://doi
.org/10.1038/s41426-018-0169-z.
30. Davies EV, James CE, Brockhurst MA, Winstanley C. 2017. Evolutionary
diversification of Pseudomonas aeruginosa in an artificial sputum
model. BMC Microbiol 17:3. https://doi.org/10.1186/s12866-016-0916-z.
31. Davies EV, James CE, Williams D, O’Brien S, Fothergill JL, Haldenby S,
Paterson S, Winstanley C, Brockhurst MA. 2016. Temperate phages both
mediate and drive adaptive evolution in pathogen biofilms. Proc Natl Acad
Sci U S A 113:8266–8271. https://doi.org/10.1073/pnas.1520056113.
32. Wang X, Kim Y, Ma Q, Hong SH, Pokusaeva K, Sturino JM, Wood TK.
2010. Cryptic prophages help bacteria cope with adverse environments. Nat Commun 1:147. https://doi.org/10.1038/ncomms1146.
33. Rokney A, Kobiler O, Amir A, Court DL, Stavans J, Adhya S, Oppenheim
AB. 2008. Host responses influence on the induction of lambda prophage. Mol Microbiol 68:29 –36. https://doi.org/10.1111/j.1365-2958
.2008.06119.x.
34. Fothergill JL, Mowat E, Walshaw MJ, Ledson MJ, James CE, Winstanley C.
2011. Effect of antibiotic treatment on bacteriophage production by a
cystic fibrosis epidemic strain of Pseudomonas aeruginosa. Antimicrob
Agents Chemother 55:426–428. https://doi.org/10.1128/AAC.01257-10.
35. López E, Domenech A, Ferrándiz MJ, Frias MJ, Ardanuy C, Ramirez M,
García E, Liñares J, de la Campa AG. 2014. Induction of prophages by
fluoroquinolones in Streptococcus pneumoniae: implications for emermsystems.asm.org 14
Modeling Control of Bacterial Infection via Proviruses
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
gence of resistance in genetically-related clones. PLoS One 9:e94358.
https://doi.org/10.1371/journal.pone.0094358.
Martínez-García E, Jatsenko T, Kivisaar M, de Lorenzo V. 2015. Freeing
Pseudomonas putida KT 2440 of its proviral load strengthens endurance to environmental stresses. Environ Microbiol 17:76 –90. https://
doi.org/10.1111/1462-2920.12492.
Kaur S, Harjai K, Chhibber S. 2012. Methicillin-resistant Staphylococcus
aureus phage plaque size enhancement using sublethal concentrations
of antibiotics. Appl Environ Microbiol 78:8227– 8233. https://doi.org/10
.1128/AEM.02371-12.
Kim M, Jo Y, Hwang YJ, Hong HW, Hong SS, Park K, Myung H. 2018.
Phage-antibiotic synergy via delayed lysis. Appl Environ Microbiol 84:
e02085-18. https://doi.org/10.1128/AEM.02085-18.
Weitz JS, Dushoff J. 2008. Alternative stable states in host-phage dynamics.
Theor Ecol 1:13–19. https://doi.org/10.1007/s12080-007-0001-1.
Payne RJ, Jansen VA. 2001. Understanding bacteriophage therapy as a
density-dependent kinetic process. J Theor Biol 208:37– 48. https://doi
.org/10.1006/jtbi.2000.2198.
Sinha V, Goyal A, Svenningsen SL, Semsey S, Krishna S. 2017. In silico
evolution of lysis-lysogeny strategies reproduces observed lysogeny
propensities in temperate bacteriophages. Front Microbiol 8:1386.
https://doi.org/10.3389/fmicb.2017.01386.
Gulbudak H, Weitz JS. 2019. Heterogeneous viral strategies promote
coexistence in virus-microbe systems. J Theor Biol 462:65– 84. https://
doi.org/10.1016/j.jtbi.2018.10.056.
Levin BR, Bull J. 1996. Phage therapy revisited: the population biology
of a bacterial infection and its treatment with bacteriophage and
antibiotics. Am Nat 147:881– 898. https://doi.org/10.1086/285884.
Leung CYJ, Weitz JS. 2017. Modeling the synergistic elimination of
bacteria by phage and the innate immune system. J Theor Biol 429:
241–252. https://doi.org/10.1016/j.jtbi.2017.06.037.
Levin BR, Bull JJ. 2004. Population and evolutionary dynamics of phage
therapy. Nat Rev Microbiol 2:166. https://doi.org/10.1038/nrmicro822.
Ankomah P, Levin BR. 2014. Exploring the collaboration between antibiotics and the immune response in the treatment of acute, selflimiting infections. Proc Natl Acad Sci U S A 111:8331– 8338. https://
doi.org/10.1073/pnas.1400352111.
Bohannan BJM, Lenski RE. 2000. Linking genetic change to community
evolution: insights from studies of bacteria and bacteriophage. Ecol
Lett 3:362–377. https://doi.org/10.1046/j.1461-0248.2000.00161.x.
Tam VH, Schilling AN, Poole K, Nikolaou M. 2007. Mathematical modelling response of Pseudomonas aeruginosa to meropenem. J Antimicrob Chemother 60:1302–1309. https://doi.org/10.1093/jac/dkm370.
Webb GF, D’Agata EMC, Magal P, Ruan S. 2005. A model of antibioticresistant bacterial epidemics in hospitals. Proc Natl Acad Sci U S A
102:13343–13348. https://doi.org/10.1073/pnas.0504053102.
Levin BR, Baquero F, Johnsen PJ. 2014. A model-guided analysis and
perspective on the evolution and epidemiology of antibiotic resistance
and its future. Curr Opin Microbiol 19:83– 89. https://doi.org/10.1016/j
.mib.2014.06.004.
Touchon M, de Sousa JAM, Rocha EP. 2017. Embracing the enemy: the
diversification of microbial gene repertoires by phage-mediated horizontal gene transfer. Curr Opin Microbiol 38:66 –73. https://doi.org/10
.1016/j.mib.2017.04.010.
Stanczak-Mrozek KI, Laing KG, Lindsay JA. 2017. Resistance gene transfer:
induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage. J Antimicrob
Chemother 72:1624–1631. https://doi.org/10.1093/jac/dkx056.
Winstanley C, Langille MG, Fothergill JL, Kukavica-Ibrulj I, Paradis-Bleau
C, Sanschagrin F, Thomson NR, Winsor GL, Quail MA, Lennard N, Bignell
A, Clark L, Seeger K, Saunders D, Harris D, Parkhill J, Hancock RE,
Brinkman FS, Levesque RC. 2008. Newly introduced genomic prophage
islands are critical determinants of in vivo competitiveness in the
Liverpool Epidemic Strain of Pseudomonas aeruginosa. Genome Res
19:12–23. https://doi.org/10.1101/gr.086082.108.
Aldred KJ, Kerns RJ, Osheroff N. 2014. Mechanism of quinolone action
and resistance. Biochemistry 53:1565–1574. https://doi.org/10.1021/
bi5000564.
Stockmann C, Sherwin CM, Zobell JT, Young DC, Waters CD, Spigarelli
MG, Ampofo K. 2013. Optimization of anti-pseudomonal antibiotics for
cystic fibrosis pulmonary exacerbations: III. Fluoroquinolones. Pediatr
Pulmonol 48:211–220. https://doi.org/10.1002/ppul.22667.
Knowles B, Silveira CB, Bailey BA, Barott K, Cantu VA, Cobián-Güemes
AG, Coutinho FH, Dinsdale EA, Felts B, Furby KA, George EE, Green KT,
September/October 2019 Volume 4 Issue 5 e00221-19
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
Gregoracci GB, Haas AF, Haggerty JM, Hester ER, Hisakawa N, Kelly LW,
Lim YW, Little M, Luque A, McDole-Somera T, McNair K, de Oliveira LS,
Quistad SD, Robinett NL, Sala E, Salamon P, Sanchez SE, Sandin S, Silva
GGZ, Smith J, Sullivan C, Thompson C, Vermeij MJA, Youle M, Young C,
Zgliczynski B, Brainard R, Edwards RA, Nulton J, Thompson F, Rohwer F.
2016. Lytic to temperate switching of viral communities. Nature 531:
466. https://doi.org/10.1038/nature17193.
Geller DE, Flume PA, Staab D, Fischer R, Loutit JS, Conrad DJ. 2011.
Levofloxacin inhalation solution (MP-376) in patients with cystic fibrosis
with Pseudomonas aeruginosa. Am J Respir Crit Care Med 183:
1510 –1516. https://doi.org/10.1164/rccm.201008-1293OC.
Proesmans M, Vermeulen F, Boulanger L, Verhaegen J, De Boeck K.
2013. Comparison of two treatment regimens for eradication of Pseudomonas aeruginosa infection in children with cystic fibrosis. J Cyst
Fibros 12:29 –34. https://doi.org/10.1016/j.jcf.2012.06.001.
Sweere JM, Van Belleghem JD, Ishak H, Bach MS, Popescu M, Sunkari V,
Kaber G, Manasherob R, Suh GA, Cao X, de Vries CR, Lam DN, Marshall
PL, Birukova M, Katznelson E, Lazzareschi DV, Balaji S, Keswani SG,
Hawn TR, Secor PR, Bollyky PL. 2019. Bacteriophage trigger antiviral
immunity and prevent clearance of bacterial infection. Science 363:
eaat9691. https://doi.org/10.1126/science.aat9691.
Burgener EB, Sweere JM, Bach MS, Secor PR, Haddock N, Jennings LK,
Marvig RL, Johansen HK, Rossi E, Cao X, Tian L, Nedelec L, Molin S,
Bollyky PL, Milla C. 2019. Filamentous bacteriophages are associated
with chronic Pseudomonas lung infections and antibiotic resistance in
cystic fibrosis. Sci Transl Med 11:eaau9748. https://doi.org/10.1126/
scitranslmed.aau9748.
James CE, Fothergill JL, Kalwij H, Hall AJ, Cottell J, Brockhurst MA,
Winstanley C. 2012. Differential infection properties of three inducible
prophages from an epidemic strain of Pseudomonas aeruginosa. BMC
Microbiol 12:216. https://doi.org/10.1186/1471-2180-12-216.
Heo YJ, Chung IY, Choi KB, Lau GW, Cho YH. 2007. Genome sequence
comparison and superinfection between two related Pseudomonas
aeruginosa phages, D3112 and MP22. Microbiology 153:2885–2895.
https://doi.org/10.1099/mic.0.2007/007260-0.
Hodson M, Butland R, Roberts C, Smith M, Batten J. 1987. Oral ciprofloxacin compared with conventional intravenous treatment for Pseudomonas aeruginosa infection in adults with cystic fibrosis. Lancet
329:235–237. https://doi.org/10.1016/S0140-6736(87)90062-6.
Hargreaves KR, Kropinski AM, Clokie MR. 2014. What does the talking?:
quorum sensing signalling genes discovered in a bacteriophage genome.
PLoS One 9:e85131. https://doi.org/10.1371/journal.pone.0085131.
Silpe JE, Bassler BL. 2018. A host-produced quorum-sensing autoinducer controls a phage lysis-lysogeny decision. Cell 176:268 –280.e13.
https://doi.org/10.1016/j.cell.2018.10.059.
Redgrave LS, Sutton SB, Webber MA, Piddock LJ. 2014. Fluoroquinolone
resistance: mechanisms, impact on bacteria, and role in evolutionary
success. Trends Microbiol 22:438 – 445. https://doi.org/10.1016/j.tim
.2014.04.007.
Valencia EY, Esposito F, Spira B, Blázquez J, Galhardo RS. 2017.
Ciprofloxacin-mediated mutagenesis is suppressed by subinhibitory concentrations of amikacin in Pseudomonas aeruginosa. Antimicrob Agents
Chemother 61:e02107-16. https://doi.org/10.1128/AAC.02107-16.
Brazas MD, Hancock RE. 2005. Ciprofloxacin induction of a susceptibility
determinant in Pseudomonas aeruginosa. Antimicrob Agents Chemother
49:3222–3227. https://doi.org/10.1128/AAC.49.8.3222-3227.2005.
Zwietering MH, Jongenburger I, Rombouts FM, van ‘t Riet K. 1990.
Modeling of the bacterial growth curve. Appl Environ Microbiol 56:
1875–1881.
Tabib-Salazar A, Liu B, Shadrin A, Burchell L, Wang Z, Wang Z, Goren
MG, Yosef I, Qimron U, Severinov K, Matthews SJ, Wigneshweraraj S.
2017. Full shut-off of Escherichia coli RNA-polymerase by T7 phage
requires a small phage-encoded DNA-binding protein. Nucleic Acids
Res 45:7697–7707. https://doi.org/10.1093/nar/gkx370.
St-Pierre F, Endy D. 2008. Determination of cell fate selection during
phage lambda infection. Proc Natl Acad Sci U S A 105:20705–20710.
https://doi.org/10.1073/pnas.0808831105.
Shapiro JW, Williams ES, Turner PE. 2016. Evolution of parasitism and
mutualism between filamentous phage M13 and Escherichia coli. PeerJ
4:e2060. https://doi.org/10.7717/peerj.2060.
De Smet J, Hendrix H, Blasdel BG, Danis-Wlodarczyk K, Lavigne R. 2017.
Pseudomonas predators: understanding and exploiting phage-host
interactions. Nat Rev Microbiol 15:517. https://doi.org/10.1038/nrmicro
.2017.61.
msystems.asm.org 15
Clifton et al.
74. Simmons M, Drescher K, Nadell CD, Bucci V. 2018. Phage mobility is a
core determinant of phage-bacteria coexistence in biofilms. ISME J
12:531. https://doi.org/10.1038/ismej.2017.190.
75. Vidakovic L, Singh PK, Hartmann R, Nadell CD, Drescher K. 2018.
Dynamic biofilm architecture confers individual and collective mechanisms of viral protection. Nat Microbiol 3:26. https://doi.org/10.1038/
s41564-017-0050-1.
76. Harper DR, Parracho HM, Walker J, Sharp R, Hughes G, Werthén M,
Lehman S, Morales S. 2014. Bacteriophages and biofilms. Antibiotics
3:270 –284. https://doi.org/10.3390/antibiotics3030270.
77. Høiby N, Bjarnsholt T, Moser C, Jensen PØ, Kolpen M, Qvist T, Aanaes K,
Pressler T, Skov M, Ciofu O. 2017. Diagnosis of biofilm infections in
cystic fibrosis patients. APMIS 125:339 –343. https://doi.org/10.1111/
apm.12689.
78. Høiby N, Bjarnsholt T, Moser C, Bassi G, Coenye T, Donelli G, HallStoodley L, Hola V, Imbert C, Kirketerp-Møller K, Lebeaux D, Oliver A,
Ullmann A, Williams C. 2015. ESCMID guideline for the diagnosis and
treatment of biofilm infections 2014. Clin Microbiol Infect 21:S1–S25.
https://doi.org/10.1016/j.cmi.2014.10.024.
79. Naber KG, Westenfelder SR, Madsen PO. 1973. Pharmacokinetics of the
aminoglycoside antibiotic tobramycin in humans. Antimicrob Agents
Chemother 3:469 – 473. https://doi.org/10.1128/aac.3.4.469.
80. Bax R, Bastain W, Featherstone A, Wilkinson D, Hutchison M. 1989. The
pharmacokinetics of meropenem in volunteers. J Antimicrob Chemother 24:311–320. https://doi.org/10.1093/jac/24.suppl_A.311.
81. Fish DN, Chow AT. 1997. The clinical pharmacokinetics of levofloxacin. Clin
Pharmacokinet 32:101–119. https://doi.org/10.2165/00003088-199732020
-00002.
82. Levin BR, Udekwu KI. 2010. Population dynamics of antibiotic
treatment: a mathematical model and hypotheses for time-kill and
continuous-culture experiments. Antimicrob Agents Chemother 54:
3414 –3426. https://doi.org/10.1128/AAC.00381-10.
83. Fisher RA, Gollan B, Helaine S. 2017. Persistent bacterial infections and
persister cells. Nat Rev Microbiol 15:453. https://doi.org/10.1038/nrmicro
.2017.42.
84. Monack DM, Mueller A, Falkow S. 2004. Persistent bacterial infections:
the interface of the pathogen and the host immune system. Nat Rev
Microbiol 2:747. https://doi.org/10.1038/nrmicro955.
85. Stewart PS, Costerton JW. 2001. Antibiotic resistance of bacteria in
biofilms. Lancet 358:135–138. https://doi.org/10.1016/s0140-6736(01)
05321-1.
86. Ptashne M. 1986. A genetic switch: gene control and phage [lambda].
Cell Press, Cambridge, MA.
87. Nanda AM, Thormann K, Frunzke J. 2015. Impact of spontaneous
prophage induction on the fitness of bacterial populations and hostmicrobe interactions. J Bacteriol 197:410 – 419. https://doi.org/10.1128/
JB.02230-14.
88. Zhang X, McDaniel AD, Wolf LE, Keusch GT, Waldor MK, Acheson DW.
2000. Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J Infect Dis 181:664 – 670.
https://doi.org/10.1086/315239.
89. Hagens S, Habel A, Bläsi U. 2006. Augmentation of the antimicrobial
efficacy of antibiotics by filamentous phage. Microb Drug Resist 12:
164 –168. https://doi.org/10.1089/mdr.2006.12.164.
90. Secor PR, Sweere JM, Michaels LA, Malkovskiy AV, Lazzareschi D,
Katznelson E, Rajadas J, Birnbaum ME, Arrigoni A, Braun KR, Evanko SP,
Stevens DA, Kaminsky W, Singh PK, Parks WC, Bollyky PL. 2015. Filamentous bacteriophage promote biofilm assembly and function. Cell
Host Microbe 18:549 –559. https://doi.org/10.1016/j.chom.2015.10.013.
91. Rapti Z. 2019. Control of bacterial infections via antibiotic-induced
proviruses. Illinois Data Bank. University of Illinois at UrbanaChampaign, Urbana, IL. https://doi.org/10.13012/B2IDB-9721455_V1.
92. Marino S, Hogue IB, Ray CJ, Kirschner DE. 2008. A methodology for
performing global uncertainty and sensitivity analysis in systems biology. J Theor Biol 254:178 –196. https://doi.org/10.1016/j.jtbi.2008.04
.011.
93. Spalding C, Keen E, Smith DJ, Krachler AM, Jabbari S. 2018. Mathematical modelling of the antibiotic-induced morphological transition of
September/October 2019 Volume 4 Issue 5 e00221-19
94.
95.
96.
97.
98.
99.
100.
101.
102.
103.
104.
105.
106.
107.
108.
109.
110.
Pseudomonas aeruginosa. PLoS Comput Biol 14:e1006012. https://doi
.org/10.1371/journal.pcbi.1006012.
Kopf SH, Sessions AL, Cowley ES, Reyes C, Van Sambeek L, Hu Y, Orphan
VJ, Kato R, Newman DK. 2016. Trace incorporation of heavy water
reveals slow and heterogeneous pathogen growth rates in cystic fibrosis sputum. Proc Natl Acad Sci U S A 113:E110 –E116. https://doi.org/
10.1073/pnas.1512057112.
Stressmann FA, Rogers GB, Marsh P, Lilley AK, Daniels TW, Carroll MP,
Hoffman LR, Jones G, Allen CE, Patel N, Forbes N, Forbes B, Tuck A,
Bruce KD. 2011. Does bacterial density in cystic fibrosis sputum increase prior to pulmonary exacerbation? J Cyst Fibros 10:357–365.
https://doi.org/10.1016/j.jcf.2011.05.002.
Price KE, Hampton TH, Gifford AH, Dolben EL, Hogan DA, Morrison HG,
Sogin ML, O’Toole GA. 2013. Unique microbial communities persist in
individual cystic fibrosis patients throughout a clinical exacerbation.
Microbiome 1:27. https://doi.org/10.1186/2049-2618-1-27.
Grillon A, Schramm F, Kleinberg M, Jehl F. 2016. Comparative activity of
ciprofloxacin, levofloxacin and moxifloxacin against Klebsiella pneumoniae, Pseudomonas aeruginosa and Stenotrophomonas maltophilia
assessed by minimum inhibitory concentrations and time-kill studies.
PLoS One 11:e0156690. https://doi.org/10.1371/journal.pone.0156690.
Dutreix M, Bailone A, Devoret R. 1985. Efficiency of induction of prophage lambda mutants as a function of recA alleles. J Bacteriol 161:
1080 –1085.
Zhanel GG, Fontaine S, Adam H, Schurek K, Mayer M, Noreddin AM,
Gin AS, Rubinstein E, Hoban DJ. 2006. A review of new fluoroquinolones. Treat Respir Med 5:437– 465. https://doi.org/10.2165/00151829
-200605060-00009.
Wingender W, Graefe KH, Gau W, Förster D, Beermann D, Schacht P.
1984. Pharmacokinetics of ciprofloxacin after oral and intravenous
administration in healthy volunteers. Eur J Clin Microbiol 3:355–359.
https://doi.org/10.1007/BF01977494.
Yu X, Xu Y, Gu Y, Zhu Y, Liu X. 2017. Characterization and genomic
study of “phiKMV-Like” phage PAXYB1 infecting Pseudomonas aeruginosa. Sci Rep 7:13068. https://doi.org/10.1038/s41598-017-13363-7.
El Didamony G, Askora A, Shehata AA. 2015. Isolation and characterization of T7-like lytic bacteriophages infecting multidrug resistant
Pseudomonas aeruginosa isolated from Egypt. Curr Microbiol 70:
786 –791. https://doi.org/10.1007/s00284-015-0788-8.
Calendar RL (ed). 2006. The bacteriophages, 2nd ed. Oxford University
Press, New York, NY.
Court DL, Oppenheim AB, Adhya SL. 2007. A new look at bacteriophage
genetic networks. J Bacteriol 189:298 –304. https://doi.org/10.1128/
JB.01215-06.
Latino L, Essoh C, Blouin Y, Thien HV, Pourcel C. 2014. A novel Pseudomonas aeruginosa bacteriophage, Ab31, a chimera formed from
temperate phage PAJU2 and P. putida lytic phage AF: characteristics
and mechanism of bacterial resistance. PLoS One 9:e93777. https://doi
.org/10.1371/journal.pone.0093777.
Schrader HS, Schrader JO, Walker JJ, Wolf TA, Nickerson KW, Kokjohn
TA. 1997. Bacteriophage infection and multiplication occur in Pseudomonas aeruginosa starved for 5 years. Can J Microbiol 43:1157–1163.
https://doi.org/10.1139/m97-164.
Ceyssens PJ, Brabban A, Rogge L, Lewis MS, Pickard D, Goulding D,
Dougan G, Noben JP, Kropinski A, Kutter E, Lavigne R. 2010. Molecular
and physiological analysis of three Pseudomonas aeruginosa phages
belonging to the “N4-like viruses”. Virology 405:26 –30. https://doi.org/
10.1016/j.virol.2010.06.011.
Garbe J, Bunk B, Rohde M, Schobert M. 2011. Sequencing and characterization of Pseudomonas aeruginosa phage JG004. BMC Microbiol
11:102. https://doi.org/10.1186/1471-2180-11-102.
You L, Suthers PF, Yin J. 2002. Effects of Escherichia coli physiology on
growth of phage T7 in vivo and in silico. J Bacteriol 184:1888 –1894.
https://doi.org/10.1128/jb.184.7.1888-1894.2002.
Heldal M, Bratbak G. 1991. Production and decay of viruses in aquatic
environments. Mar Ecol Prog Ser 72:205–212. https://doi.org/10.3354/
meps072205.
msystems.asm.org 16
|
|
https://openalex.org/W4289878331
|
https://discovery.ucl.ac.uk/id/eprint/10160287/1/s13054-022-04104-y.pdf
|
English
| null |
Clarithromycin as an immunomodulator in sepsis: still a (IN)CLASS act
|
Critical care
| 2,022
|
cc-by
| 1,136
|
© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Dear Editor, whole blood transcriptomic data reflect changes spe-
cifically in monocytes is questionable. This may explain
why RNA sequencing data did not reveal differences in
pathways regulating monocyte HLA-DR, even though
HLA-DR is typically regulated at the transcriptional
level via Class II transactivator (CIITA) [4]. Intuitively, a
reduction in HLA-DR expression would be expected as
clarithromycin inhibits protein translation via bacterial
ribosomal inhibition, with human ribosomes affected at
higher concentrations [5]. We read with great interest the INCLASS study by
Karakike et al [1]. We commend the authors on conduct-
ing a clinical trial providing mechanistic insights, under-
pinned by a sound scientific rationale. While the primary
objective, 28-day mortality, was unaffected by addition
of clarithromycin to a beta-lactam in the management
of community-acquired pneumonia, the authors report a
significant reduction in sepsis recurrence associated with
increased monocyte HLA-DR. The lack of mortality ben-
efit associated with clarithromycin use is consistent with
a previous study from the authors in patients with venti-
lator-associated pneumonia [2]. Although clarithromycin use was associated with a
significantly lower day- 28 sepsis recurrence (67.9% vs. 30.4%), acute kidney injury was in fact, more common in
patients receiving clarithromycin and mortality rates (at
both 28 and 90 days) were similar between groups. Persistent downregulation of monocyte HLA-DR
expression, and lymphopenia are characteristic of sepsis-
induced immunosuppression. The authors suggest a four-
day course of clarithromycin may expedite recovery of
monocyte HLA-DR expression by day- 10 which may, in
turn, be responsible for reduced recurrence of sepsis. It
would be intriguing to know if monocyte co-stimulatory
molecule CD86 increased in tandem, as reported by the
authors in a previous study [3]. Assessment of effector
cell receptor expression (e.g., T-lymphocyte CD28 and
CTLA4) would provide greater insight. Finally, it is worth commenting on antimicrobial resist-
ance issues. Antimicrobial resistance is now endemic in
many parts of the world. The SENTRY Antimicrobial
Surveillance Program, collected between 2015 and 2017,
reported that 32.4% of S. pneumoniae isolates were resist-
ant to azithromycin. The authors report that the patho-
gens identified in their patient population were already
highly resistant (multi-drug resistant 24.4%; extremely
drug-resistant 47.4%; pan-drug resistant 7.7%) on study
enrollment, but do not provide data on macrolide resist-
ance. Additionally, the emergence of macrolide-resistant
organisms following clarithromycin treatment is worthy
of reporting. Snow et al. Critical Care (2022) 26:238
https://doi.org/10.1186/s13054-022-04104-y Snow et al. Critical Care (2022) 26:238
https://doi.org/10.1186/s13054-022-04104-y Open Access Open Access This comment refers to the article available online at https://doi.org/10.1186/
s13054-022-04055-4.
*Correspondence: timothy.snow@doctors.net.uk
Bloomsbury Institute of Intensive Care Medicine, University College London,
Gower Street, London WC1E 6DH, UK Dear Editor, Transcriptomics were performed from total RNA iso-
lated from whole blood, in which monocytes constitute
a small proportion of the leukocyte population. Whether Whilst this trial may not have achieved its primary
clinical outcome, it adds to our understanding of mac-
rolide-induced immunomodulation. Identification of the
optimal dose, time, duration and patient cohort to benefit
from macrolide-induced immune modulation are yet to
be realised. References 1. Karakike E, Scicluna BP, Roumpoutsou M, Mitrou I, Karampela N, Karageor-
gos A, et al. Effect of intravenous clarithromycin in patients with sepsis,
respiratory and multiple organ dysfunction syndrome: a randomized
clinical trial. Crit Care. 2022;26(1):183. 1. Karakike E, Scicluna BP, Roumpoutsou M, Mitrou I, Karampela N, Karageor-
gos A, et al. Effect of intravenous clarithromycin in patients with sepsis,
respiratory and multiple organ dysfunction syndrome: a randomized
clinical trial. Crit Care. 2022;26(1):183. 2. Giamarellos-Bourboulis EJ, Pechere JC, Routsi C, Plachouras D, Kollias S,
Raftogiannis M, et al. Effect of clarithromycin in patients with sepsis and
ventilator-associated pneumonia. Clin Infect Dis. 2008;46(8):1157–64. 3. Spyridaki A, Raftogiannis M, Antonopoulou A, Tsaganos T, Routsi C, Bazi-
aka F, et al. Effect of clarithromycin in inflammatory markers of patients
with ventilator-associated pneumonia and sepsis caused by Gram-nega-
tive bacteria: results from a randomized clinical study. Antimicrob Agents
Chemother. 2012;56(7):3819–25. 4. Ting JP, Trowsdale J. Genetic control of MHC class II expression. Cell. 2002;109(Suppl):S21-33. 5. Svetlov MS, Koller TO, Meydan S, Shankar V, Klepacki D, Polacek N, et al. Context-specific action of macrolide antibiotics on the eukaryotic ribo-
some. Nat Commun. 2021;12(1):2803. Competing interests Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. Received: 1 July 2022 Accepted: 9 July 2022 Received: 1 July 2022 Accepted: 9 July 2022 Page 2 of 2 Snow et al. Critical Care (2022) 26:238 Snow et al. Critical Care (2022) 26:238 Snow et al. Critical Care (2022) 26:238 Snow et al. Critical Care (2022) 26:238 •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress.
Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from: Author contributions NA and MS conceived the manuscript and provided critical review. TACS
and AC drafted the manuscript. All authors read and approved the final
manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub-
lished maps and institutional affiliations. •
fast, convenient online submission
•
thorough peer review by experienced researchers in your field
•
rapid publication on acceptance
•
support for research data, including large and complex data types
•
gold Open Access which fosters wider collaboration and increased citations
maximum visibility for your research: over 100M website views per year
•
At BMC, research is always in progress. Learn more biomedcentral.com/submissions
Ready to submit your research
Ready to submit your research ? Choose BMC and benefit from:
? Choose BMC and benefit from:
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.